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An International Online Open Access

Original Research Paper
Publication group

Municipal Solid Waste (MSW) production and management in Kovilpatti

Municipality
Journal of Research in Biology

Authors: ABSTRACT:
Govindarajan B1
Senthilkumar P2 and
Prabakaran V3.

Studies were performed to appreciate the present status to municipal solid

waste (MSW) in Kovilpatti town. MSW is a complex waste. It is a classic example
Institution: where many different types of wastes aggregate from domestic, commercial and
1
Department of Zoology, industrial sources within a single waste stream. Solid waste problem is universal but
Manonmaniam Sundaranar recycling and utilization of wastes according to local conditions is an important task.
University, Thirunelveli- Kovilpatti is an important town for match box industries. The present study is aimed to
627012, Tamil Nadu, India. find out possible ways of utilization of MSW of Kovilpatti town.
2
Entomo Pathology Lab,
Institute of Forest Genetics
and Tree Breeding,
Coimbatore-02, TamilNadu,
India.
3
Department of Zoology,
Government Arts College,
Karur-639005, Tamil Nadu, Keywords:
India. Municipal Solid Waste (MSW), Kovilpatti municipality.

Corresponding author:
Govindarajan B.

Article Citation:
Govindarajan B Senthilkumar P and Prabakaran V.
Municipal Solid Waste (MSW) production and management in Kovilpatti Municipality.
Journal of research in Biology (2011) 5: 307-311.

Web Address: Dates:

http://jresearchbiology.com/ Received: 25 Aug 2011 /Accepted: 27 Aug 2011 /Published: 01 Sep 2011
Documents/RA0091.pdf.

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

307-311 | JRB | 2011 | Vol 1 | No 5

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Research Journal www.ficuspublishers.com www.jresearchbiology.com

INTRODUCTION
India generates about 210 million tons of
Municipal solid wastes (MSW) each day and the
quantum of wastes produced increases at a rate of
1.5% per year (Abbasi and Ramasamy, 2001).The
urban population in India is increasing explosively
with proportionate increase in the solid waste,
causing great stress on environment. Improper
management of urban solid wastes causes pollution
of ground and surface water, land and air covering
biosphere problems. These problems are already
acute in cities, towns and villages as the disposal
facilities are not keep in pace with the quantum of
wastes being generated.
Garbage is a heterogeneous bulky material
of solid wastes generated from human dwelling and
other city habitation, which includes both
biodegradable and non-biodegradable components.
At all levels of development human beings produce
domestic, agricultural, industrial, hospital and
wastes at the public places. For designing the solid
waste management system of an area first important
aspect is to be known how much solid waste is
generated at a specific area.
Solid waste problem is universal but
recycling and utilization of wastes according to
local conditions is a difficult task. Kovilpatti is an
important town for match box industries, and
vegetable market. It has a population of 87450
(2001 census) in 36 wards with an extent of 6.59
sq.km (www.municipality.tn.gov.in). The solid
wastes produced by the city at present are disposed
of by dumping the wastes in the dumping yard in
Alampatti Village, 3 km away from the town
centre, which occupy an area of 3.81 acres. Since
improper disposal of municipal wastes cause severe
environmental pollution, the present study is aimed
to find out possible ways of utilization of MSW of
Kovilpatti town.
The sanitary infrastructure of the
municipality, sanitary staff, population density, area
covered, type of area, total quantity of waste
produced, vehicles used for collection, frequency of
collection, sources of waste and present disposal
method of municipality are the important aspects to
be considered for planning effective solid waste
management. Hence efforts must be taken for
proper scientific management of MSW in cities and
towns. The present study was to assess the solid
waste handling by Kovilpatti municipality.
308
Govindarajan et al.,2011
MATERIALS AND METHODS
Collection and Physical characterization:
Physical characterization of municipal solid
waste components in various sanitary divisions of
Kovilpatti was carried out according to the method
of Tchobanoglous et al (1977). To assess the
individual components present with in the mass of
the heterogeneous waste mix of the MSW, a truck
full of waste was collected from each sanitary
division (1-6) and transported to a corner of the
dumping yard where the waste was segregated into
different components by hand sorting and listed.
RESULTS
The municipality town of Kovilpatti is
divided into 36 wards and based on the
geographical location of these wards, grouped
together to form 6 sanitary divisions. Number of
population, streets, garbage bins, garbage collection
per day and culverts in Kovilpatti (Table 1), wards
covered under each sanitary division and the
population density are given in Table 2. Only 124
dust bins have been placed for the total area of 6.59
sq.km of Kovilpatti municipality. Slum area details
are given in Table 3. The various kinds of waste
composition in Kovilpatti municipality was debited
in Table 4. A sullage truck is used to collect the
night soil from the public toilets when the tank is
full. In addition to these tricycle is used to collect
and dump the bio-medical wastes from various
hospitals, dispensaries and clinics.
The profile of the current sanitary manpower
of Kovilpatti municipality involved in solid waste
disposal is given in Table 5. The staffs grouped into
different categories, viz., general administrative
staff, revenue department, accounts section,
engineering division, town planning section, and
public health section. In all these sanitary divisions
the organic materials such as food waste and the
plant residues dominated the other wastes. The
percentage presence of glass, iron products, rubber
and leather were found to be negligible. At present
dumping site was not fully improved to carry out
any composting activities. Therefore the municipal
authorities are trying to solve the problem.
DISCUSSION
Municipal solid waste management is a
complex problem and it demands creative solutions
from many disciplines. Studies on solid waste
management have been carried out by many
workers in various cities and towns: Nanda et al
(2000 a, b) in Burla Town, Orissa, Goswami et al
Journal of Research in Biology (2011) 5: 307-311
Govindarajan et al.,2011

Table: 1 Municipal solid waste management at Kovilpatti town

Garbage
No. of No. of
Ward No. Population collection per Culverts
Streets Garbage bins
day (MT)
1. 2629 5 2 0.815 2
2. 1768 5 3 0.522 5
3. 3633 1 2 1.033 6
4. 1684 9 5 0.476 27
5. 3487 7 3 0.9522 9
6. 2633 8 4 0.790 9
7. 2460 5 2 0.678 7
8. 2964 5 3 0.942 19
9. 3321 3 4 1.016 12
10. 2104 4 3 0.662 16
11. 2883 11 5 0.855 22
12. 2940 9 2 0.862 7
13. 3006 6 2 0.917 12
14. 1812 8 5 0.562 10
15. 2190 9 4 0.702 17
16. 1530 8 3 0.514 6
17. 2380 5 4 0.722 7
18. 2272 6 5 0.739 10
19. 2219 4 4 0.678 4
20. 1480 11 4 0.467 5
21. 1845 4 6 0.551 10
22. 1366 5 2 0.473 2
23. 1059 5 4 0.896 5
24. 1845 4 6 0.551 10
25. 2348 6 2 0.854 10
26. 3067 6 5 0.714 5
27. 2880 8 3 1.104 7
28. 3047 7 3 0.923 6
29. 2958 19 2 0.889 7
30. 3158 5 4 0.878 13
31. 2225 5 3 0.741 10
32. 2047 4 4 0.613 8
33. 2751 11 3 0.862 16
34. 2543 5 3 0.760 11
35. 3061 4 4 0.641 3
36. 2106 7 4 0.606 10

(2001) in Gauhati, Assam,; Saxena & Joshi (2002)
in Hardwar, Uttranchal,; Daniel & Paul (2004 a);
Paul (2005) and Paul & Daniel (2007) in Dindigul,
Tamil Nadu, India. The quantity of waste produced
in the developing countries like India is lesser than
that in the developed countries and is normally
observed to vary between 0.2 to 0.5 kg/head/d
(CPHERJ 1973; Ahsan 1999; Palnitkar 2000).
The present study has shown that the
average per capita waste production of Kovilpatti
town is 27m.t/d. It is high because of largest match
industry area. Villages around the town regularly
visit this area on all days for various commercial
purposes. Munnoli and Bhosale (2002) also
reported that the quantity of solid waste generation
shoots up because of the floating population in Goa.
Similar type of results was observed by
Cailas et al (1996) and Beede and Bloom (1995) in
their studies on the rate of waste production with
reference to the population size. The wards covered
under the thickly populated domestic area, i.e.,
ward no 10 is a slum area and hence the population
is dense. The residential household waste
dominated the non-residential sector because of the
large population, but the non-residential source
contained more quantity of organic fractions and

Journal of Research in Biology (2011) 5: 307-311 309

 Govindarajan et al.,2011
Table: 2 Zone wise (Sanitary Division) Details
Sanitary Division Wards Covered No of individual Waste Generation
1. 1,2,3,4,23,24 12,144 3.64
2. 5,6,7,8,12,13,14 19006 5.81
3. 15,16,17,18,19 11,023 3.30
4. 20,21,22,25,26,27 12,291 3.68
5. 28,29,30,31,32,33 15,134 4.54
6. 9,10,11,34,35,36 21,108 6.33
Total 90,706 27.30

Table: 3 Slum Areas and Population

Name of the Slum Ward No. Population No. of Household
Bharathi Nagar 30 2225 556
Stalin Colony 31 3065 766
Natarajapuram 35 1633 408
Subramaniapuram 36 2107 536
Anna Nagar 29 2958 740
Valluvar Nagar 10 3428 851
Total 13661 3857

that was due to the presence of the match box in the whole Kovilpatti town with a 6.59 sq.km.
industries and vegetable wastes and large quantities area. Park (1997) has explained the advantage of
of leaves in the wastes, i.e., the wastes produced by house to house collection and it has resulted in a
the markets, hotels and wedding halls. Some of the simultaneous reduction in the number of public
waste produced by the wedding halls and the hotels bins. Plastics are the major compounds that pollute
are collected and used as dog feed by those who the environment, but comparatively its presence
rear them around the Kovilpatti municipality limits. was very less. The same type of observation has
Open and unregulated dumping is the been made by Munnoli & Bhosale (2002) in Goa,
predominant method of waste disposal in, and Reddy et al (2002) in Bangalore.
Kovilpatti. The waste piles are exposed to wind and The composition of the hospital wastes of
rain and also to rats, flies, insects, pigs and other Kovilpatti town showed only 0.2 mt was found
animals. In some places the people set fire to the highly infectious. The WHO (2002) also has
wastes. The rag pickers spend their days sorting observed the presence of 20% of hazardous
through the garbage for reusable and recyclable materials and 80% of benign materials in bio-
materials. The trashes were found to be medical wastes. For planning an effective solid
accumulated along the road sides, in vacant places, waste management by Kovilpatti municipality, a
particularly in the poorer sections of the town. newly modified profile recommended. Recycling is
Placing dust bins may lead to reduction in a major play in MSW reduction. Glass, aluminium
the problem of waste accumulation along the cans, plastics, newspapers and organic materials can
roadsides, but only 124 dust bins have been placed be recycled, the construction waste can be used for
Table: 4 Details of Solid Waste Composition landfills and the hospital waste can be incinerated.
It is essential to get public support for a sound
Waste Composition Quantity %
(MT) Generation
Table: 5 Man power involved in Kovilpatti
Hoseholds, Petty shops Municipality
21.00 77.7
and Establishments
Vegetable, Fruit, S.No Position Numbers
3.00 11.11 1 General Administration 19
Flower market
Meat, Fish, and 2 Revenue Department 12
0.5 1.8
Slaughter house 3 Account Section 1
Construction 2.5 9.39 4 Engineering Division 34
Hospital waste 0.200 Negligible 5 Town Planning Section 3
Total 27 100 6 Public health Department 26
310 Journal of Research in Biology (2011) 5: 307-311
Govindarajan et al.,2011

waste management programme. Creation of Nanda SN, Mishra B and Tiwari TN 2000-a.
awareness among the public on waste management Municipal solid wastes in Burla Town (Orissa): (1)
will improve sanitary conditions and the scenic Preliminary Survey. Indian J. Environ. and
beauty of Kovilpatti. Ecoplan., 3(3):643-646.

ACKNOWLEDGEMENT Nanda SN, Mishra B and Tiwari TN. 2000-b.

Authors sincerely thank kovilpatti municipality Municipal solid wastes in Burla town (Orissa): (II)
staffs and heartful thanks to Mr K. Naveenkumar, Physico-chemical characteristics. Indian J. Environ.
B.E., and Ecoplan., 3(3):647-652.

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Delhi. P. 1-7. Palnitkar S. 2000. Municipal solid wastes. In:
manual on municipal solid waste. Ed: Palinitkar S
Ahsan N. 1999. Solid waste management plan for Pub: All India Institute of local self-government. pp
Indian megacities Indian J. Environ. Protection. 19 9-19.
(2):90-95.
Paul JA, Karmagam N and Daniel T. 2005.
Beede DN and Blom DE. 1995. The economics of Effect of worm population on biomass of Eudrilus
municipal solid waste. The World Bank Observer. eugeniae in municipal solid waste and the status of
10(2):113-150. microorganisms. J. Ecobiol., 17(6):567-570.

Cailas MD, Kerzee RG, Bing-Canar J, Mehash Paul JA and Daniel T. 2007. Standardization of
EX, Croke KG and Swager RR 1996. An sampling method for physical characterization of
indicator of solid waste generation potential for municipal solid waste. Asian J. Water Environment
Illinois using principal components analysis and and Pollution.
geographic information systems of the air and waste
management association. 466:414-421. Saxena P and Joshi N 2002. Organic Waste
Decomposition by using Mucor sps. and
Daniel T and Paul JAJ. 2004. Solid waste Penicillium sps. in combination. Indian J. Environ
management options for Class-I Towns. In: and Ecoplan., 6(3):583-586.
Proceedings of the National conference on
Environment awareness and pollution impacts, Tchobanoglous G, Hillary Theisen and Roif
Centre for Energy and Environmental Science and Eliassen 1977. In: Solid Wastes: Engineering-
Technology, NIT, Trichy, Tamil Nadu. India. 157- Principles and Management Issues. McGraw Hill
165. International Students Edition, New York.

Goswami B, Kalita MC and Talukdar S. 2001 WHO 2002. Wastes from health care activities,
Bioconversion of municipal solid waste through World Health Organization Information, Fact
vermicomposting. Asian J. Microbiol. Biotech. Sheet No. 253.
Environ. Sci., 3:205-207.
www.municipality.tn.gov.in.
Kawata K. 1963. Environmental sanitation in
India. Christian Medical College, Ludhiana, Punjab.

Munnoli P and Bhosale S. 2002. Solid waste

disposal in Goa. Proceedings ofNational seminar on
Solid waste management-current status and
Strategies for future (Eds) R.K. somashekar and
M.A.R. Iyengar, Bangalore, India. 9-13.

Journal of Research in Biology (2011) 5: 307-311 311

 Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Effect of prebiotic (GroBiotic-A) on the growth performance and intestinal

microflora on rainbow trout (Oncorhynchus mykiss Walbaum)
Journal of Research in Biology

Authors: ABSTRACT:
Azari AH1, 2, Hashim R1,
Azari Takami G3, Farabi
SMV2, Darvish M4 and
Safari R5.
The use of prebiotics as feed supplements that improve efficiency of
intestinal bacteria is becoming de rigueur in animal husbandry in many regions
Institution: worldwide. We tested the effects of a commercial prebiotic (GroBiotic-A) including
1
Laboratory of Feeds and yeast and dairy fractions in different levels on survival, growth, carcass composition
Feeding Management, and intestinal microflora on rainbow trout (Oncorhynchus mykiss Walbaum). In the
Aquaculture Research present study, we were able to detect high amounts of lactic acid bacteria (LAB) in the
Group, School of Biological intestine after 12 weeks of prebiotic supplementation, conducted. Ultimately, when
Sciences, Universiti Sains all supplementation diets are considered, GroBiotic-A inclusion to diets for appearing
Malaysia, 11800 Penang, on increase on growth, survival, and carcass composition on O. mykiss.
Malaysia
2
Department of Aquaculture,
Caspian Sea Research
Institute of Ecology, Sari,
Iran
3
Department of Aquatics
Disease and Health, Faculty Keywords:
of Veterinary Science, Rainbow trout, prebiotics, GroBiotic-A and lactic acid bacteria (LAB).
Tehran University, Tehran,
Iran
4
Department of Marin
Chemistry Science and
Technology, Islamic Azad
University, North Tehran
Branch, Tehran, Iran
5
Department of
Biotechnology, Caspian Sea
Research Institute of
Ecology, Sari, Iran.
Article Citation:
Azari AH, Hashim R, Azari Takami G, Farabi SMV, Darvish M and Safari R.
Corresponding author: Effect of prebiotic (GroBiotic-A) on the growth performance and intestinal microflora
Abdolhamid Azari on rainbow trout (Oncorhynchus mykiss Walbaum).
Journal of research in Biology (2011) 5: 325-334

Email:
ahazaritakami@yahoo.com
Web Address:
http://jresearchbiology.com/
Documents/RA0055.pdf.
Dates:
Received: 03 Jul 2011 /Accepted: 08 Jul 2011 /Published: 12 sep 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

325-334 | JRB | 2011 | Vol 1 | No 5

Journal of Research in biology
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INTRODUCTION:
The culture of salmonids particularly
Atlantic salmon, Salmo salar and rainbow trout
(Oncorhynchus mykiss) is one of the most
important examples of commercially successful
intensive aquaculture in the world. Rainbow trout is
a large economic fish in Iran and artificial breeding
farms of this fish species are increasing apparently.
It is also the second cultured fish species in Iran
(FAO, 2006). The increasing economic and social
concerns by decreasing the use of antibiotics and
other chemicals used in fish farming have
encouraged more environment friendly approaches
for increasing growth (Verschuere et al., 2000).
Prebiotic, unlike probiotic, is not an organism and
has less influence in natural environment. These so-
called prebiotic carbohydrates are defined as
nondigestible food ingredient(s) that beneficially
affect host health by selectively stimulating the
growth and/or activity of one or a limited number of
bacteria in the colon (Gibson and Roberfroid
1995). Therefore, the need for alternative
techniques is increasing, and the contribution of
prebiotics such as xylooligosaccharides (XOS) may
be considerable. XOS, fructooligosaccharides
(FOS), inulin, and other related carbohydrates have
received considerable attention because of the
health benefits they are believed to confer on the
host (Mussatto and Mancilha 2007; Cerezuela et al.,
2008). However, it is evident that they play an
important role in generating a colonic microflora
that comprises predominantly bifidobacteria in
young mammals (Houdijk et al., 1998; Erney et al.
2000; Costalos et al., 2008).
A functional nutrient can be further
defined as a dietary ingredient that employs
possible positive effects on health in addition to its
direct role as a nutrient. Therefore, the present study
was conducted to explore growth performance,
survival and microflora into intestine of juvenile
rainbow trout, that feed the variety levels of dietary
prebiotic, GroBiotic-A which is a commercial
mixture of partially autolyzed brewers yeast, dairy
ingredient components and dried fermentation
products (with 5, 10, 15, 20, 25 and 30g kg-1
GroBiotic-A).
MATERIAL AND METHODS:
Source and maintenance of fish
The experimental system was used here for
an experiment to optimize the feeding regime of
rainbow trout, Oncorhynchus mykiss. Apparently
healthy O. mykiss with an average body weight of
326
Azari et al.,2011
4.5g were obtained from private farm at Haraz,
Niyak Ghezel. The fish were acclimated for one
week in four fiberglass tanks (441 m, 8 m3) to
fish house conditions before the start of the trial.
Fish were kept in fiberglass tanks in dimension of
1.51.50.4 m, 1 m3 with an open system of supply
located in the north of Iran. Fishes randomly were
divided into 7 groups which were placed in
fiberglass tanks as described in above and in fresh
water-flow at 9-12oC for 84 days prior to use in
experiments.
Experimental Procedure
The present work was performed at the
Department of Aquaculture, Ecological Academy of
Caspian Sea, Sari, Iran. The trial was performed for
12-weeks using fish in 3 replicates per treatment.
Before the start of the feeding trial, three fish were
randomly sacrificed with an overdose of clove oil,
and triplicate pooled samples were taken for the
determination of initial whole body composition. At
the end of the trial, all fish were weighed and
counted and three fishes from each fiberglass tank
were collected for resolution of whole carcass
composition.
Experimental diets
The fish were fed with a standard
commercial food (Chine Co.) at a rate of 2-5 % of
the biomass per day. Commercial dry food served
as a basis in which the various concentrations of
prebiotic, GroBiotic-A were added, in 20 ml of
sunflower oil, to commercial dry feed followed by
mixing with a blender or by handy. Six
experimental diets were formulated with increasing
levels of GroBiotic-A (1, 1.5, 2, 2.5 and 3% of
basal diet) and non-supplemented diet (control): as
diet 1, 2, 3, 4, 5, 6 and 7 containing 43.30 0.07%
and 40.56 0.06% protein, 13.57 0.15% and
15.19 0.04% lipid and an estimated gross energy
level of 20.31 0.03 and 20.61 0.04 kj.g-1 (FFT,
Fingerling Feed Rainbow Trout and GFT1, Growth
Feed Rainbow Trout), respectively.
Analytical methods
Chemical analyses
The food, experimental diets and fish
carcass were analysed for proximate composition of
dry matter, crude protein, crude lipid, fibre and ash
using standard AOAC methods (1997). Briefly, dry
matter was determined by drying at 100 0C to
constant weight; crude protein was determined by
the Kjeldahl procedure (Nitrogen6.25); crude fat
by chloroform methanol extraction (2:1, v/v); crude
ash content by determining the residue after heating
in a muffle furnace at 550 0C for 5 h and crude fibre
Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011
by loss on ignition of dried residue after successive
digestion with 5% H2SO4. Nitrogen free extract
(NFE) was calculated by subtracting the sum of
crude protein, crude fat, ash, and crude fibre from
the total dry matter content. Proximate analyses of
the trial diets are described in Table 1.
Bacterial analysis
The microbial analyses were done twice;
before the trial and at the end of the fourth week.
Ten fish from each group were sacrificed by
immersion in a tank containing MS-222 at a
concentration of 150 mg L-1 of water for 15 min,
and they were eviscerated aseptically and the whole
intestine was removed. The intestine was dissected
and the contents were collected by carefully
scraping with a rubber spatula and weighed. The
microbial analyses were performed by spreading
appropriate dilutions in PBS from 101 to 106 on
tryptic soy agar (TSA, Merck, Darmstadt,
Germany), a general media for facultative aerobic
bacteria. The plates were incubated aerobically at
30C for 2 days. For determination of anaerobic
bacteria was used from anaerobic jars with
disposable Anaerocult A bags (Merck, Darmstadt,
Germany) for the generation of an anaerobic
medium. For lactic acid bacteria (LAB)
determination, diluted samples were plated on
deMan, Rogosa, and Sharpe (MRS) agar (Merck,
Darmstadt, Germany) and incubated at 30C for 2
3 days in an anaerobic jars with disposable
Anaerocult C bags (Merck, Darmstadt, Germany)
(Jones et al., 2008).
Calculation and Statistical Analysis
The following formulae were applied to the data:
Feed intake (FI) = [total feed intake / number of fish (g)]
Specific growth rate (SGR %) = [((ln Wf ln Wi) / T) 100]
Feed efficiency (FE) = [total wet weight gain (g) /
total feed intake (g)]
Protein Efficiency Ratio (PER) = [wet weight gain
(g) / total protein intake]
Average Daily Growth (ADG = [((wf wi) / wi) (Tf-Ti)]
Survival rate (%) = [(Number of fish which survived /
initial number of fish) 100]
Where Wf refers to the mean final weight, Wi is the
mean initial weight of fish, T is the feeding trial
period in days.
Journal of Research in Biology (2011) 5: 325-334
All data are expressed as mean SD, where n
represents the number of fish. The differences in
parameters were tested for significance using a one-
way analysis of variance (ANOVA) which was
performed using SPSS.V11.5 Subsequent
significance between groups was delineated by
Duncans test. A value of P<0.05 were taken for
significance in all statistical tests.
RESULTS:
Growth, Survival and Feed Performance
No external clinical sign occurred in any
treatment during the period of this experiment. The
statistical analysis of different growth parameters of
Oncorhynchus mykiss at the end of experimental
period (Table 2) indicated a significant increase in
body weight gain percent (WG %) between the six
different groups and control diet (P<0.05). O.
mykiss in group G-A5 kept on diet supplemented
with (GroBiotic-A2.5%) were the fast grower
followed by the O. mykiss in the groups G-A4 and
G-A6 received diet supplemented with (GroBiotic-
A 2 and 3% respectively) in comparison to other
groups. The specific growth rate (SGR) takes
almost the same pattern of WG% in which O.
mykiss in the group G-A5 (2.5%) have the highest
SGR followed by O. mykiss in groups G-A4 (2%)
and G-A6 (3%) in comparison to other groups.
Average daily growth (ADG) of fish under
G-A (2, 2.5 and 3%) feeding were significantly
greater (P<0.05) than the basal diet and other
groups.
Survival also was affected significantly
(P<0.05) by dietary treatments as the fish fed
control diet had lower survival 93.33
2.31compared to an overall survival rate of 98.67
2.31 among G-A5 and G-A6 fed the remaining diets
(Table 2).
Apparent Protein Utilization
Protein utilization efficiency, measured in
term of protein efficiency ratio (PER) and net
protein utilization (NPU) is summarized in Table 3.
These were the same for protein efficiency ratio
(PER) in which the O. mykiss in the groups treated
with prebiotic 2, 2.5 and 3% supplemented diets
exceeded the value of other groups and even
control. The feed efficiency (FE) of O. mykiss takes
almost the same patterns of PER in which in the
group G-A5 (2.5%) have the highest FE followed
by O. mykiss in group G-A2 and G-A3% in
comparison to other groups and basal diet.
Apparent net protein utilization (NPU) the groups
327
328
Table 1 Proximate analyses of the diets used in varying levels -response experiments in rainbow trout (Oncorhynchus mykiss) during 84 days
trial1.
Proximate composition

Treatments Experiment Moisture (%) Protein (%) Lipid (%) Fiber (%) Ash (%) NFE9 (%) GE10 (kj.g-1)
Diets
Control FFT1 5.83 0.14b 43.30 0.07a 13.57 0.15b 6.57 0.09b 9.12 0.03a 27.44 0.13b 20.31 0.03g
GFT12 6.85 0.15a 40.56 0.06b 15.19 0.04a 6.59 0.07a 9.22 0.02b 28.44 0.07a 20.61 0.04b
G-A13 FFT+G1 5.79 0.12b 43.19 0.11a 13.70 0.15b 6.52 0.11b 9.12 0.17a 27.47 0.44b 20.34 0.06fg
GFT1+G1 6.91 0.07a 40.42 0.10b 15.15 0.06a 7.18 0.06a 8.58 0.12b 28.67 0.13a 20.44 0.01de
G-A24 FFT+G2 5.89 0.05b 43.29 0.21a 13.60 0.21b 6.48 0.15b 9.14 0.04b 27.43 0.22b 20.38 0.07efg
GFT1+G2 6.91 0.03a 40.35 0.06b 15.15 0.03a 7.22 0.09a 8.57 0.18a 28.70 0.25a 20.41 0.02def
G-A35 FFT+G3 5.85 0.11b 43.18 0.04a 13.65 0.30b 6.49 0.20b 9.15 0.06b 27.53 0.42b 20.43 0.06de
GFT1+G3 6.92 0.06a 40.58 0.28b 15.17 0.11a 7.31 0.05a 8.50 0.17a 28.44 0.30a 20.40 0.01def
G-A46 FFT+G4 5.76 0.11b 43.19 0.11a 13.69 0.12b 6.51 0.07b 9.13 0.03b 27.47 0.19b 20.49 0.10cd
GFT1+G4 6.83 0.05a 40.38 0.06b 15.18 0.15a 7.33 0.10a 8.51 0.04a 28.59 0.09a 20.76 0.04a
G-A57 FFT+G5 5.92 0.10b 43.21 0.20a 13.68 0.06b 6.47 0.17b 9.20 0.10b 27.43 0.10b 20.67 0.03b
GFT1+G5 6.80 0.10a 40.37 0.05b 15.19 0.15a 7.20 0.11a 8.51 0.08a 28.72 0.26a 20.49 0.05cd
G-A68 FFT+G6 5.73 0.16b 43.30 0.13a 13.68 0.03b 6.56 0.06b 9.14 0.09b 27.31 0.27b 20.53 0.03c
GFT1+G6 6.74 0.01a 40.36 0.17b 15.16 0.05a 7.24 0.08a 8.52 0.09a 28.73 0.22a 20.48 0.03cd
1
Fingerling Feed Rainbow Trout (commercial Rainbow Trout food, Chinne)
2
Growth Feed Rainbow Trout (commercial Rainbow Trout food, Chinne)
3
GroBiotic-A (A commercial prebiotic) 0.5% of diet
4
GroBiotic-A (A commercial prebiotic) 1% of diet
5
GroBiotic-A (A commercial prebiotic) 1.5% of diet
6
GroBiotic-A (A commercial prebiotic) 2% of diet
7
GroBiotic-A (A commercial prebiotic) 2.5% of diet
8
GroBiotic-A (A commercial prebiotic) 3% of diet
9
Nitrogen free extract {100 (protein + lipid + ash + fiber)}
10
Gross energy content (Brafield 1985).
Azari et al.,2011

Journal of Research in Biology (2011) 5: 325-334

Azari et al.,2011

which receiving G-A feeding were vary

Table 2 Initial weight, final weight, percentage weight gain, specific growth rate, average daily growth and survival of rainbow trout(Oncorhynchus significantly (P < 0.05) with these supplements

724.04 41.60c
level in the diets, similarly to PER. FI (feed intake)

93.33 2.31b
36.76 2.14c

2.51 0.06c
38.45 2.50c
4.46 0.05
Control
for G-A feeding tended to have better values than
control diet with increasing supplements level and
also feeding to G-A produced a better productive.
The protein productive values (PPV) of the
fingerlings fed with different experimental diets are

Survival rate (%) = (Final fish number / Initial fish number) 100
presented in Table 3. Fish fed 25 g G-A kg- 1 diet (G

97.33 2.31ab
Values are mean SD (n=3). Mean values within columns not sharing the same superscript are significantly different (P<0.05)
871.51 29.04b
43.24 1.13ab

46.18 1.36ab
2.71 0.04b
-A5) had maximum PPV and there was no
4.45 0.05
G-A 67

improvement (P>0.05) in PPV beyond this level in

comparison to basal diet.
Body Composition
In order to determine the nutritional effects
GroBiotic-A (A commercial prebiotic) 3% of diet
GroBiotic-A (A commercial prebiotic) 2% of diet
GroBiotic-A (A commercial prebiotic) 1% of diet

of administered prebiotic on rainbow trout fry, the

SGR% = {(LnWf LnWi) / Total days} 100
biochemical composition of carcass was analyzed.
933.09 14.86a
45.21 1.46a

2.78 0.02a
48.60 1.62a
98.67 2.31a
4.40 0.11

The results are presented in Table 4. At the end of

G-A 56

the experiment, in comparison to initial values, all

the experimental groups within control showed
higher percentage of protein and ash but a lower
mykiss) fed diets containing varying Concentrations of GroBiotic -A and control for 84 days1.

percentage lipid and moisture. Protein values of

carcass in all treatments were significantly higher
847.08 18.41b

(P<0.05) than controls. The best result was obtained

41.93 1.12b

2.68 0.02b
44.64 1.29b
98.67 2.31a
4.43 0.03
G-A 45

from G-A5 (17.06 0.06%). Significantly (P<0.05)

different lipid values of carcass in prebiotic groups,
Wf = Final weight

compared to the controls, were indicated.

GroBiotic-A feeding resulted in a decrease in body
lipid (fat) with increase in dietary supplements.
Moisture values of G-A2, G-A3, G-A4, G-A5 and
770.08 33.11c

97.33 2.31ab
38.24 1.17c

2.57 0.05c
40.29 1.42c
4.40 0.06

G-A6 indicated a significant (P<0.05) difference as

G-A 34

well (Table 4). Fish fed the diet (commercial

11
9

13
7
3
5

rainbow trout food, CHINE) under feeding received

G-A 2, 2.5 and 3% showed significant (P<0.05)
difference that had higher body ash than fish from
GroBiotic -A (A commercial prebiotic) 1.5% of diet
GroBiotic -A (A commercial prebiotic) 2.5% of diet
GroBiotic-A (A commercial prebiotic) 0.5% of diet

the control and other treatments (Table 4).

768.86 39.36c

94.67 2.31ab
38.92 1.84c

2.57 0.05c
41.00 2.17c
4.48 0.04

Bacterial Analysis
G-A 23

In all treatments, lactic acid bacteria (LAB)

ADG (%) = {(Wf Wi) / Total days} 100

successfully colonized the O. mykiss digestive tract

on the end of trials (12-weeks). At the beginning of
the study, we observed that all the fish were not
significantly different on the aerobic, anaerobic and
774.09 18.14c

97.33 2.31ab
38.98 0.74c

2.58 0.02c
41.10 0.86c
4.46 0.07

LAB media count (P>0.05; Table 5). Total bacterial

Wg = {(Wf Wi)/Wi} 100
Treatments
G-A 12

counts among prebiotic supplemented groups were

significantly different from total bacterial counts in
controls in digestive tract of fish (Table 5; P<0.05).
Wi = Initial weight,

The mean of total bacterial counts of intestine with

control diet in aerobic and anaerobic condition in
Survival(%)13

Parameters

TSA media were 4.46 0.01 CFU/g and 4.80

ADG (%)12
SGR (%)11
Wg (%)10

0.01 CFU/g, respectively increased exponentially

Wf (g)9
Wi (g)8

from the experimental groups in intestine (P<0.05).

On the other hand, LAB colonization was detected
10
12
1
2
4
6
8

artificially dominant in experimental groups,

Journal of Research in Biology (2011) 5: 325-334 329

 Azari et al.,2011

Table 3 Feed intake, feed efficiency, protein efficiency ratio, Nett protein utilization and productive protein
value of rainbow trout (Oncorhynchus mykiss) fed diets containing varying Concentrations of GroBiotic -A and
control diet for 84 days1.
Parameters
Treatments Feed intake FE8 PER9 NPU (%)10 PPV11
G-A 12 40.24 1.78bc 0.86 0.03cd 1.99 0.06cd 4.77 2.61cd 89.50 6.14a
G-A 23 41.20 0.23ab 0.84 0.04de 1.97 0.09d 2.40 0.01d 86.21 0.48a
G-A 34 41.77 1.07ab 0.81 0.01e 1.89 0.03d 7.46 0.92bc 88.46 1.61a
5 ab b bc b
G-A 4 41.55 0.85 0.90 0.01 2.08 0.02 8.27 1.28 89.88 2.73a
G-A 56 42.99 0.65a 0.95 0.02a 2.19 0.04a 13.84 1.29a 91.44 1.17a
G-A 67 41.36 1.16ab 0.94 0.00ab 2.16 0.01ab 9.97 2.79b 90.33 2.73a
Control 39.00 0.86c 0.83 0.04de 1.89 0.08d 2.24 0.61d 88.23 1.69a
1
Values are mean SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic-A (A commercial prebiotic) 0.5% of diet 3
GroBiotic-A (A commercial prebiotic) 1% of diet
4 5
GroBiotic -A (A commercial prebiotic) 1.5% of diet GroBiotic-A (A commercial prebiotic) 2% of diet
6 7
GroBiotic -A (A commercial prebiotic) 2.5% of diet GroBiotic-A (A commercial prebiotic) 3% of diet
8
Feed efficiency = weight gain (g) / food intake (g)
9
Protein efficiency ratio = weight gain (g) / protein intake (g)
10
Nett Protein Utilization (NPU=Wf Protein Muscle Final Wi Protein Muscle Initial/Protein Consumed)
11
Productive protein value = protein gain (g) 100 / protein intake (g).

however, the opposite pattern was observed for the
digestive tracts of O. mykiss, in which the mean of
total LAB counts among prebiotics administered
groups were more than control (P<0.05). The
results of identification according to media on to
aerobic, anaerobic and lactic acid bacteria
condition, the highest count revealed the
experiment that was under G-A5 (4.92 0.01, 7.21
0.02 and 6.60 0.01 CFU/g, respectively; Table
5).
DISCUSSION:
Growth Performance and Protein utilization
Fish growth performance is affected by
different factors including water quality, stresses
and diseases, diet quality and quantity etc. Fish
under intensive culture conditions will be badly
affected and often fall target to different microbial
pathogens that have been conducted with
chemotherapeutic substances of which antibiotics
were used. The use of biological products namely
the probiotic either alone or in combination with
prebiotics is recently the aim of the disease
biocontrol program in aquaculture as they improve
the fish health and alter the fish associated
microbial population (Gibson and Roberfroid,
1995). While Gibson and Roberfroid (1995)
defined, prebiotic term as A nondigestible foods
ingredient (s) that beneficially affects the host by
330
selectively stimulating the growth and/or activity of
one or a limited number of bacteria in the colon,
and thus improves host health. Besides FAO
experts A prebiotic is a non-viable food
component that presents a health benefit on the host
a s s oc ia t ed w it h mo du la t i o n o f t h e
microbiota (FAO 2007). The data of prebiotic
research is still young, yet the progress made in
elucidating the useful health effects of specific
prebiotics is significant not only for humans and
animals, but for fish species also as well. In the
present study, growth performance was increased
about 23 % in fish fed prebiotic (GroBiotic-A) diet
compared to fish fed control diet; growth
performance was significantly higher (P<0.05)
exhibited in O. mykiss juvenile maintained on the G
-A diet supplemented with (prebiotic diet). The
specific growth rate (SGR) illustrated almost the
same pattern of W.G% in which O. mykiss in the
group that received G-A5 have the highest SGR
followed by O. mykiss in group G-A4 and G-A6
content in comparison to control group, G-A1, G-
A2 and G-A3. These were also true for protein
efficiency ratio (PER), NPU (protein utilization)
and survival in which the O. mykiss in groups
treated with prebiotic supplemented diets was more
than the amount of control group. In our study,
survival of fish in the treatment which produced the
highest growth performance was 5.7 % than the
Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011
lowest survival value. Since the feed efficiency
(FE) of O. mykiss kept on a basal diet (control) was
lower than the diets supplemented with prebiotics,
while the highest FE was belonged to group which
had G-A 2.5 % and a 14.5 % increased on feed
efficiency, corroborating results of other previous
studies. The PER results indicated that
supplementing diets with prebiotics significantly
improved protein utilization in rainbow trout.
Similar results have been reported by Li et al.,
(2005) who observed increased weight gain and
feed efficiency which were generally observed in
hybrid striped bass fed diets supplemented with
partially autolyzed brewers yeast and GroBiotic-
A at each sampling time (4, 8, 12 and 16 weeks).
Staykov et al., (2007) have conducted rainbow trout
(O. mykiss) with supplementation of prebiotic Bio
Mos (0.2%) in standard commercial extruded
feeds which were raised in net cages and raceways,
at the end of the six weeks trial period, the mean
body weight of fish receiving prebiotic was 13.7%
and 9.97%, respectively higher, compared to the
control groups (P<0.01). The supplementation of
prebiotic in the diet significantly decreased FCR
(P<0.05) and mortality (P<0.01). This result was in
agreement with the results expressed by Bogut et
al., (2006) which demonstrated improvements in
growth with the use of prebiotic MOS (Bio-Mos)
to the diet of other freshwater species, European
catfish (Silurus glanis) juveniles from 22 to 76 g in
the control groups and 83 g in the Bio-Mos
groups, a 9.7% higher average body weight
(P<0.01). Here the FCR was also lower to 11.6%
Table 4 Carcass proximate compositions of rainbow trout (Oncorhynchus mykiss) fed control and varying
Concentrations of GroBiotic-A and control diet for 84 days1.
Protein (%)
At the start 14.68 0.13
2 d
At the end G-A 1 15.50 0.43
G-A 23 15.10 0.00e
G-A 34 15.85 0.16c
G-A 45 16.17 0.21bc
G-A 56 17.06 0.06a
G-A 67 16.23 0.06b
Control 15.06 0.11e
1
Values are mean SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic-A (A commercial prebiotic) 0.5% of diet 3
4 5
GroBiotic -A (A commercial prebiotic) 1.5% of diet
6 7
GroBiotic -A (A commercial prebiotic) 2.5% of diet
Journal of Research in Biology (2011) 5: 325-334
(P<0.01) and mortality decreased from 28.33 to
16.67% (P<0.01). These data supported the findings
of Hanley et al., (1995) who also have shown that
hybrid red tilapia juveniles, fed 0.6% prebiotic
(Aqua-Mos, Alltech Inc., KY, USA) in their
hatchery diets, that have had a 22.5% improved
survival with 27.2% increase in weight gain. The
results revealed that both groups received prebiotic-
supplemented diets showed higher growth rate than
those kept on a basal diet, suggesting that the
addition of prebiotics enhanced the growth have
performance and feed utilization.
In our study observed proximate analysis at
the end of the experiment indicated significant
(P<0.05) differences in muscle protein, lipid, ash
and moisture contents of all the treatments.
Although the diets included containing different
levels of G-A, had increased effect on total muscle
protein and ash (13.3% and 20.6%), respectively
however decreased influence on muscle lipid and
moisture (20.6% and 1.3%), respectively content of
the fish. This study has found that supplementation
with G-A may help to positively change body
composition by increasing lean body mass (which
includes muscle mass) and decreasing fat mass.
The fillets (MR%) of the groups of rainbow
trout differed with regard to the received of G-A
and non- G-A; the groups that fed G-A
supplemented diets exhibited higher values than of
basal diet (P< 0.05). Results demonstrated that
prebiotic G-A can be an influence device for the
enhancement of growth of performance, health
status and feed efficiency of rainbow trout grown in
Lipid (%) Ash (%) Moisture (%)
9.40 0.05 1.87 0.03 75.10 0.36
a b
8.23 0.06 1.22 0.02 75.01 0.02a
7.59 0.45b 1.21 0.06b 74.93 0.05ab
7.44 0.12b 1.23 0.23b 74.89 0.63ab
7.44 0.03b 1.38 0.11ab 74.49 0.10bc
7.05 0.04c 1.46 0.05a 74.24 0.09c
7.09 0.05c 1.44 0.05a 74.51 0.18bc
8.02 0.07a 1.21 0.02b 75.16 0.13a
GroBiotic-A (A commercial prebiotic) 1% of diet
GroBiotic-A (A commercial prebiotic) 2% of diet
GroBiotic-A (A commercial prebiotic) 3% of diet
331
 Azari et al.,2011

Table 5 Total bacterial levels (log CFU/mg intestinal contents) in TSA medium (Aerobic, Anaerobic) and LAB
(Lactic Acid Bacteria) condition in the juvenile O.mykiss fed with test diets for 12 weeks1.

Initial Final
Anaerobic Anaerobic
Source Aerobic (TSA) LAB Aerobic (TSA) LAB
(TSA) (TSA)
G-A12 4.49 0.02 4.80 0.01 0.00 0.00 4.49 0.02e 4.81 0.01e 3.63 0.01f
G-A23 4.49 0.02 4.80 0.01 0.00 0.00 4.65 0.02d 5.22 0.01d 3.90 0.03e
G-A34 4.48 0.02 4.80 0.01 0.00 0.00 4.49 0.02c 6.82 0.01c 6.06 0.05d
G-A45 4.48 0.03 4.81 0.01 0.00 0.00 4.86 0.02b 7.20 0.02a 6.46 0.01c
G-A56 4.46 0.02 4.80 0.01 0.00 0.00 4.92 0.01a 7.21 0.02a 6.60 0.01a
G-A67 4.47 0.01 4.81 0.02 0.00 0.00 4.87 0.01b 7.15 0.01b 6.55 0.03b
Control 4.46 0.02 4.80 0.00 0.00 0.00 4.46 0.01e 4.80 0.01e 0.00 0.00j
1
Values are mean SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic-A (A commercial prebiotic) 0.5% of diet 3 GroBiotic-A (A commercial prebiotic) 1% of diet
4
GroBiotic-A (A commercial prebiotic) 1.5% of diet 5 GroBiotic-A (A commercial prebiotic) 2% of diet
6
GroBiotic-A (A commercial prebiotic) 2.5% of diet 7 GroBiotic-A (A commercial prebiotic) 3% of diet.

a variety of production systems. On other hand, the
beneficial influence of G-A supplemented on
growth may be due to a change of the intestinal
microflora by mannanoligofructose, lactose or other
carbohydrates from the dairy ingredient, partially
autolyzed yeast and/or dried fermentation products.
In the present study, it is given the evidence of
improvement in the health and growth performance
of fish in spite of the differences in methods and
species used.
Intestinal microflora
At the end of trial, it was noticed that the
prebiotic supplementation into diet (extruded pellet
food) in these studies resulted significant (P<0.05)
increases in total bacterial counts among prebiotic
supplemented groups, which were significantly
different in compared to total bacterial counts in
controls at the digestive tract of O. mykiss (P<0.05).
In the present study, we were able to detect high
amounts of lactic acid bacteria (LAB) in the
intestine after 12 weeks of prebiotic
supplementation. However the values noted on
different media such as: (Aerobic; 4.92 vs. 4.46
CFU/g), (Anaerobic; 7.21 vs. 4.80 CFU/g) and
(LAB; 6.60 vs. 0 CFU/g) G-A group treatments and
basal which these present data the beneficial
influence of prebiotic on growth was perhaps due to
an alteration of the intestinal microflora. After 12
weeks of G-A-feeding in the present study, we were
able to detect high amounts of LAB and total
bacterial counts on aerobic and anaerobic media in
the intestine, which demonstrates that G-A
332
supplemented diets and basal group, isolated from
the inner digestive tract bacteria of rainbow trout,
have a big capacity to adhere to and survive in the
intestinal tract. LAB supplementation, however,
demonstrated an ability to challenge the resident
microbiota, since the increase in LAB observed was
possibly the result of a fall in intestinal pH motive
by lactic acid or other fermented products produced
by LAB strains. The previous studies (Ring et al.,
1998; Ring and Olsen 1999) indicated that dietary
fatty acids and carbohydrates changed the bacterial
flora of the gastrointestinal tract of fish. Similar
findings were pointed out by Li and Gatlin (2004,
2005), who investigated the effect of commercial
prebiotics GrobioticTM AE and GrobioticTM
supplemented in diets of hybrid striped bass
(Morone chrysops M. saxatilis) growth and
exhibited that the prebiotic promoted the growth
performance. The results of this present study
clearly indicated that diet supplemented with G-A
at a suitable concentration (20, 25 and 30 g kg-1)
could improve the total bacterial counts and LAB of
all intestine O. mykiss (Table 5). Lactic acid
bacteria had been considered beneficial residents of
the fish intestinal function because of producing
bacteriocins and hence positively affecting the
hosts microflora (Ring et al., 1998; Irianto and
Austin 2002; Ring et al., 2006). Prebiotic
oligosaccharides such as inulin and oligofructose
are fermented in the colon where they promote the
growth of bacterial populations associated with a
healthy, well functioning colon. This selective
Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011
stimulation occurs because oligosaccharides are
readily fermented by beneficial types of colonic
bacteria and are not used effectively by potentially
pathogenic bacteria species (Flickinger et al.,
2003). Our results have demonstrated that G-A, as
well as other prebiotic ingredients, may promote the
maintenance of lactic acid-production as some
reports have also supported the point (Mussatto and
Mancilha 2007; Moura et al., 2007). Based on our
data, the beneficial influence of prebiotic on growth
was possible due to an alteration of the intestinal
microflora.
ACKNOWLEDGMENTS
We express our best sincere gratitude to
Ghezelalaparvar Trout farming (private fish
farming), Caspian Sea Research Institute of
Ecology of Iran and IIC, International Ingredient
Corporation of USA for providing the fry and
facilities for the study.
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334 Journal of Research in Biology (2011) 5: 325-334

 Journal of Research in Biology
Isolation, screening and induction of mutation in strain for extra cellular
lignin peroxidase producing bacteria from soil and its partial purification
Journal of Research in Biology
Authors: ABSTRACT:
Renugadevi R,
Ayyappadas MP,
Preethy PH and Savetha S.
Institution:
Assistant Professors,
Department of
Biotechnology
RVS College of Arts
and Science, Sulur.
Corresponding author:
Renugadevi R
Email:
renugadevi@rvsgroup.com
Web Address:
http://jresearchbiology.com/
Documents/RA0085.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper Publication group
The Lignin peroxidase enzyme production was carried out through bacteria
isolated from sample soil collected around decayed wood at Sulur, Coimbatore. In this
report, a mutant of Bacillus subtilis LPTK was obtained after chemical mutagenesis
with EMS and screened for better Lignin peroxidase production. In the production
optimization studies, the bacterial strain needs temperature around 37OC, pH.6,
lactose as a carbon source, peptone as nitrogen source and incubation time for 36
hours for its higher enzyme productivity. The partial purification was also done.
Keywords:
Lignin, LP, Bacillus subtilis and enzyme activity.
Article Citation:
Renugadevi R, Ayyappadas MP, Preethy PH and Savetha S.
Isolation, screening and induction of mutation in strain for extra cellular lignin
peroxidase producing bacteria from soil and its partial purification
Journal of research in Biology (2011) 4: 312-318
Dates:
Received: 17 Aug 2011 /Accepted: 27 Aug 2011 /Published: 12 Sep 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
312-318 | JRB | 2011 | Vol 1 | No 5
Submit Your Manuscript
www.ficuspublishers.com www.jresearchbiology.com

Introduction
Lignin is the most abundant renewable
carbon source on earth (Bo Zhang et al., 2008).
When plants die, it will rot and degrade in soil.
Researches are widely done on fungi which degrade
lignin. While bacteria grow and multiply faster, it is
anticipated to be better in production of lignin
degrading enzymes. There are many types of lignin
degrading enzymes such as lignin peroxidase,
manganase peroxidase, laccase and glyoxal oxidase
are produced by fungi as well as bacteria such as
lignin peroxidase by the bacteria Streptomyces
viridosporus (Ramachandra et al., 1987).
Lignin peroxidase is an enzyme that is used
to degrade lignin. It was first discovered in 1983.
Lignin peroxidases are produced by many wood
degrading fungi as a family of isoenzymes (Kirk
and Farrell, 1987). Recent researches also showed
that it can be produced from bacteria such as
Streptomyces viridosporus. These hemeproteins are
similar to the more familiar plant peroxidases in
structure and mechanism, and utilize hydrogen
peroxide and organic peroxides to oxidize a variety
of substrates (Tien et al., 1985, 1986). Some of the
most important features distinguishing these
enzymes from other oxidoreductases (such as
horseradish peroxidase), for example, are their very
low pH optima and much higher redox potentials.
The substrates of lignin peroxidase include
both phenolic and non-phenolic aromatic
compounds; the phenolic substrates are oxidized to
yield products similar to those produced by
classical peroxidases, while the oxidation of the non
-phenolic methoxybenzenes are unique to the lignin
peroxidases (Korsten L and Cook N, 1996), the
oxidation of these substrates yield aryl cation
radicals. Lignin peroxidase can catalyze the
oxidation of substrates with a reduction potential
greater than 1.3 volts. The enzyme is capable to
oxidizing lignin monomers, dimers and trimers as
well as polycyclic aromatic compounds such as
benzopyrene (Haemmerli et al., 1996). The
powerful and relatively non-specific nature of this
enzyme has lead to the investigation of its potential
use in diverse fields of biodegradation of toxic
chemicals, pulp, paper processing, and in the textile
industry.
METHODS AND MATERIALS
Sample Collection and Isolation of Bacteria
The sample soil was collected around
decayed wood at Sulur, Coimbatore in a sterile
container. The collected sample was serially diluted
313
Renugadevi et al.,2011
up to 107 dilutions using sterile saline as a blank and
the diluted samples were plated into the sterile
nutrient agar using spread plate method. The
nutrient agar plates were incubated at 37C for 24
hours. The isolated colonies were further purified
by streak plate method using sterile media plates.
The pure cultures were inoculated into sterile
nutrient agar slants and nutrient broth for further
use.
Screening for the Enzyme Production
The isolated pure strains were screened for
the production of extra cellular lignin peroxidase
using screening medium which contains lignin as a
substrate (Yeoh Teik Loon et al., 2006). The pure
cultures were streaked at the lignin agar plates and
the plates were incubated at 37C for 24 hours. The
observation was made to see the substrate utilized
zone around the colony. Only the strain showing
positive and better zone was taken for further study.
Strain Improvement by Mutation
The chemical mutagenesis was carried out
according to Kotchoni and D. Bartels (2003). Ethyl
methanesulphonate (EMS) was used as the
mutagenic agent. The better zone formed strain was
grown to mid logarithmic phase of growth in LB
broth and from that cells were centrifuged at room
temperature. The pellet was re-suspended in normal
saline and a suspension of (v/v) of EMS was made.
Appropriate dilutions were made and incubated
overnight at room temperature to allow mutational
segregation. The culture was then spread on agar
media and incubated overnight at 37C. Single
mutant clones were further grown in new plates till
the third generation. The third generations (M3) of
the mutants were replica-plated in nutrient agar
plates. The wild and mutant strain was screened
again using lignin screening medium. The selection
of mutants was based on the diameter of the clear
zone surrounding the colonies.
Enzyme Production
Preparation of Inoculum
The inoculum for further production of
enzyme and other studies was prepared using Luria
broth (Zoltan PraGai, 2002). The pure culture was
inoculated into sterile inoculum broth and was
incubated at 37C in a rotary shaker for overnight.
The fresh over night culture was used as inoculum
for the production of enzyme.
Production
The enzyme production was carried out by
shake flask fermentation using production medium
with pH 8. The inoculated medium was incubated
at 37C for 48 hours. The medium was agitated at
Journal of Research in Biology (2011) 5: 312-318
Renugadevi et al.,2011
200 rpm for better aeration and growth of the
organism.
Lignin peroxidase Assay
Bacterial crude culture prepration
Aliquots of 10ml of the culture suspension
were centrifuged at 5000 rpm for 15 minutes and
cell free extract was subjected to enzyme assay. The
extract was stored at 4C for further analysis.
Plate Assay
The plate assay was performed using agar
plates amended with Lignin. The agar plates were
prepared by mixing of 1% Lignin with 1.7% agar.
After solidification of agar, around 10 mm
diameters of wells were cut out aseptically with the
help of cork-borer. The well was filled with the
culture filtrate and incubated at 37C for overnight.
The observation was made to see the hydrolytic
zone around the wells. Two wells were filled with
distilled water and production medium without
culture acts as a control (Yeoh Teik Loon et al.,
2006)
Chemical Assay
Lignin peroxidase activity was determined
by estimating the reducing sugar produced during
enzymatic reaction by dinitro salicylic acid method.
Assay for lignin peroxidase were performed using
substrate composed of alkaline lignin dissolved in
0.1M phosphate buffer with pH of 7.0. After
incubation at 33C for 24 hours, the broth is
transferred for centrifugation. Centrifugation was
done for 5 minutes. The supernatant obtained was
used as a crude enzyme. The crude enzyme was
incubated with substrate at 33C. The reaction
mixtures were tested for its simple sugar using DNS
method.
Estimation of Total Protein
The chemical assay for the total protein
content from the sample was determined using
Bradford method (Bradford, 1976). To the culture
filtrate Bradford reagent was added. The tube was
gently tilted once for mixing and the absorbency
was taken at 595nm in UV- VIS spectrophotometer.
The blank was prepared by mixing distilled water
reagent. The protein concentration was determined
by comparing the value with standard graph
prepared using Bovine serum albumin.
PARAMETER OPTIMISATION STUDIES
Effect of incubation time on lignin peroxidase
production
Around 500ml of sterile production medium
for bacterial were prepared and 5% bacterial
inoculum was added aseptically. The inoculated
Journal of Research in Biology (2011) 5: 312-318
medium was incubated at 37C temperature with
shaking around 150 rpm. After incubation, around
20 ml of culture was aseptically withdrawn
periodically at 6 hours intervals up to 48 hours. The
culture filtrate was tested for the total protein
content and lignin peroxidase activity (Tuncer et al,
2004).
Temperature
Hundred ml of sterile production medium
for bacteria was prepared in different conical flask
at pH 8.0 and inoculated with 5% inoculum. Each
flask was incubated at different temperatures such
as 28C, 32C, 37C, 42C, 47C, and 52C for 36
hours. The protein estimation and enzyme activity
were estimated followed by Tuncer et al (2004).
pH
Hundred ml of sterile production medium
was prepared for bacterial strain in different
conical flasks and each flask was adjusted to
different pH such as 4, 5, 6, 7, 8 ,9 using 0.1N
NaOH and 0.1N HCl. After sterilization, flasks
were inoculated with 5% inoculum. The flasks were
incubated at 37C for 36 hours. The protein
estimation and enzyme activity were estimated
(Tuncer et al, 2004).
Carbon Sources
Hundred ml of sterile production medium
(pH.6) for bacteria was prepared in different conical
flasks. Each flask was amended with different
carbon sources such as Glucose, Fructose, Maltose,
Lactose, Sucrose and Mannitol. The flasks were
inoculated with 5% inoculum and incubated at 37C
for 36 hours. The culture filtrate was collected for
protein estimation and enzyme activity (Tuncer et
al, 2004).
Nitrogen source
Hundred ml of sterile production media for
bacteria (pH 6) was prepared in different conical
flasks. Each flask was amended with different
nitrogen sources such as Beef extract, Casein,
Peptone, Gelatin, Ammonium chloride, Ammonium
sulphate and Potassium nitrate. The flasks
containing bacterial medium were inoculated with
5% inoculum and incubated at 37C for 36 hours.
The culture filtrate was collected for Protein
estimation and Enzyme activity (Tuncer et al,
2004).
Partial Purification
Ammonium Sulphate Fractionation of Proteins
The enzyme separation from the exhausted
medium was done by 70% Ammonium sulphate
saturation (Elba et al, 1999). The mixture was then
314
 Renugadevi et al.,2011

stored in a cold room for 24 hours to precipitate all culture was withdrawn for enzyme activity. The
the proteins and the precipitation was separated by bacterial cultures were withdrawn once in every six
centrifugation for 10 minutes. The supernatant was hours. The results revealed that there is gradual
discarded and the remaining precipitate was increasing of production has occurred from 18 th
dissolved with 5 ml of 1M-citrate phosphate buffer hour to 36th hours and higher production has
(pH.8). Then the mixture was subjected to dialysis. occurred at 36th hours (Table.1). This shows that
Dialysis bacterial isolate should have maintained its log
The purpose of dialysis is to remove phase from around 18th hour to 36th hour. Besides, it
undesired small molecular weight molecules from a is believed that the higher production of lignin
mixture in which the desired species of molecules peroxidase has occurred in extreme log phase
are too large to travel across the membrane. because even though the log phase was maintained
Ordinarily this process is utilized during protein around 18th to 36 hours, the followed drop of
purification in which salting out procedure has been production has indicated that the organism should
employed as the initial step with ammonium have entered into stationary phase of growth (Fig
sulphate. After the protein is precipitated from the 1).
initial source, it is re-dissolved in buffer and then Effect of Temperature
poured into a dialysis bag. The environmental parameters are showing
great influence in the growth of the organisms and
RESULTS the production of enzymes. The optimum
Naturally occurring microorganisms are temperature for the better production was made in
having the ability to produce various enzymes. Now various temperatures. It was found that like other
a days most of the enzymes are important for mesophilic organisms, the higher lignin peroxidase
human welfare and industry. In this study, the activity was found (41U/ml) at 37C from bacteria
bacterial strains were isolated from the sample soil
at Sulur area, Coimbatore because most of the
natural wastes are degraded by the native microbes
that are growing over that waste. In such a way, it is
fact that the microbes which are isolated from
decayed wood soil may have ability to produce
lignin peroxidase. From the samples, around 15
bacterial strains were isolated and screened; it was
found that four bacterial strains showed positive
results on lignin peroxidase production. The better
zone formed bacterial strain was considered for
further strain improvement study.
Strain improvement was done by Kotchoni
and Shonukan (2002) method. In this experiment
Figure 1. Enzyme production and total protein
the better positive strain for lignin peroxidase was content of bacterial isolate at different incubation time
treated with Ethyl methanesulphonate and the M3
generation mutant strain produced much better (Table.2). These indicate that the optimum
yellow zone then compared to wild strain. This temperatures for better production of bacterial
strain was used for further studies. isolates are 37C (Fig.2). The temperature
In plate assay the lignin peroxidase activity requirement of the organism is based on the nature
was identified by a clear zone when compared with of organisms. Many thermophilic bacteria like
the control and the chemical assay determined by Clostridium sp. needs 55C for better production of
DNSA (3, 5- dinitro salicylic acid) method using lignin peroxidase (Tuncer et al, 2004).
starch as a substrate.
Effect of Time Effect of pH
The growth study of the organism is The pH is the important parameter which
essential for the production of enzyme because determines the growth of the organism and lignin
most of the extra cellular enzymes are produced peroxidase production. The study results showed
during log phase of the organisms. Generally during that the optimum pH around six is better for
growth, the bio mass of the cells was estimated. The bacterial isolate (Table. 3 & Fig.3). Like
315 Journal of Research in Biology (2011) 5: 312-318
Renugadevi et al.,2011

temperature, different organisms need different pH Table.3 Effect of pH on total protein and enzyme
ranges for its lignin peroxidase production. The production of bacterial isolate
Thermophilic bacterium has produced high amount Enzyme
of lignin peroxidase between pH 4-6 (Tuncer et al, S. pH
Total protein content
activity
2004). NO (g/ ml)
(U/ml)
1 4 9 4
Effect of Carbon source 2 5 31 21
Table.2 Effect of Temperature on total protein and 3 6 73 41
enzyme production by bacterial isolate 4 7 92 33
5 8 70 15
Total protein Enzyme
S. Temperatur 6 9 62 12
content activity
NO e (C)
(g/ml) (U/ml)
1 28 49 15
2 32 50 20
3 37 52 41
4 42 40 26
5 47 31 12
6 52 28 4

Figure.3 Effect of pH on total protein and enzyme

production of bacterial isolate

nitrogen sources are of secondary energy sources

for the organisms, which play an important role in
the growth of the organism and the production
(Table.5 & Fig.5).

Figure 2. Effect of Temperature on total protein and DISCUSSION

enzyme production by bacterial isolate The Lignin peroxidase enzyme production
Table. 4 Enzyme production and total protein
The carbohydrates are soul energy source for oduction on different carbon sources
most of the heterotrophic organisms. These shows
great influence on the production of many enzymes. Bacteria
Carbon Total protein Enzyme
In this study, lactose was found to be a right carbon S.No
sources content activity
source for bacterial strain for higher production of (g/ ml) (U/ml)
lignin peroxidase (Table. 4 & Fig.4). Lignin 1 Glucose 48 6
peroxidase production will be low in the medium 2 Fructose 57 18
amended with glucose, fructose, maltose,sucrose 3 Maltose 38 9
and mannitol when compare to lactose (Tuncer et 4 Lactose 86 31
al, 2004). 5 Sucrose 51 15
Effect of Nitrogen source 6 Mannitol 47 10
The nitrogen sources are of secondary
energy sources for the organisms, which play an was carried out through bacteria isolated from
important role in the growth of the organism and sample soil collected around decayed wood at
the production. The nature of the compound and the Sulur. Trichoderma harzianum isolates (D1/4)
concentration that we are using may stimulate or proved to be the highest producer for the two
down modulate the production of enzymes. The enzymes such as 160% and 186% of CMCase and

Journal of Research in Biology (2011) 5: 312-318 316

 Renugadevi et al.,2011

Figure.4 Enzyme production and total protein

production on different carbon sources
-glucosidase respectively, more than the original
strain (Ahmed M. El-Bondkly et al., 2010). In this
report, a mutant of Bacillus subtilis LPTK was
obtained after chemical mutagenesis with EMS and
screened for better Lignin peroxidase production.
The effect of carbon and nitrogen sources on lignin
peroxidase (LiP) activity of Aspergillus sp., showed
that glucose (1%) and ammonium sulphate were the
best carbon and nitrogen sources (Ahammed S and
Prema P,2002). In the production optimization
studies, the bacterial strain needs temperature
around 37OC, pH.6, lactose as a carbon source,
peptone as nitrogen source and incubation time for
36 hours for its higher enzyme productivity.
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Ahammed S and Prema P. 2002. Influence of
media nutrients on synthesis of lignin peroxidase
from Aspergillus sp., Appl Biochem Biotechnol,
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Figure. 5 Enzyme and total protein production on
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Construction of -Glucosidase Hyperproducers of
Trichoderma Harzianum Using Microbial
Biotechnology Techniques, J. Microbial &
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quantization of microgram quantities of protein
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Ramaswamy. 2008. Reaction Kinetics of the
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Ahmed M. El-Bondkly AAM, Aboshosha NH. Elba P S. Bon, Hilton J Nasecimento, jacyara M,
B Macedo and Jose G, silva Jr. 1999. Lignin
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different nitrogen sources viridosporus T7A: are they a monomer based
Bacteria structure, Biotechnology Techniques 13:289-293.
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(g/ ml) (U/ ml) Fiechter A. 1996. Oxidation of benzo(a) pyrene by
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ligninases, J. Biochem, 261:6900-6903.
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5 38 24 Kirk TK and Farrell. 1987. Enzymatic
chloride
Ammonium combustion: the microbial degradation of lignin,
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Potassium
7 31 23
nitrate Korsten L and Cook N. 1996. Optimizing
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culturing conditions for bacillus subtilis, South

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Regualtory mutations affecting the synthesis of
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Ramachandra M, Crawford DL and Hertel G.

1988. Characterization of an extracellular lignin
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Tien M, Kersten PJ, Kalyanaraman B and Kirk

TK. 1985. The ligninase of Phanerochaete
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2612.

Tien M, Kirk TK, Bull C and Fee JA. 1986.

Steady state and transient state kinetics on the
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Tuncer M, Kurul A, Isikli M and elenk FGC.

2004. Optimization of extracellular endoxylanase,
endoglucanase and peroxidase production by
strptomyces sp. F2621 isolated in turkey, J. Applied
Microbiology 97:783-791.

Yeoh teik loon. 2006. Screening of lignin degrader

from soil, thesis. Submitted for the degree of
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Journal of Research in Biology (2011) 5: 312-318 318

 Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Relationship between Anemia and Parasitic Infections in Shekhan District,

Iraq
Journal of Research in Biology

Authors: ABSTRACT:
Bushra H. Al-Naemi, A total of 431 of stool samples were collected from patients consulting the
Zohair IF. Rahemo*, governmental health center in Al-Shakhan district (Mosul, Iraq) , their ages range from
Sundus N. Al-Kallak. 1-50 years, the period of collection lie between January 2009-October 2009. The
highest percentage of the occurrence of cysts was those of Entamoeba histolytica
(35.8.0%) followed by eggs of Enterbius vermicularis (32.60%), then cysts of Giardia
lamblia (26.81%), then the eggs of Ancylostoma dudenale (2.17%), then eggs of
Ascaris lumbricoides (1.44 %) and the lowest was eggs of Trichuris trichura (1.08%).
The percentage of anemia is higher in infected persons compared with uninfected as
infection with E. histolytica (38.98% and 35% respectively), similarly G. lamblia
Institution: (28.18% and 26.2% respectively) , A. duedenale ( 8.47% and 0.4% respectively)T.
Department of Basic trichura (5.08% and 0.0 respectively, and in A. lumbricoides (1.69% and 1.3%
Medical Sciences, College of respectively), while infection with E. vermicularis the percentage of infection is higher
Nursing, Mosul University, in unanemic persons(36.8% and 16.9% respectively). F u r t h e r m o r e , the
Iraq highestpercentage of infection by E. histolytica occurs in the age 11-20(40%) while the
*Department of Biology, lowest was in the age 41-50 and more(21.4 %). As regard infection of G. lamblia the
College of Science, highest infection was in the age 1-10 (39.5%) while the lowest was in the age 31-40
University of Mosul, Mosul, (11.4%). The infection with E. vermicularis , the highest was in the age 11-20(43.4%)
Iraq. and the lowest in the age 1-10(19.7%). The highest infection with A. duedenale was in
the age 11-20(3%) while the lowest in the age 1-10(2 %). As concern infection with A.
lumbricoides the highest infection rate (3.1%) was in the age 1-10 while the lowest
was in the age 11-20(1%). Infection with T. trichuris the highest infection rate (2%) was
in the age 1-10 while the lowest ( 1%) in the age 11-20. As regards haemoglobin
concentration and the type of the parasite, the highest concentration was in case of
infection by G. lamblia (11%), then E. histolytica(10.3%), A. lumbricoides(10%) then E.
Corresponding author: vermicularis (9.5%) then in case of T. trichura( 9 ), then the lowest was in A. duodenale
Zohair IF. Rahemo (8%).while concentration of hemoglobin in different age group: the highest
concentration was in age group41-50(10.5 g/dl) and then the 31-40(10 g/dl) then the
age group 1-10(9.7% g/dl) then in age group 11-20( 9.2 g/dl) and the lowest was in the
age group 21-30(8.1% g/dl).

Web Address:
http://jresearchbiology.com/
Documents/RA0087.pdf.
Article Citation:
Bushra H. Al-Naemi, Zohair IF. Rahemo, Sundus N. Al-Kallak.
Relationship between Anemia and Parasitic Infections in Shekhan District, Iraq
Journal of research in Biology (2011) 5: 319-324
Dates:
Received: 17 Aug 2011 /Accepted: 27 Aug 2011 /Published: 12 Sep 2011

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

319-324 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION
Anemia is a common disease observed in
tropics. Iron deficiency anemia affects about 1.3
billion people with the highest prevalence and
morbidity in young children and pregnant women
(Gillespie et al.1991).The only study investigated
the relationship between intestinal parasites and
anemia was done persons in Nineveh Governorate
(Al-Heali,2009), she examined 499 stool samples
and found six species of parasites and she
concluded that the haemoglobin decreased in the
blood of infected persons with 22% . However
anemia is considered a serious public health and
Schistosoma mansoni is considered a risk factor in
Egypt (Curtale et al. 1998).Food and drinks are the
main routes in which intestinal parasites can inter
the body and to certain extent other routes such as
flies and mosquitoes (Greets and Gryseels, 2000).
Furthermore, bad hygiene and social behavior in
addition to general health can increase the
possibility of anemia occurrence(El Kichaoi and
Abd Rabou, 2004). Intestinal helminthes such as
Ancylostoma duedenale infect 9000 million people
in the world, while Ascaris lumbricoides and
Trichuris trichura infect 800 million, and
Enterobius vermicularis infect 42 million in
developed countries(Lobo et al. 1998). Most of
persons infected with parasitic infection suffering
from nutrition, nausea, weakness, diarrhea, anemia
(Pothinpak et al.2005). Patients with heavy
infection may lose up to 200 ml of blood per day,
but around 40% of iron may be reabsorbed before it
leaves the intestine. Nevertheless, a moderate
Ancylostoma doudenale infection will gradually
produce an iron-deficiency anemia as body reserves
of iron are used. Severity of anemia depend on
worm load and dietary iron intake of patient
(Roberts and Janovy, 2005).
Anemia is the most common disease among
malnutrition and among the main causes is
insufficient protein , folic acid , B6 or B12 and
vitamine C, while the other causes of anemia are
intestinal helminthes, amoebiasis and malaria
(Koukounari et al. 2008). The most common
symptoms are bale blood, eye white, feeling of tire,
nausea, lips fractures(Shubair et al.2005).
The aim of the present study is to investigate
the relationship between the anemic persons and the
presence of intestinal parasites in different age
group in a sample taken from Shekhan district,
Mosul, Iraq.
320
Al-Naemi et al.,2011
MATERIALS AND METHODS
A total of 431 of stool samples were
collected from patients consulting the governmental
Health Center in Al-Shekhan district. Al Shekhan
district situated about 50 Km northern of mosul
city, it is a hily area with continental climate similar
to mosul climate especially in spring and autumn,
while in winter temperature range for -5 to 8c and
in summer 40-50 c. Its population can be estimation
to be more than 70.000 person including the center
out its suburbs. The water supply come from wells
situated in the hilly area. Most of Shekhan people
are working in agriculture especially wheat and
barley in addition to animal breeding such as sheep,
goats, and poultry. Their ages range between 1-50
year, the period of collection lie between January
2009- October 2009.Patients attending this
governmental outpatient clinic are usually suffering
from common cold, diarrhea, renal colic,
dermatological problems, diabetes, heart pressure,
breast cancer, gynecological diseases, and sterility.
The patient who suffer from diarrhea, and
abdominal pain, and vomiting were selected for this
study. Stool specimens were preserved in a 15 cc
vial containing 10% formaline as a fixative with
fitted cover and the age of the patient was recorded
for each sample, and stored for two three days.
Two to three slides from each vial were prepared, a
drop of Lugols iodine was added, then examined
under the microscope by direct method according
to Neimeister et al. (1990). Haemoglobine was
estimated using Sahli method (Lewis et al., 2001).
The normal haemoglobine levels:
Age Hemoglobin levels
Less than 7 days 17 to 22 h/dl
1 Week 15 to 20 g/dl
1 Month 11 to 15 g/dl
Children 11 to 13 g/dl
Adult males 14 to 18 g/dl
Elderly males 12.4 to 14.9 g/dl
Adult females 12 to 16 g/dl
Elderly females 11.7 to 13.8 g/dl
RESULTS
As indicated in Table (1), the highest
percentage of the occurrence of cysts was those of
Entamoeba histolytica (35.8.0%) followed by eggs
of Enterbius vermicularis (32.60%), then cysts of
Giardia lamblia (26.81%), then the eggs of
Ancylostoma dudenale (2.17%), then eggs of
Ascaris lumbricoides (1.44 %) and the lowest was
eggs of Trichuris trichura (1.08%).
Journal of Research in Biology (2011) 5: 319-324
Al-Naemi et al.,2011

Table (1): the different intestinal parasites and their lowest in the age 1-10(2 %). As concern infection
percentages. with A. lumbricoides the highest infection rate
Total number Percentage of (3.1%) was in the age 1-10 while the lowest was in
Parasite
of infection infection the age 11-20(1%). Infection with T. trichuris the
Entamoeba highest infection rate (2%) was in the age 1-10
99 35.8
histolytica while the lowest ( 1%) in the age 11-20.
Giardia lamblia 74 26.81 As indicated in Table (4), showing
Enterobius haemoglobin concentration and the type of the
90 32.60
vermicularis parasite, the highest concentration was in case of
Ancylostoma
6 2.17 infection by G. lamblia (11%), then E. histolytica
duodenale
Ascaris
(10.3%), A. lumbricoides(10%) then E.
4 1.44 vermicularis (9.5%) then in case of T. trichura( 9 ),
lumbricoides
Trichuris then the lowest was in A. duodenale(8%).
3 1.08 Table(5), showing the concentration of
trichura
total 276 100.0 hemoglobin in different age group. The highest
concentration was in age group41-50(10.5 g/dl) and
As shown in Table(2), the percentage of then the 31-40(10 g/dl) then the age group 1-10
anemia is higher in infected persons compared with (9.7% g/dl) then in age group 11-20( 9.2 g/dl) and
uninfected as infection with E. histolytica (38.98% the lowest was in the age group 21-30(8.1% g/dl).
and 35% respectively), similarly G. lamblia
(28.18% and 26.2% respectively), A. duedenale DISCUSSION
( 8.47% and 0.4% respectively)T. trichura (5.08% As seen in Table(1), six parasites have been
and 0.0 respectively, and in A. lumbricoides (1.69% encountered in persons consulting Shekhan district
and 1.3% respectively), while infection with E. health center including two protozoans( E.
vermicularis the percentage of infection is higher in histolytica and G. lamblia) and four nematodes
unanemic persons(36.8% and 16.9% respectively). namely E. vermicularis, A. duodenale , A.
Table(3), indicated the revealed parasites lumbricoides, and T. trachura. In the present study
listed with their age group . The highest percentage E. histolytica was the most abundant parasite
of infection by E. histolytica occurs in the age 11- (35.8%) while that with lowest infection was T.
20(40%) while the lowest was in the age 41-50 and trichura(1%).
more(21.4 %). As regard infection of G. lamblia the Similar parasites were previously reported
highest infection was in the age 1-10 (39.5%) while from the school pupils and food handler by Al-
the lowest was in the age 31-40(11.4%). Daoody(1998) the most abundant one was E. coli
The infection with E. vermicularis, the 24.7% Al- Shirifi (2000) found that the most
highest was in the age 11-20(43.4%) and the lowest abundant parasites was G. lamblia (15.69%) while
in the age 1-10(19.7%). The highest infection with E. histolytica was 8.45% . Al-Abbady (2001) found
A. duedenale was in the age 11-20(3%) while the the abundant parasites was E. coli while E.
Table (2): intestinal parasite in patients with or histolytica was only 11.07%. Hama(2007)found
without anemia that E. vermicularis was the predominant
Parasite With anemia Without anemia helminthes with high rate of infection (29.8%)
Entamoeba while G. lamblia was the most abundant among
23 38.9 76 35.0 protozoa in schoolchildren in Erbil province
histolytica
Giardia (37.44%). Anyhow the abundance of E. histolytica
17 28.18 57 26.2
lamblia is likely because its life cycle is simple as there is
Enterobius no intermediate host, and the infection comes
10 16.95 80 36.8
vermicularis usually through polluted food and drinks and the
Ancylostoma house flies also is very known mechanical
5 8.47 1 0.4
duodenale transmitter in such cases, similar was drawn was
Ascaris conclusion by Niazi et al.1976.The lowest infection
1 1.69 3 1.3
lumbricoides
by T. trichura is expected as the worm is one of the
Trichuris
3 5.08 0 0.00 geohelminths which need some period about 10
trichura
days in the soil to be developed to infected stage
total 59 100.00 217 99.7

Journal of Research in Biology (2011) 5: 319-324 321

 Al-Naemi et al.,2011

Table (3): prevalence of intestinal parasites by age in 276 patients

Age (year)
parasite 1-10 11-20 21-30 31-40 41-50
n % n % n % n % n %
E. histolytica 35 35.4 40 36.7 10 32.3 11 40.7 3 30.0
G. lamblia 38 38.4 21 19.3 9 29.0 4 14.8 2 20.0
E.vermicularis 19 19.2 43 39.4 12 38.7 11 40.7 5 50.0
A. duodenale 2 2.0 3 2.8 --- --- 1 3.7 --- 0.0
A.lumbricoides 3 3.0 1 0.9 --- --- --- 0.0 --- 0.0
T. trichura 2 2.0 1 0.9 --- ---- --- 0.0 --- 0.0
total 99 100.0 109 100.0 31 100.0 27 100.0 10 100.0

(Markell et al.1999) such soil may be not available,
in other hand using chemical fertilizer instead of
stool in farming may lead to reduction of infection.
As indicated in Table(2), the percentages of
anemia were more in infected persons with E.
histolytica, G. lamblia, A. duodenale and T.
trichuris, A. lubmricoides. The presence of the
parasite which accompanied by anemia is likely due
to the effect of parasitism, or the person is in low
nutritional level. On the other hand the presence of
parasite and the absence of anemia may be because
the parasitic infection is in its early stage. Such
finding is similar to the results found by Le et al.
(2007) as she found more prevalence of anemia in
schoolchildren with intestinal parasite infection in
rural Vietnam especially in case of Trichuris
infection(76%). In other study conducted in Kenya ,
Koukounari et al.(2008) found the children heavily
infected with S. mansoni were more likely to be
anemic compared with uninfected children.
However, in a group study of youngster in
northern Brazil, Ferrerria et al.(1998) found no
statistical difference when the association was made
between each parasite (A. duodenale, T. trichuris,
A. lumbricoides) and anemia. similar study was
conducted in Paroguoy( Labiano abello et al.1999).
However, in a study conducted by
Mupfasoni et al. (2009) in northern Rwanda
concerning polyparasite in helminth infections and
their association to anemia nor the odds of stunting
were found to be significantly different in the three
polyparasite infection profiles.
In Mexico, Brenllinger et al.(2003) after a
comparative study of infection and anemia in adult
women found haemoglobin levels in hookworm-
infected women - were significantly lower than
uninfected woman anyhow anemia even if it is
evident in the present population in Shekhan district
it is not clear whether it is due to falate and vitamin
B12 deficiencies and haemoglobinopathies like
sickle-cell anemia and thalassemia. The cause of
anemia in our sample is more likely due to mild
iron deficiency which does not display microcytosis
or other nutritional deficiencies similar to the
results of Tysuyuoka et al. (1999) in Aracaju
sergipe Brazil.
As seen in Table(3), the highest percentages
of infection was in the age 11-20 and 1-10 by E.
histolytica , G. lamblia , E. vermicularis, A.
dudenale, T. trichura. The highest percentage was
in this age means that the highest infection was in
the childhood and adolescent group which mean the
persons are highly active and do not care about their
hygiene as persons are in close contact with
pollutant with soil as they play on the ground and
less care about their hygiene. Similar results of
children infection especially by S. mansoni was
observed by Koukounari et al.(2008) in Kenyan
schoolchildren similar conclusion was drawn by
Erosie etal. (2001), (see Le et al.2007) when studies
the relation between hookworm infection and
haemoglobin status in rural elementary school

Table (4): concentration rate of hemoglobin according Table (5): concentration rate of hemoglobin according
to parasites species to age group
parasite n Haemoglobin g/dl Age group n Haemoglobin g/dl
E histolytica 23 10.3
1-10 19 9.7
G. lamblia 17 11
11-20 17 9.2
E. vermicularis 10 9.5
21-30 10 8.1
A. dudenale 5 8
31-40 8 10
A. lumbriciodes 1 10
T. trichura 3 9 41-50 5 10.5
322 Journal of Research in Biology (2011) 5: 319-324
Al-Naemi et al.,2011
children in southern Ethiopia as they claims that
farming and playing in moist soil resulting in higher
exposure to infective egg and filariform larvae in
the soil. Scholchildren in rural Vietnam was also
highly infected with the highest prevalence for T.
trichura , A. lumbricoides as concluded by Le et al.
(2007). while Table(4),showing the highest
concentration of hemoglobin was in case of G.
lamblia.These results are puzzling as it is well
known that G. lamblia cause fatty stool with no
blood (Roberts and Janovy,2005). Anyhow possibly
a virulent strain ( see Roberts and Janovy, 2005)
may exist in Shekhan and caused such anemia .
On the other hand in the present results A.
duodenale seem to cause less anemia compared to
G. lamblia. However, it was also known that blood
loss per worm is about 0.03 ml per day in N.
americanus and 0.26 ml per day for A. duodenale
(Roberts and Janovy, 2005) Possibly these
fluctuations of anemia depends on nutritional status
of individuals which were not estimated in the
present investigation, as nutritional status is an
important criterion in determination of anemic
persons.
As regard Table (5), the anemia in older
group is expected as immunity usually decrease and
intestinal parasites with more percentage while the
lowest anemia was in the age 21-30 which is the
climax of young stage . It puzzling that the highest
concentration of hemoglobin in age 41-50 while
lower in age 21-30 . These seem to contradict with
the above results in Table(6) and Fig(6). These
results need more confirmation by examining the
nutritional status of individual examined in health
center, and make more larger sample of Shekhan
district.
It is suggested that further study searching
for anemia in Shekhan especially as related to
nutritional status and searching other causes of
anemia such as chromic infection ,inflammatory
disease and hemoglobin pathies like sickle-cell
anemia and thalassemia are needed of individual
which could reveal more information about their
occurrence, which later on facilitate its treatment.
Al Abbady AIA. 2001. Epidemiology of intestinal
parasites among pupils of a number of primary
schools and kinerhation children in mosul city and
Attempt to infection laboratory mice with
Enterobius vermicularis. A thesis submitted by
college Sciences, mosul university .
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 Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Vermiwash mixed diet effect on growth of Oreochromis mossambicus (Tilapia)

Journal of Research in Biology

Authors: ABSTRACT:
Rameshguru G1,
Senthilkumar P2 and
Govindarajan B3. Oreochromis mossambicusfries were collected from Kullursanthai reservoir
near Virudhunagar. They were acclimatized to the lab conditions at 291C for one
week. O. mossambicus receiving CFF (Control Fish Feed) diet exhibited a growth of
Institution:
1
Department of Zoology, 14010.58 mg in 21 days. Maximum growth was observed in fish receiving FFV (Fish
VHNSN College, Feed with 100% Vermiwash) 100 diet and it was 460.6706.03 mg. Thus vermiwash
Virudhunagar-626001, plays asignificant role in enhancing the growth.
Tamilnadu, India.
2
Entomo Pathology Lab,
Institute of forest genetics
and tree breeding,
Coimbatore-641002.
3
Manonmaniam Sundaranar
University, Thirunelveli- Keywords:
627012, Tamil Nadu, India. Oreochromis mossambicus, Vermiwash.

Corresponding author: Article Citation:

Rameshguru Rameshguru G, Senthilkumar P and Govindarajan B.
Vermiwash mixed diet effect on growth of Oreochromis mossambicus (Tilapia)
Journal of research in Biology (2011) 5: 335-340
Web Address:
http://jresearchbiology.com/ Dates:
Documents/RA0099.pdf. Received: 07 Sep 2011 /Accepted: 10 Sep 2011 /Published: 14 Sep 2011

Ficus Publishers.
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cited.

335-340 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION
Fish is the major source of protein for over
one billion people around the world (Cathay et al.,
2004). In aquaculture field, the edible fish has
earned a significant position in the nutrition of man.
It is rich in protein and amino acids like lysine,
methionine. It contains unique unsaturated fatty
acid like eicosapentaenoic acid which is known to
control cholesterol in the blood. It is easily
digestible and cheaper than other animal flesh
(Santhanam et al., 1987). Food intake is a vital
factor for fish growth and is dependent upon the
quality or chemical composition of food
(Elankumaran et al., 1992). Fish requires diets
relatively higher in protein than those of other
commercially cultured animals (NRC, 1981).
With the increasing research thrust on this
aspect during recent years there has been much
interest on the possibility of using various non-
conventional feed sources. Experiments with the
incorporation of materials of animal origin like
silkworm pupae (Venkadesh et al., 1986;
Nandeesha et al., 1988; Hossain et al., 1992), and
vermicompost (Mahalakshmi., 2004) hormones like
t h yr ox i n e ( M a ha la ks h mi , 2 0 0 4 ) a nd
diethylstilbestrol (Nanjundappa & Varghese, 1989)
and Earthworm meal (Keshavappa et al., 1989;
Kostecka & Paczka, 2006) as substitutes for meal
has also been found encouraging. The objective of
the present study was to assess the growth
ofO.mossambicus by vermiwash adding diet.
MATERIALS AND METHODS
Animal collection
Healthy O.mossambicus fries were collected
from Kullursanthai reservoir near Virudhunagar.
They were acclimatized to the lab conditions at
291C for one week. During acclimatization, the
fish were fed ad libitum with fish meal contained
control diet daily once at 10 AM and water also
changed daily.
Vermiwash preparation
The vermiwash can be collected from the
body cavity of earthworms without causing any
harm to them. In this method of collecting the fluid,
three to four earthworms were taken in an
approximately 10 cm diameter petri plate. Holding
the plate in a slanting position and keeping
earthworms pointing downwards, cold shock was
given to earthworms by gently moving a small
beaker containing a few ice cubes over the body of
worms. The vermiwash released due to cold shock
drips and gets collected at the lower side of the petri
336
Rameshguru et al.,2011
plate. The fluid can be pipetted out using a treated
pipette with fine nozzle. This is the pure vermiwash
(Radha D.Kale., 2006).
Preparation of feed
Three different fish feeds, CFF (Control Fish
Feed), FFV75 (Fish Feed with 75% Vermiwash),
FFV100 (Fish Feed with 100% Vermiwash), were
prepared by using the same quantity of ingredients
(Table 1).To the ingredients 1g of sodium alginate
was added. It acts as a binder and does not allow
the compounded feed to dissolve in water. The only
difference in the feed was the quantity of
vermiwash mixed. In the control diet the feed
ingredients were mixed with water. But in FFV75,
75% of vermiwash and FFV100, 100% of
vermiwash was used.
Experimental Design
The experiment was carried out to find out
the influence of different concentration of
vermiwash on growth, food consumption,
assimilation, metabolism and feeding rate in fish.
All the experiments were carried out in the
laboratory at 29C1C and 12:12 photoperiod. Fish
was reared in round glass troughs containing 1 liter
of water. 15 fish, of similar size were chosen from
the stock. They were weighed and divided into five
groups each containing three fish. Before the
commencement of experiments, the fish were
starved for 24 hours in order to facilitate to
evacuate their alimentary contents. They were
weighed to find out the initial live weight. The fish
were subjected to five types of experimental food
CFF, FFV75, FFV100 and all experiments were
conducted simultaneously.
Fish in all groups were fed with 100mg of
dry pelleted food. The fish were fed daily once at
10AM, the feed remains and faeces were collected
separately using pipettes and dried in a hot air oven
and weighed in a monopan balance. Water was
changed daily. The rearing experiment was carried
out for a period of 21 days. The weight of the fish
was found out on 0, 7th, 14th, and 21stday to assess
Table: 1 Feed preparation composition
Ingredients Weight (g)
Dried fish powder 42g
Ground nut oil cake powder 20g
Blood meal powder 5g
Tapioca powder 15g
Wheat flour 15g
Mineral mix 2g
Vitamins 1g
Sodium alginate 1g
Journal of Research in Biology (2011) 5: 335-340
Rameshguru et al.,2011
the growth. The fish were not subjected to any
disturbances except during feeding and water
changing operations.
RESULTS
Mass budget
O. mossambicus were reared at 291C for
21 days. The fish were divided into five groups,
each group with three triplicates. The first group of
fish received control diet (CFF). The second, third,
fourth and fifth group of fish received FFV75 and
FFV100 diets. Mass budgets were prepared for all
groups of fish (Table 2, 3 and 4).
Growth
O. mossambicus receiving CFF diet
exhibited a growth of 14010.58 mg in 21 days.
Maximum growth was observed in fish receiving
FFV100 diet and it was 460.6706.03 mg.Fish
receiving CFF diet consumed 685.670.6.11 mg of
dry food. The fish receiving FFV100 diet consumed
maximum food and it was 1088.3305.86 mg and
showed an assimilation of 774.6705.77 mg.
Feeding rate
Feeding rate of fish differed in different
groups of fish. Fish receiving CFF diet exhibited a
feeding rate of 105.3300.47 mg dry wt/g live wt/
day. Fish receiving FFV75 diet exhibited a feeding
rate of 149.3301.15 mg dry wt/g live wt/day and
FFV100 diet exhibited a feeding rate of
159.3301.53 mg dry wt/g live wt/ day.
Assimilation rate
Assimilation rate of fish differed in different
groups of fish. The fish receiving CFF diet showed
Table: 2 Mass budget of O. mossambicus fed with
control diet (CFF) at 291C for 21 days. Food assimilation (A)
Parameters (mg dry weight/individual) Control
Growth (P) 140.0010.58
Food consumption (C) 685.6706.11
Faces (F) 155.6710.26
Food assimilation (A) 530.0007.65
Metabolism (R) 390.0007.21
Feeding rate (Cr) (g live weight/day) 105.3300.47
Assimilation rate (Ar) (g live weight/day) 81.6701.53
Conversion rate (Pr) (g live weight/day) 21.6701.53
Metabolism rate (Mr) (g live weight/day) 60.0001.00
Assimilation efficiency (Ae) (%) 7701
Gross conversion efficiency (K1) (%) 2002
Net conversion efficiency (K2) (%) 1701
Note: Each value ( S D) represents an average of 3
individuals.
Journal of Research in Biology (2011) 5: 335-340
an assimilation rate of 81.6701.53 mg dry wt/g
live wt/day.The assimilation rates for fish receiving
FFV75 and FFV100 diets were 106.3302.08 and
113.6700.47 mg dry wt/g live wt/day,
respectively. Assimilation efficiency of fish in
different groups did not differ much (Table 2, 3,
and 4).
DISCUSSION
Fish growth is influenced by the nutrients
levels of food. The optimum and maximum feeding
levels are influenced by the nature of the diet. It is
dependent on the type of the food offered. Increase
in availability of food having higher protein that
plays a major role in growth. From the culturists
point of view, a good diet would be one which is
readily available, cost effective, simple and
versatile in application (Vigneeswaran 2005).
Surendra et al 2005 reported vermiwash very much
useful in growth of legumes plants.
Several research peoples have studied the
growth of fish with conventional and
supplementary diet. Food supplement includes
Table: 3 Mass budgets of O. mossambicus fed with diet
containing 75% vermiwash (FFV75) at 291C for 21
days.
Parameters (mg dry weight/
FFV75
individual)
Growth (P)
Food consumption (C)
Faces (F)
Metabolism (R)
Feeding rate (Cr)
(g live weight/day)
Assimilation rate (Ar)
(g live weight/day)
Conversion rate (Pr)
(g live weight/day)
Metabolism rate (Mr)
(g live weight/day)
Assimilation efficiency (Ae) (%)
Gross conversion efficiency
(K1) (%)
Net conversion efficiency
(K2) (%)
Note: Each value ( S D) represents an average of 3
individuals.
337
 Rameshguru et al.,2011

earthworm (Stafford & Tacon 1984), earthworm receiving FFV100 diet showed 2.3 times more
powder and krill meal (Toshio Akiyama et al., growth than control.Radha D.Kale 2006 has
1984), earthworm meal (Nandeesha et al., 1988), observed that vermiwash is rich in amino acids,
earthworm and chara plant (Elankumaran et al., vitamins, and minerals. Similarly, in this research
1992), frozen earthworms (Oscar Pereira & Emidio fish receiving FFV75 and FFV100 over fish
F.Gomes 1995) and diets containing artemia, receiving control diet. This observation reveals that,
earthworms and liver mixed diet (James & coelomic fluid mixed with balanced diet to prepare
Kunchitham Sampath 2004). fish feed does not in any way act as a repellent in
In the present work O. mossambicus fries fish.
were fed with pelleted diet containing different However, Stafford& Tacon 1984 while
concentrations of vermiwash. However fish rearing trout on earthworm reported poor growth.
This is attributed to the coelomic fluid present in
Table: 4 Mass budgets of O. mossambicus fed with diet the earthworm which, according to him is
containing 100% vermiwash (FFV100) at 291C for unfavorable to fish. However, in the present study
21 days. instead of feeding the fish with earthworm, the
Parameters (mg dry weight/ coelomic fluid of earthworm has been used to
FFV100
individual) prepare balanced diet and this has proved to be a
Growth (P) good diet improving consumption, conversion and
assimilation in O. mossambicus. Satia 1974 and
Food consumption (C) Austrong & Reftsio 1979 have reported that
increase in body protein in rainbow trout with
Faces (F) increasing dietary protein levels.
Sakthivel 1994 reported 14% increase in
Food assimilation (A)
body protein with 16% increase in dietary
Metabolism (R) protein.In the present work also, increase in the
protein, lipid and carbohydrate in the diet, increased
Feeding rate (Cr) (g live the protein, lipid and carbohydrate of the carcass.
weight/day) Gross and net conservation efficiency also
Assimilation rate (Ar) (g live increased with increase in the amount of vermiwash
weight/day)
Conversion rate (Pr) (g live
(CFF) exhibited a gross and net conservation
weight/day) efficiency of 20% and 17%, respectively. From the
Metabolism rate (Mr) (g live above observation it is concluded that vermiwash
weight/day) can be used to prepare fish diets. It does not repel
Assimilation efficiency (Ae) the food consumption and decrease growth. Instead
(%) it enhances consumption, assimilation, growth and
Gross conversion efficiency conservation efficiency in fish. As it is rich in
(K1) (%) vitamins, minerals and amino acids, vermiwash can
Net conversion efficiency (K2) be used a safe and suitable medium for the
(%) preparation of fish diets.
Note: Each value ( S D) represents an average of 3
individuals.

Table: 5Growth of O. mossambicus reared in diet containing different concentrations of vermiwash.

Days CFF FFV75 FFV100
1st day 3018.6719.86

7th day 3071.0021.59

14th day 3111.6711.24

21th day 3185.6715.70

338 Journal of Research in Biology (2011) 5: 335-340

Rameshguru et al.,2011

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 Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Temporal distribution of the transferrin alleles (Tf aand Tf b) in Amazon

Turtle (Podocnemis expansa Schweigger, 1812)
Journal of Research in Biology

Authors: ABSTRACT:
Moura AS1, Oliveira
PHG2, Klein GN3,
Tancredi NS4, Silva CA1 This study aims to evaluate the temporal distribution of allele frequencies at
and Teixeira AS1.
the codominant transferrin (Tf ) gene locus in three population samples of Amazon
turtle (Podocnemis expansa) collected from the Rio Uatum, Rio Trombetas and Rio
Institution: Tapajs in the Amazon Basin, Brazil, over a period of 16 to 24 years ago. Confirming
1. Instituto Nacional de previous research, the Tf locus which was analyzed by starch gel electrophoresis
Pesquisas da Amaznia showed a diallelic polymorphism with the presence of theoretically expected
(INPA), Avenida Andr genotypes (Tfaa, Tfab and Tfbb), presumably encoded by the codominant alleles (Tfa and
Arajo 2936, 69060-001, Tfb). A good genetic fit according to Hardy-Weinberg expectations within and among
Manaus, AM, Brasil. all the population samples was also noticed. Contingency tests on the overall
2. Centro de Preservao e transferrin allele distributions by calculating the chi-square (x2) showed no statistically
Pesquisas de Quelnios significant temporal variation (x2 = 3.05, d.f. = 5, 0.70 > P > 0.50) in the population
Aquticos (CPPQA), BR samples of P. expansa in the surveyed areas.
174, km101, AM 240, km
77, S/N, 69736-000,
Presidente Figueiredo, AM,
Brasil.
3. Instituto Chico Mendes de Keywords:
Conservao da Podocnemis expansa, Amazon Basin, transferrin allele frequencies,
Biodiversidade (ICMBio), eletrophoresis, temporal analysis.
Reserva Biolgica (Rebio)
do Rio Trombetas, Praa da
Feirinha, S/N, 68.275-000,
Porto Trombetas, PA, Brasil.
4. Instituto Brasileiro do
Meio Ambiente e dos
Recursos Naturais
Renovveis (IBAMA),
Avenida Tapajs, 2267,
Bairro do Laguinho, Article Citation:
68040-000, Santarm, PA, Moura AS, Oliveira PHG, Klein GN, Tancredi NS, Silva CA and Teixeira AS.
Brasil. Temporal distribution of the transferrin alleles (Tfa and Tfb) in Amazon turtle
(Podocnemis expansa Schweigger, 1812)
Journal of research in Biology (2011) 5: 346-351
Corresponding author:
Teixeira AS
Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 15 Sep 2011
Email:
saturn@inpa.gov.br
Ficus Publishers.

Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://
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Documents/RA0082.pdf. cited.

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INTRODUCTION
The community of Amazon aquatic turtles
has 14 species, of which the representatives of the
genus Podocnemis are edible, commercially
exploited, and act as an important source of protein
for local people (Vogt, 2008). The Podocnemis
species are listed in the Appendix II of the
Convention on International Trade in Endangered
Species of Wild Fauna and Flora (CITES),
published as Annex II, Normative Instruction
Number 11/2005, available at the Brazilian Institute
of the Environment and Renewable Natural
Resources (IBAMA). The Podocnemis expansa
(Schweigger, 1812) known as the Amazon turtle
and the largest freshwater turtle of the genus
Podocnemis in South America (Alfinito, 1975;
Pritchard & Trebbau, 1984) is no longer threatened;
however, as it still shows low risk of extinction, it is
considered a conservation dependent species
according to the criteria of the International Union
for Conservation of Nature and Natural Resources
(IUCN). Of the three levels of biodiversity
conservation recognized by the IUCN (biological
diversity, ecosystem diversity and genetic
diversity), the genetic diversity is the result of
evolutionary history of populations, in which a loss
of genetic diversity can reduce their ability to adapt
in response to environmental changes (evolutionary
potential) (Frankham et al, 2002).
Research focusing on the population
structure of Podocnemis expansa has already been
carried out by the plasma iron binding protein,
transferrin (Teixeira et al, 1996), allozymes (Bock
et al, 2001), mitochondrial DNA and microsatellites
(Sites et al, 1999; Pearse et al, 2006).
Analysis involving temporal variations in
allele frequencies of molecular genetic markers can
be extremely useful for investigating genetic
variability, effective population size (Ne), founder
Table 1. The sampling localities from where population samples of the Amazon turtle (P. expansa) were caught
in the Amazon Basin.
Stage of
Locality and position of catch
development
Maracarana, Rio Uatum, AM
Hatchling
(0213S; 5815W)
Trombetas, Rio Trombetas, PA
Hatchling
(0404S; 5538W)
Monte Cristo, Rio Tapajs, PA
Hatchling
(0120S; 5645W)
Total - -
347
Moura et al.,2011
effects and processes associated with colonization
and population-genetic structure (Waples, 1989;
Kambhampati et al, 1990; Shikano et al, 2001;
Williams et al, 2003; Jacquemyn et al, 2006;
Chevolot et al, 2008; Lucentini et al, 2009).
However, this type of study has never been
performed on P. expansa.
The aim of this study was to evaluate the
temporal distribution of transferrin allele
frequencies from three population samples of P.
expansa collected from the Rio Uatum, Rio
Trombetas and Rio Tapajs in the Amazon Basin,
Brazil.
MATERIAL AND METHODS
After obtaining license given by the
Brazilian Institute of Environment and Renewable
Natural Resources (IBAMA) through the
Biodiversity Authorization and Information System
(SISBIO License No. 13081-1 of 19/12/2007),
ninety five specimens of the Amazon turtle (P.
expansa) were collected from three geographical
areas in the Amazon region, totalizing three
population samples: Maracarana, Rio Uatum-AM
(30); Trombetas, Rio Trombetas-PA (34) and
Monte Cristo, Rio Tapajs-PA (31) (Table 1, Fig.
1). Blood samples were taken from the femoral
artery and placed in 5 ml BD vacutainer tubes,
containing 0.5 ml of 3.8% sodium citrate as
anticoagulant, and blood plasma specimens were
separated by centrifugation at 3,000 rpm for 20
minutes and stored at -25C, until electrophoresis
run (Teixeira et al,1996). A rivanol precipitation
method was used for isolating plasma transferrin
molecules prior to starch gel electrophoresis
(Jamieson & Tuner, 1978), with modifications
performed by Teixeira et al. (1996), where the
blood plasma specimens were treated with rivanol
solution (2%) at a ratio of 1:1. For detecting the
Number of specimens
Date of catch
tested
10.01.2008 30
04.02.2008 34
30.01.2008 31
95
Journal of Research in Biology (2011) 4: 346-351
Moura et al.,2011
Fig. 1. Map of the approximate locations from which the population samples of P. expansa, were captured from
the Amazon region: Maracarana, Rio Uatum (1); Trombetas, Rio Trombetas (2) and Monte Cristo, Rio
Tapajs (3). Map modified from Teixeira et al. (1996).
transferrin molecules, the starch gels were stained
in 1% Amido Black for five minutes (Jamieson &
Tuner, 1978). The Amido Black was diluted in a
solution composed of water, methanol and acetic
acid (5:5:1), respectively. Then the gels were
washed by successive immersions in plastic
containers containing the above-mentioned solution
(water, methanol and acetic acid) for approximately
15 hours, and the Tf alleles were identified and
classified according to Teixeira et al. (1996). At the
end of the experiments all the animals were
returned to their respective collection sites.
Chi-square (x2) and maximum log-likelihood
(G) tests for genetic balance assuming Hardy-
Weinberg equilibrium were applied to compare the
observed and expected numbers of transferrin
genotypes within and among the Amazon turtle
Journal of Research in Biology (2011) 4: 346-351
population samples examined. Moreover, chi-
square contingency tests were applied on the overall
transferrin allele distributions to compare the
population samples tested in this study with those
tested previously by Teixeira et al. (1996).
RESULTS AND DISCUSSION
The transferrin gene locus (Tf) of P. expansa
population samples (Uatum, Trombetas and
Tapajs), which was analyzed by starch gel
electrophoresis, showed a diallelic polymorphism
with the presence of the genotypes (Tfaa, Tfab and
Tfbb) theoretically expected, presumably encoded by
codominant alleles (Tfa and Tfb) (Fig. 2). A good
genetic balance within and among all the population
samples was noticed (Table 2). The present
findings are in accordance with the previous report
348
 Moura et al.,2011

over a period of 16 to 24 years ago (Table 3).

Although the population samples of P. expansa
during this time have been suffering some degree of
predation for ma i nt ena nc e a n d/ or
commercialization (Alho, 1985; Alves & Santana,
2008), the locus Tf has preserved its integrity for
Tf a about two generations, since, according to Vogt
b
Tf (2008) this species takes eight to 12 years to reach
its reproductive maturity. This means that in theory,
during this interval of time (two generations),
Tf a neither the anthropogenic action mentioned above,
Tf b nor the evolutionary processes that normally
contribute to change the genetic composition of
populations: mutation, migration, genetic drift,
natural selection, among others (Ayala & Kiger,
1980; Hartl, 2008) were sufficient to cause
Fig. 2. Electrophoregram of Tf locus in P. expansa. significant changes at the locus Tf in P. expansa.
The genotypes are: lanes 1 and 5 (Tfaa); lanes 2, 4 and Apparently conservation programs of the Amazon
6 (Tfab) and lane 3 (Tfbb). turtle are contributing to maintaining the genetic
of Teixeira et al. (1996), whose data were variability of its natural populations over time, at
consistent with a free flow of genes among the least for the Tf locus.
population samples, supporting the hypothesis of a Routine analysis with historical series of
single stock of P. expansa in the geographic area molecular genetic markers examined so far in P.
examined. expansa, such as, transferrin (Tf) (Teixeira et al,
Contingency tests revealed no temporal 1996), allozymes (Bock et al, 2001), nuclear and
variation on the overall Tf allele distributions in the mitochondrial DNA (Sites et al, 1999; Pearse et al,
populations samples of P. expansa in the surveyed 2006) will serve as an effective tool in obtaining
areas, when the present data were compared with information about the genetic composition of its
those previously reported by Teixeira et al. (1996) natural stocks. This kind of approach is very
Table 2. The genotypes and transferrin allele frequency distributions in three population
samples of Amazon turtle (P. expansa). Chi-square (x2) and maximum log-likelihood (G)
statistical values assuming Hardy-Weinberg equilibrium are shown. The observed numbers in
each transferrin genotype showed good agreement with expected numbers shown in
brackets.
Population samples tested
Rio Uatum Rio Trombetas Rio Tapajs Total
N = 30 N = 34 N = 31 N = 95
Tf genotypes
Tfaa 16 (16.0339) 22 (22.1642) 20 (19.2787) 58 (57.5556)
Tfab 12 (11.9322) 11 (10.6716) 9 (10.4426) 32 (32.8889)
Tfbb 2 (2.0339) 1 (1.1642) 2 (1.2787) 5 (4.5556)
Tf allele frequencies
Tfa 0.7333 0.8088 0.7903 0.7789
Tfb 0.2667 0.1912 0.2097 0.2211
Hardy-Weinberg test
d.f. 1 1 1 1
x2 0.001022 0.0345 0.6332 0.0708
P 0.9745 0.8527 0.4262 0.7902
G 0.001024 0.0355 0.5824 0.0697
P 0.9745 0.8504 0.4454 0.7918
349 Journal of Research in Biology (2011) 4: 346-351
Moura et al.,2011

Table 3. Chi-square contingency tests used to examine the temporal distribution of the transferrin alleles (Tfa
and Tfb) among the population samples of P. expansa from three geographical areas in the Amazon region.
Expected numbers of alleles are shown in parentheses.

Rio Uatum Rio Trombetas Rio Tapajs

Teixeira Teixeira Teixeira
This This This
et al. (1996)1 et al. (1996)2 et al. (1996)3 Total
report report report
46 44 85 55 98 49
Tf a 377
(47.92) (47.92) (81.47) (54.31) (95.85) (49.52)
14 16 17 13 22 13
Tf b 95
(12.08) (12.08) (20.53) (13.69) (24.15) (12.48)
Total 60 60 102 68 120 62 472
(x2 = 3.05, d.f. = 5, 0.70 > P > 0.50)
1
Sample collected in the Rio Uatum (Balbina Hydroelectric Power Plant Dam) from 13.04.1988 to 22.05.1988
(adults).
2
Samples collected in the Rio Trombetas in 1984 (juvenile), in 24.11.1988 (adults), in 10.01.1990 and 04.01.1992
(hatchlings).
3
Sample collected in the Rio Tapajs in 04.01.1992 (hatchlings).

important for monitoring, preservation and Alho CJR. 1985. Conservation and management
management programs of this species which are strategies for commonly exploited Amazonian
now being carried out by the Chico Mendes turtles. Biol Conserv., 32:291-298.
Institute for Biodiversity (ICMBIO) and the Center
for Conservation and Management of Reptiles and Alves RRN and Santana GG. 2008. Use and
Amphibians (RAN) in the Amazon region. commercialization of Podocnemis expansa
(Schweiger 1812) (Testudines: Podocnemididae)
ACKNOWLEDGMENTS for medicinal purposes in two communities in
This research was partially financed by the North of Brazil. J Ethnobiol Ethnomed., 4:1-6.
Instituto Nacional de Pesquisas da Amaznia
(INPA), Manaus, AM, Brasil through the Ayala FJ and Kiger Jr. JA. 1980. Modern
Institutional Project Programme (PRJ12.01). The Genetics. The Benjamins/Cummings Publishing
authors wish to thank the Coordenao de Company, Inc., Menlo Park, California.
Aperfeioamento de Pessoal de Nvel Superior
(CAPES), Braslia, DF, Brasil and Fundao de Bock BC, Pez VP and White MM. 2001. Genetic
Amparo Pesquisa do Estado do Amazonas population structure of two threatened South
(FAPEAM), Manaus, AM, Brasil for the Master of American river turtle species, Podocnemis expansa
Science (MSc) scholarship given to Arlisson Silva and Podocnemis unifilis. Chelonian Conserv Bi.,
de Moura, the Centro de Preservao e Pesquisas de 4:47-52.
Quelnios Aquticos (CPPQA), Presidente
Figueiredo, AM, Brasil, the Instituto Brasileiro do Chevolot M, Ellis JR, Rijnsdorp AD, Stam WT
Meio Ambiente e dos Recursos Naturais and Olsen JL. 2008. Temporal changes in allele
Renovveis (IBAMA), Santarm, PA, Brasil and frequencies but stable genetic diversity over the
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the turtle specimens in the field, and Mr. George Frankham R, Ballou JD and Briscoe DA. 2002.
Nakamura who kindly revised the preliminary Introduction to conservation genetics. Cambridge
English version of the manuscript. Univerity Press, Cambridge, UK.

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351 Journal of Research in Biology (2011) 4: 346-351

 Journal of Research in Biology
Study on display sequences in Cotton Pygmy-Goose Nettapus
Coromandelianus Coromandelianus Gmelin
Journal of Research in Biology
Authors:
Upadhyaya S1 and
Saikia PK2.
Institution:
1. Asstt. Prof., Dept. of
Zoology, Tyagbir Hem
Baruah College,
Karchantola, Dist.-Sonitpur
(Assam), India. PIN-784189.
2. Department of Zoology,
Gauhati University, Assam
(India).
Corresponding author:
Sanjib Upadhyaya
Email:
sanjib1970@sify.com
Web Address:
http://jresearchbiology.com/
Documents/RA0089.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper Publication group
ABSTRACT:
The pairing and display sequence of Cotton Pygmy-goose (Nettapus
coromandelianus coromandelianus Gmelin) in Eastern Assam was observed from April
2007 to July 2008. The frequency of head movement (up-down and left-right) are
found to be 0.288 and 0.28 per second. Courtship bouts occurred in groups averaging
5.7 birds, with a male: female ratio of 1.1:1. Several new displays and vocalizations are
described. Early pair bonds appeared tenuous and were continually tested until nest-
searching activities began in May. Birds in groups displayed and vocalized more than
paired birds, and birds displayed and vocalized more or less equally throughout the
period with mild rainy climate. Early pairing may be related to a birds condition,
climatic condition, success in obtaining a nesting site, and increased productivity.
Keywords:
Burping, coquette call, chin-lifting, display shake, inciting, preening,
vocalizations, whistling jerks.
Article Citation:
Upadhyaya S and Saikia PK.
Study on Display Sequences in Cotton Pygmy-Goose Nettapus Coromandelianus
Coromandelianus Gmelin.
Journal of research in Biology (2011) 5: 341-345
Dates:
Received: 21 Aug 2011 /Accepted: 27 Aug 2011 /Published: 15 Sep 2011
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 Upadhyaya et al.,2011

with the river Brahmaputra.

INTRODUCTION Scan and ad libitum sampling methods were
Most anatids confine their displays to the used to study the nesting behavior of the Cotton
water (or land) surface, since their heavy weight Pygmy-goose, as per Altmann (1974) and
relative to their wing area dictates continuous Brockelman (1975). Field observations were
flapping and makes complex maneuvers, such as conducted continuously for three days from April
hovering and soaring, difficult or impossible. Aerial 2007 to July 2008 on every week in each month of
communication is thus largely restricted to short, the year at Kadamani wetland and Dagaon.
ritualized flights (ordinarily close to the water Courtship was recorded from a portable tree blind
surface) and vocalizations, including contact calls or while concealed in vegetation or in the open. The
that help to maintain flock coherence in these rapid day samplings were taken total of 2 5 hours and
fliers that often go long distances between landings. totaling 50 hours devoted in a week. The sampling
Most of the studies on display behavior of anatids patterns followed for the study time viz., first day:
are confined to Mallards and a few other species. 05:00 to 08:00 hours and 09:00 to 11:00 hours,
The ethogram of the Cotton Pygmy-goose second day: 08:00 to 09:00 hours, and 11:00 to
(CPG, Nettapus coromandelianus coromandelianus 14:00 hours, third day: 14:00 to 16:00 hours.
Gmelin) is incomplete, so to say not studied at all. Cotton Pygmy-goose behavior before and after
A description of Wood duck vocalizations and courtship and were observed using a pairs of
courtship displays is given by Heinroth (1910) and binoculars (10 X 50 and 20 x 50) and data were
Lorenz (1951 - 1953). Display behavior in Blue- recorded and sketches were drawn. No birds were
billed Dick has been studied by Johnsgard and marked. The frequency of head movement during
Nordeen (1981). Males court one female with social feeding and searching of nesting trees were also
display relative to Anas species (Lorenz 1951 recorded. The display behaviors were categorized
1953, Wishart 1983). Pair formation, courtship and as per Lorenz (1951-1953).
display in musk duck and mallards were also
studied (Bossema and Roemers 1985, Fullagen and RESULTS & DISCUSSION
Carbonell 1986). The Cotton Pygmy-goose generally found
Information is lacking on the chronology of to select big hole or cavity bearing trees for nesting
pair formation and strength of pair bonds on CPG, purposes during the breeding season. The initiation
because most published studies have concerned of searching the nesting tree was made by the pair
only spring courtship behavior in Wood ducks before 10 12 days of egg laying. Both the male
(other than CPG), Mallards (Lebret 1961; and female birds circling in an around the selected
Weidmann & Darley 1971) and a few other species. nesting tree, might be to confirm the security status
CPGs breeds during the monsoon season (Ali & of the trees and as well as the anthropogenic
Ripley 1983). This study was designed to determine activities. They perform several rounds of flights in
the chronology and mechanism of pair formation various hours of the day near nesting trees and then
and display in CPG. they select the nest sites. The monitoring flight rate
STUDY AREA & METHODS was found to be high on a rainy day between 07:00
The study was carried out in the Sonitpur 10:00 hours. They move around the nesting tree
district of Assam (240 09/ N to 27058/ N and 89042/ for nesting territory with a mean duration of 3.28
E to 96001/ E) during 2007 - 2008. The Sonitpur hour/day (n = 7, Table 1). The selection of nesting
district of Assam, with an area of 5,324 km2, is tree was an unique phenomenon of CPG which
located in between 9302/80// E to 930 57/1// E prefers to move out during rainy or mild raining
longitude and 260 22/1// N to 260 42/ 2// N latitude. condition and the frequency was found to be 0.056
The district is bounded by Hawajan tributary in the rounds per seconds.
east, Pachnoi tributary in the west, the mighty river Display behavior and pair formation
Brahmaputra in the south and the state Arunachal During field observations for display
Pradesh (previously North Eastern Frontier Area or behavior and pair formation, altogether 110 Cotton
NEFA) in the north. Physiographically, major parts Pygmy-goose were encountered on 10 occasions.
of the district are plain area with a number of Courtship was observed twice (once at Kadamani
tributaries like Pachnoi, Mora-Bhoroli, Jia-Bhoroli, on 21st May 2007 for 1.4 min and once in an
Ghiladhari, Burigang, Borgang, Buroi, and Satrang agricultural field on 12th June 2008 for 1.1 min near
arose from the hills of Arunachal Pradesh and joins Dagaon, Biswanath Chariali). Display bouts
342 Journal of Research in Biology (2011) 5: 341-345
Upadhyaya et al.,2011

Table 1 Movement pattern of Cotton Pygmy-goose during nesting period (prior to nest selection) 2007;
(location- Kadamani; Tree species- Sowalo Litsea polyantha)
Freq. of
Weather Active period Total time Birds Mov. Social
Obs. Dates movement
condition (in hours) (in hours) (no. of tips) Structure
/sec.
1 06/7/ 2007 Cloudy 06.30 -09.30 3.00 hrs 8 P 0.044
2 14/7/ 2007 Raining 07.10 -10.00 2.40 hrs. 11 P 0.069
3 15/7/ 2007 mild raining 09.45 -11.45 2.00 hrs 8 P 0.067
4 15/7/ 2007 mild raining 07.30 -10.30 2.00 hrs 5 P 0.042
5 16/7/ 2007 --do-- 11.00 -14.00 3.00 hrs 0 0 0
6 07/8/ 2007 mild raining 07.00 -09.00 2.00 hrs 7 S (male) 0.058
7 07/8/.2007 --do-- 09.30 -11.30 2.00 hrs 0 0 0
Average --- --- 3.28 hrs/day 7.8 tips/day --- 0.056 / second

consisted of a series of sequences in which the
intensity of sexual behavior progressively
increased. Various types of display bouts were
observed, which were categorized as: (i)
vocalizations, (ii) head-up-down movement, (iii)
head-turn, (iv) burping, (v) Whistling jerks or bill
shaking, (vi) Coquette call, (vii) chin-lifting, (viii)
display shake, (xi) wing-and-tail-flash, (x) inciting
and (xi) preening and feeding etc.
A typical display sequences were usually
starts with vocalizations or agonistic postures from
male (Fig. 1a). The male sat quietly on the water;
with the head turn back (Fig. 1b) and slowly
changed positions around the female. The male
starts whistling or possess bill jerks or shakes their
bills (Fig. 1c). The female produce the Coquette
call (Fig. 1d). The female move the bill down and
up as it drinking water and lifts the chin up (Fig.
1e), and make a display shake and whistled
frequently along with the wing-and-tail-flash
(Fig.1f) until a male began to inciting or males
starts fighting among them. Following an agonistic
possession between them, both usually bathed. This
behavior leads to a session of preening on breast or
back or feeding, and then they completed display
(Fig. 1gi; Fig. 2). Similar types of behavioral
observations were also made by Lorenz (1951 - 53),
Johnsgard (1960), McKinney (1970), Armbruster
(1982) and others in various species of anatids.
Increased intensity of vocalizations and aggressive
behavioral postures increases the sexual excitement
again. The length of display sequences ranged
between 2 - 6 minutes interspersed with 10 - 65
minutes of preening or feeding. These types of
behavioral displays were frequently seen during
breeding season. The display behaviors were
observed during early hours of the morning and late
afternoon. The displayed vocalization was almost
similar with that of domestic pigeon before mating,
viz. gor...r...gor...r...gor...r.....r........The up-down
postures, left-and-right side movement of head are
the common display behavior of CPG. The
frequency of head movement was found to be 0.288
and 0.28 per second (total duration =204.29
minutes; n =7) respectively during the study period
(Table 2). Fox and Madsen (1981) published more
or less similar results in Greenland White-fronted
goose (Anser albifrons) while studying its pre-
nesting behavior.

Table 2. Frequency of head movement of Cotton Pygmy-goose during breeding season

(Location: Kadamani wetland, date: 02/07/2007 and 03/07/2007).
Sl. Nos. Distance of Duration of Up and down movement Lt. and Rt. movement of
obs. (m) obs. (sec.) of the head the head
No. of times Freq/sec No. of times Freq/sec
1 18.00 60 33 0.55 31 0.517
2 20.00 120 40 0.333 36 0.300
3 22.00 120 31 0.258 36 0.300
4 26.00 150 36 0.24 33 0.220
5 22.00 150 49 0.327 47 0.313
6 23.00 230 37 0.161 37 0.161
7 32.00 600 87 0.145 88 0.147
Average 23.29 204.29 44.71 0.288 44 0.280

Journal of Research in Biology (2011) 5: 341-345 343


Fig. 1 Diagrammatic representation of courtship behavior of Cotton Pygmy-goose observed during the
study period.
The results of the present study shows
that, in Cotton Pygmy-goose the pair formation
starts from the month of May and retains up to the
month of July, though they pass most of the time of
the year in flocks, they form pair with a male and a
female during the breeding season. The Cotton
Pygmy-goose moves out of the foraging site during
breeding period in search of nesting tree. The
frequency of group formation was found to be 5.4
birds with maximum 8 pairs during the third week
of May and second week of June 2007 (Table 3).
The study shows that, the Cotton Pygmy-
goose prefers to nest during June September.
Higher frequency of nesting was found in the
month of August, though the pairing and courtship
display starts in the months of May to July during
the study period (n =52). The nest site selection and
display behavior in Cotton Pygmy-goose are related
344
Upadhyaya et al.,2011
with the breeding season and availability of habitats
for the growing ducklings. This might be due to
their requirement of extra as well as stored energy
for nesting period. Pair formation takes place
throughout the year for the adult Cotton Pygmy-
goose. However, the most active period of display
and copulation found during the present observation
was May to July. It is important to mention here
that, the growing of breeding plumage will
encourage to formation of pairs. The Cotton Pygmy
-goose acquires alternate plumage in November
retains up to February, pair by May to July, lay first
eggs in second half of the May. The breeding
plumage might help in courtship. Though early
pairs are frequently unstable, but firm bonds are
distinguishable only after many weeks of pairing
activity.
Journal of Research in Biology (2011) 5: 341-345
Upadhyaya et al.,2011

Table 3. Pair formation in Cotton Pygmy-goose around the Kadamani wetland, 2007.
Months Duration No. of pairs No. of solitary male/female
May 2007 1/5/2007 to 7/5/2007 3 -
8/5/2007 to 14/5/2007 4 -
15/5/2007 to 21/5/2007 8 -
22/5/2007 to 28/5/2007 6 1M
29/5/2007 to 31/5/2007 6 1M
June 2007 1/6/2007 to 7/6/2007 6 1M
8/6/2007 to 14/6/2007 8 -
15/6/2007 to 21/6/2007 4 1M
22/6/2007 to 28/6/2007 6 1M
29/6/2007 to 30/6/2007 6 1M
ACKNOWLEDGEMENT 101-156.
Authors are very much thankful to Mr. C. K.
Bora, the then DFO, Sonitpur Forest Division Johnsgard PA. 1960. Comparative behaviour of
(East), and Mr. Biren Sahani, Kadamani for their the Anatidae and its evolutionary implications.
active support during the study period. Authors are Wildfowl 11:31-45.
also thankful to UGC (NERO) for providing
financial assistance in the form of Minor Research Johnsgard PA and Nordeen C. 1981. Display
Project. behavior and relationships of the Argentine Blue-
billed Duck. Wildfowl 32:5-9.
REFERENCES
Ali S and Ripley SD. 1983. Handbook of the Birds Lebret T. 1961. The pair formation in the annual
of India and Pakistan. Compact Edn. Oxford Univ. cycle of the Mallard (Anas platyrhynchos L.).
Press, New Delhi (revised edition). xlii+737. Ardea 49:97 -158.

Altmann J. 1974. Observational study of Lorenz K. 1951-1953. Comparative studies on the

behaviour: sampling methods. Animal Behav., behavior of Anatidae. Aviculture Manage., 57:157-
49:227-267. 182; 58:8-17, 61-72, 86-94, 172-184; 59:24-34.

Armbruster JS. 1982. Wood Duck displays and McKinney F. 1970. Displays of four species of
pairing chronology. The Auk., 99:116-122. blue-winged ducks. Living Bird, 9:29-64.

Bossema I and Roemers E. 1985. Mating strategy Weidmann U and Darley J. 1971. The
including mate choice including mate choice in synchronization of signals in the social display of
Mallards. Ardea 73:147-157. Mallards. Rev. Comp. Anim., 5:131-135.

Brockelman WY. 1975. Competition, the fitness of Wishart RA. 1983. Pairing chronology and mate
o f f s p r i n g a n d o p t i m a l c l u t c h - s i z e. selection in the American Wigeon (Anas
American Naturalist 109:677-699. americana). Can. J. Zool., 61:1733-1743.

Fox AD and Madsen J. 1981. The pre-nesting

behavior of the Greenland White-fronted
Goose. Wildfowl 32:48-54.

Fullagen PA and Carbonell M. 1986. The display

postures of the male Musk Duck, Wildfowl. 37:142
-150.

Heinroth O. 1910. Beobachtungen bei

Einbiirgerungsversuch mit der Brautente
(Lampronessa sponsa). Journal of Ornithology 1:

Journal of Research in Biology (2011) 5: 341-345 345

 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Some observations on demography and edible plants of Lion-tailed Macaques

(Macaca silenus) in the rain forest fragmented habitats of Anamalai Hills, Western Ghats.
Journal of Research in Biology

Authors: ABSTRACT:
Narasimmarajan K, Some observations on demography and edible plants species of endangered
Nagarajan R and Lion-tailed Macaques (LTM) (Macaca silenus) were studied between January 2008 and
Kumaraguru A. July 2008 in the rain forest fragments of Annamalai Hills, Southern India. Totally, 14
different fragments were surveyed, in which LTM existence was observed in 10
fragments. LTM were noticed in Iyyerpady, Uralikkal, Chinnakalar, Manompoly,
Sekalmudy, Korangumudi, Pannimedu, Varattuparai, Puthuthottam and Andhiparai.
18 different troops of LTM numbering 279 individuals were observed in 10 different
fragments. The group size ranged from smallest of five individuals at Pannimedu to
highest number of 62 individuals at Puthuthottam. In total, 48 adult males, 86 adult
Institution: females, 24 sub-adult males, 41 sub-adult females, 49 juveniles and 30 infants were
Deportment of Zoology and observed for the entire study period. It was observed that the group size and number
Division of Wildlife of troops varied in these fragments which could be attributed to the food availability
Biology, AVC College, in each fragments and other factors such as anthropogenic pressures, competition
Mayiladuthurai 609001, between sympatric primates. Other variations such as diversity of vegetation, mean of
South India. tree density, Girth at Breast Height (GBH), canopy spread and tree height noticed in
these fragments were also analyzed for their implication on the distribution pattern.
Based on this study we give some essential suggestion (1) Ecotourism is one of the
serious threats for the LTM populations, (2) People knowingly feeding the plastics and
food items to LTMs, which are yet to have existing on the road side. (3) We suggest
that tourists traveling between Azhiyar to Valparai should be checked properly with
mobile patrol and spot penalty is to be needed (Fig; 2) for the control of these
Corresponding author:
activities.
Narasimmarajan K
Keywords:
Demography; Lion-tailed Macaque, Fragmentation, Edible plants, Ecotourism
and Anamalai
Abbreviations:
LTM Lion-tailed Macaque.
Email:
narasimma@wii.gov.in Article Citation:
Narasimmarajan K, Nagarajan R and Kumaraguru A.
Some observations on demography and edible plants of Lion-tailed Macaques
(Macaca silenus) in the rain forest fragmented habitats of Anamalai Hills, Western
Ghats.
Journal of research in Biology (2011) 5: 352-362

Web Address: Dates:

http://jresearchbiology.com/ Received: 29 Aug 2011 /Accepted: 16 Sep 2011 /Published: 20 Sep 2011
Documents/RA0093.pdf.

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commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

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INTRODUCTION
Tropical rain forests are the centers of bio-
diversity. These forests cover about 6% of the land
surface of the world (McNeely et al., 1990).
Extensive removal of these forests is therefore a
matter of global concern, and especially isolation
process continuous the progressive lose an
endangered species from these fragments
(Kumaraguru, 2005). Furthermore the effects of
fragmentations on many forests could ultimately
have the same consequences as extinction (Cutler,
1991). Wilcox and Murphy (1985) stated that,
Habitat fragmentation is the most serious threat to
biological diversity and forms the primary cause of
the present extinction crisis. At the current rate of
habitat loss and fragmentation of tropical rain
forests, the survival of significant components of
bio-diversity would depend on the retention and
management of a network of fragmented forest
(Saunders et. al., 1991).
Ecological and behavioural changes form
the first phase of the interactions between a species
and fragmentation of its habitat (Umapathy, 1998).
These include alterations in the activity pattern,
feeding ecology, changes in predation pressure,
inter-specific competitions and host-parasite
relationships (Strunsaker, 1976). The demography
consequences are the final response of the species
to habitat fragmentation leading earlier to its
decline and gradually to the extinction. The
demographic changes in primates might be due to
habitat degradation and severe resource depression
(Umapathy and Kumar, 2000b; Struhasker, 1976;
Sugiama and Oshawa, 1982; Decker, 1994; Johns
and Skorupa, 1987). Parasites and pathogens
(Holmes, 1996), stochastic and inbreeding (Quinn
and Hastings, 1987) also cause demographic
changes in fragmented population. Dispersal can
increase the persistence and stability of fragmented
population through various ways (Hanski and
Gilpin, 1991, Arnold et. al., 1993). However, there
is a considerable variation among species in their
ability to disperse among habitat islands (Singh, et.
al. 2001).
Hence we studied the demographic and food
plant species of endangered, close to extinct (IUCN,
2007), Lion-tailed Macaque (LTM) (Macaca
silenus) due to habitat fragmentation. We examined
major demographic parameters in selected
fragments. Fourteen fragments of the Indira Gandhi
Wildlife Sanctuary in the Anamalai hills were
selected as the main study area. Anamalai hills have
the largest extent of rain forests in Western Ghats
353
Narasimmarajan et al.,2011
(Singh, et.al, 2002). Due to extensive plantation of
beverages; these rain forests are highly fragmented.
Hence, the LTMs habitat is highly fragmented and
surrounded by tea estates (Singh, et.al. 2006). Some
of the basic Demographic parameters and edible
plants species were studied based on the following
objectives.
1. To identify some of the major demographic
parameters and distribution pattern of Lion-
tailed macaque.
2. To assess the vegetation characteristic features
for selected fragmented habitats.
Study Area
The study was conducted in rain forest
fragments in and around Anamalai Tiger Reserve
(formaly Indira Gandhi Wildlife Sanctuary) in
Anamalai hills, Southern India. It was extensively
clear felled between 1860s and 1930s for
cultivation of tea, coffee and cardamom; later teak
and eucalyptus plantations leaving behind
fragments varying in area from less than 10 hectares
to more than 200 hectares (Singh et.al, 2002). The
Anamalai Tiger Reserve (101108N to 10
3327N and 764902E to 772109E) isolated
in the state of Tamil Nadu is one of the largest
sanctuaries in south India (Fig.1) with an area of
987sq km. Rainfall varies from 800 mm to 1000
mm. The day temperature varies from 23C to 40C
at the foothills, and from 20C to 30C at higher
elevations (above 1800m).
Extensive clear-felling has reduced the
tropical rain forest in the sanctuary and adjoining
areas to 30 fragments ranging in the area from less
than 10 hectare to about 2500 hectares (Singh et.al,
2002). Only five fragments are more than 200
hectares in area. Most of the smaller fragments
occur as islands surrounded by tea estates and are
private owned viz., the larger fragments (more than
100 hectares) of the sanctuary are bordered by teak
and eucalyptus plantations and in some cases viz.,
by tea plantations on one side. This area has a town
(Valparai) with a human population of nearly 2,
00,000 people (Umapathy and Kumar, 2000a).
METHODS
The Valparai town was taken as the centre
and 14 fragments within the radius of 30Km were
taken for the intensive study. The fragments were
Iyyerpady, Uralikkal, Chinnakalar, Manompoly,
Sekal mudy, K or angumudi , Panni medu,
Varattuparai, Puthuthottam, Andhiparai, Solayar
Dam, Kavarakkal, Sangarankudi and Akkamalai
respectively. The survey was conducted following
Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011
(Merenlender et al. 1998), the method, which that
relies on repeatedly identifying social groups, and
obtaining demographic data on all the identified
groups. At most sites, data were collected on pre-
existing forest trails, abundant forest roads and
nallah (marked at 5 km intervals) that had either
been created by forest Dept. or by researchers
working in the area. Each trail roads and nallah
were surveyed from early morning to early
afternoon, at a slow pace. During the study period,
all detectable groups were identified to count total
numbers and determine the age, sex classes (adults,
sub adults, juveniles, and infants), whenever
encounters LTM, the details of feeding activities
were also recorded. Data collections were repeated
4-5 times at each site, over 3-day period intervals
(R. J. Rao & Abhishek Bhatnagar 2001).
Size of these 14 fragments and altitude were
recorded from the survey maps and earlier literature
(Umapathy & Kumar, A 2000b). The group counts
were taken when the entire group had to move one
or very few canopy, across the road, steams or
canopy openings. The age/sex classification was
based on a comparison with individuals of known
Fig 1. Map showing the rain forest fragments of Anamalai Hills, Western Ghats.
Journal of Research in Biology (2011) 5: 352-362
approximate age in groups that have been studied
for many years. Individuals in a group were
classified as infants (up to 1year old), juveniles (1
to 4 years), sub-adults (4 years to first birth at about
6.5 years for females; 5 years to about 8 years old
for males), adult females (females that have given
birth at least once) and adult males (>8 years old)
(Singh, et.al, 2002; Umapathy and Kumar, 2000b).
Vegetation in these fragmented habitats was
assessed using parameters such as tree density,
Girth at Breast Height (GBH) of the trees, canopy
spread, and tree height. These were estimated from
forty to fifty 1010 meters quadrates in selected
eight fragmented habitats. The vegetation plots
were done in Iyyerpady, Uralikkal, Chinnakalar,
Varattuparai, Korangumudi, Puthuthottam,
Andhiparai and Akkamalai fragments respectively.
In each quadrates all trees were enumerated the
measurement of GBH in each quadrate the number
of tree species, number of food species, number of
individuals of each species and basal area were
assessed. The various edible plant portions such as
leaf, flower, fruit, mesocarp, seed coat and seed
were recorded from all the food trees by observing
354
 Narasimmarajan et al.,2011

LTM. The trees used by LTM for roosting were
also recorded. LTM were observed through Nicon
Action series binoculars 10X.
RESULTS
Among the 14 fragments LTM was observed
in only 10 fragments. Troop size of LTM observed
in these fragments is given in Table 1. The altitude
of the fragment ranged from 750 1300m,
fragments ranged from 24 ha 2000 ha (Table 1).
On the other hand, the LTM was not observed in
Solayar Dam, Kavarakkal, Sangarankudi and
Akkamalai. These fragments had different
vegetations and forest types such as evergreen
forest, rain forest, moist deciduous forest, wet
evergreen, dry evergreen forest, and also the
Cardamom (Elettaria cardamomum) and Coffee
(Coffea arabica) plantations. Totally 18 different
troops were observed in 10 different fragments. The
fragments Chinnakalar, Manompoly, Korangumudi
and Varattuparai had one troop each. A maximum
of four troops were observed in Iyyerpady whereas
in Puthuthottam three troops were observed. The
fragments viz., Uralikkal, Sekalmudy, and
Andhipparai had two troops each (Table 1).
The demography of LTM in different
fragments is given in Table 2. A total of 279
individuals were observed in 10 different
fragments. The group size ranged from smallest of
five individuals at Pannimedu to highest number of
62 individuals at Puthuthottam. In total, 48 adult
males, 86 adult females, 24 sub-adult males, 41 sub
-adult females, 49 juveniles and 30 infants were
observed for the entire study period. Pannimedu
had the smallest troop (5 individuals) followed by a
troop in Puthuthottam which had six individuals in
one troop. No sub-adult female was observed in one
troop of Iyyerpady, one troop of Puthuthottam, and
Varatupparai and Pannimedu. In Andhipparai, both
troops didnt have any sub-adult females. No
juvenile was observed in one troop of Uralikkal,
one troop of Sekalmudy and troops of Manompoly,
Varatupparai, and Pannimedu. The highest number
of juveniles was observed in one troop of
Puthuthottam (15 juveniles). Two troops of
Iyyerpady, Andhipparai and Sekalmudy, one troop
of Uralikkal, Varatupparai and Pannimedu didnt
have any juveniles. Two troops of Iyyerpady and
Puthuthottam, one troop of Sekalmudy,
Andhipparai and Manompoly didnt have any
infants (Table 2).
A total of 51 edible plant species and one
unknown species were recorded from 8 different
fragments of Anamalai Hills during this study
period. Chinnakalar had maximum species of plants
(23 species) followed by Iyyerpady (19 species),
Akkamalai (18 species) and Korangumudi (15
species). Uralikkal had the lowest number of
species of (12 species). Cullenia excellrata was the
only species found in all the fragments except
Akkamalai. Agalia roxburghina, Cardia mixa,
Mimusops elengii, was observed only in

Table. 1: List showing the Fragments, with Forest type, Area, Altitude, Presence / Absence of Lion -
tailed Macaque and the Number of Troops observed
S. Fragment Area No. of Total no. of
Forest type Altitude (m)
No Name (ha) Troops Individuals
1 Iyyerpady Evergreen 750 - 850 1800 4 47
Cordomomum
2 Uralikkal 850 - 950 400 2 44
Plantation
3 Chinnkalar Riveraine 1000 - 1100 1300 1 15
4 Monamboly Evergreen 1000 - 1100 500 1 13
5 Sekalmudy Evergreen 1000 - 1100 600 2 25
6 Korangumudi Coffee plantation 1100 - 1150 35 1 27
7 Pannimedu Moist deceduous 1100 - 1150 50 1 5
8 Varattupparai Coffee plantation 1100 - 1150 24 1 8
9 Puthuthottam Coffee plantation 1100 - 1200 65 3 77
10 Andhiparai Wet evergreen 1200 - 1300 185 2 20
11 Soliyar Dam Dry evergreen 850 - 900 700 0 0
12 Kavarakkal Evergreen 900 - 1100 350 0 0
13 Sangarankudi Evergreen 1100 - 1200 180 0 0
14 Akkamalai Evergreen 1200 - 1300 2000 0 0
355 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Table. 2: Demography of Lion -tailed Macaque in different forest fragments in Annamali Hills
Sub Sub
Adult Adult Group
Fragment Name Adult Adult Juvenils Infants
Male Female Size
Males Females
1 2 1 2 1 1 8
4 2 1 1 2 0 9
Iyyerpady
2 2 0 2 4 0 12
4 8 2 0 3 1 18
2 4 2 5 0 4 17
Uralikkal
4 9 2 5 3 4 27
Chinnkalar 2 5 1 2 2 1 13
2 5 1 3 2 2 15
Sekalmudy
3 5 0 2 0 0 10
Monamboly 3 5 0 3 2 0 13
Varattupparai 1 4 1 0 0 2 8
Pannimedu 1 2 0 0 0 2 5
Korangumudi 3 6 2 5 6 5 27
1 1 0 1 3 0 6
Puthuthottam 1 2 0 0 6 0 9
10 15 7 10 15 5 62
2 6 2 0 0 3 13
Andhiparai
2 3 2 0 0 0 7

Akkamalai, Varatupparai and Puthuthottam
respectively (Table 3).
The mean tree density, Girth at Breast
Height (GBH), Diameter at Breast Height (DBH),
Canopy spread and Tree height of different
fragments are given in Table 4. Among the
fragments Akkamalai had the highest tree density
(1200 424.26/ha) followed by Iyyerpady (1140
24.64/ha) and the lowest plant density was observed
in Andhiparai (560 80.07/ha). The GBH (239
20.22cm), Tree height (68 19.94ft) were highest
in Iyyerpady. On the other hand the lowest GBH
(51 10.58cm), and Tree height (12 1.73ft) were
observed in Varatupparai. The canopy spread was
highest in Iyyerpady, Uralikkal and Puthuthottam.
The canopy spread was lowest in Varatupparai (5
2.64cm) (Table 4).
The portions of different plant species which
were fed by LTM was tabulated in Table 5. LTM
consumed leaves of seven different plant species,
flower and fruits of eight different plant species,
mesocarp of 17 different plants, seed coat of four
different plant species and seed of 15 different plant
species were given in (Table 5).
DISCUSSION
The number of troops and group size are
mainly determined by the food resource availability
(Umapathy and Kumar, 2000b) and competition
between two males which are equal in size and
strength which leads to the splitting up of groups
(Kumar, 1995). Hence the variations in the group
size and number of troops in these fragments were
assessed with reference to food availability and
dominance of individual males in the group.
In Chinnakalar, with an area of 1300 ha,
only one troop was observed. Such low population
maybe attributed due to less availability of edible
plants. Another reason could be of competition
between LTM and Nilgiri Langur (Trachypithecus)
recorded in this area (Kumaraguru 2005).
Furthermore this area faces huge disturbance due to
ecotourism, human settlements, and monoculture
practices tea plantation in vast areas by private
estates for commercial uses (Sharma, 2002).
Earlier, Umapathy and Kumar (2000a) found
that the size of the area would be important when
the area was small. But in Chinnakalar, in spite of
vast area, group size and number of troops was
found to be less, which could explain size of the
area was not a determining factor for the group size
and number of troops were found to be less, which
could explain size of the area was not a determining
factor for the group size and number of groups.
Furthermore Umapathy and Kumar (2000b)
confirmed that the density of trees and canopy

Journal of Research in Biology (2011) 5: 352-362 356

 Narasimmarajan et al.,2011

Table. 3: Presence/ Absence of Tree species in different Fragments of Annamali Hills.

''- indicates presence the respective species in the fragament where as 'x'- indicates
the absence.
Plants species 1 2 3 4 5 6 7 8
Aglaia roxburghina X X x x x X
Artocarpus heterophyllus X X x x x
Beasa indicum X X x X
Bischofia javanica X x x x
Cardia mixa X X x x X x
Calophillium sp. x x X x X
Cambendra sp. X X x x X x X
Carcinia cambogia X X x x X x
Cassia sp. X X x x x X x X
Colikkarna sp. X x x X x X
Cinnamomum malabaricum X X x X x X
Cinnamomum zeylanicum X x x X x
Coffea Arabica X X x x
Cullenia excellrata X
Dalbergia sissu X x x x X x X
Disocyleum sp. X X x x X X
Divesperous sp. X x x X x X
Eucalyptus sp. X x x x x X
Eleaocarpus tuberculatus X X x x x X
Ficus bengalensis X X x x x x X
Ficus glomerata X X x x X x X
Ficus microcarpa X X x X x X
Harppulia imbricate X X x x x X x X
Kitnocarpus sp. X X x X X
Lechea globerata X x x X x X
Litsea deccanenis X x X X
Litsea oleoides X X x x X X
Maccaranga peltata X X x x X
Maccaranga roxburghii X X x x x
Mealeosma sp. X x X x X
Measa indigum X x x X X
Measua ferrea X x
Mimusops elengii X X x x x x
Mimusops eleatum X X x x x X x X
Myristica beddomi X x x X x
Myristica indicum X x X X
Myristica malabaricum X x x x X X
Palaquium ellipticum x x x
Randia dumetorum X X x x X x
Sandalam album X X x x x X x
Sembcarprus sp. X x x X x
Semicarpus anacardium X x x X x X
Sheleichera oleosa X
Shorea talura X x x x X X
Sterculia guttata X X x x x X x X
Syzygium cumini X x x x x X
Syzygium lanceolatum X X x x x X x
Toona ciliate X x x x X
Tripatacean sp. X X x X x
Vateria indica X X x x x X x X
Vernonia sp. X x x X x X
Unknown X
1=Iyyerpady; 2=Uralikkal; 3=Chinnakalar; 4=Korangumudi; 5=Varatupparai;
6=Puthuthottam; 7=Andhiparai; 8=Akkamalai.

357 Journal of Research in Biology (2011) 5: 352-362

Narasimmarajan et al.,2011

Table. 4: List showing the Mean ( S.D) Density of Tree, and Girth at Breast Height
(GBH), Canopy spread and Tree Height in 8 fragments of Anamalai Hills.
Tree GBH of the Canopy Tree height
Fragment Name
Density / ha tree (cm) spread (m) (m)
Iyyerpady 1140 24.66 239 201.29 3.05 1.49 20.72 6.07
Uralikkal 620 116.61 101 44.64 3.05 1.79 14.93 3.40
Chinnakalar 1080 213.54 167 114.77 2.74 1.02 17.37 7.20
Varatupparai 620 231.51 51 10.58 1.52 0.80 3.65 0.52
Korangumudi 820 240.01 117 46.68 2.13 0.52 15.84 5.48
Puthuthottam 960 412.79 179 139.30 3.04 1.87 18.28 6.58
Andhipparai 560 80.07 123 112.86 2.74 1.38 11.58 7.65
Akkamalai (Shola) 1200 424.26 104 76.66 2.13 1.10 13.10 6.18

height were the best predictors of the presence of
LTM in a forest area. Although the Chinnakalar had
high density of trees, the canopy height was shorter
than that of the other LTM preferred fragments such
as Iyyerpady, and Puthuthottam.
The mesocarp of Cullenia excellrata, a plant
recorded in all of these fragments, was one of the
most preferred food items of LTM. The dispersal of
Cullenia excellrata in different fragments might be
due to LTM. Cullenia excellrata, Eleaocarpus
tuberculatus, Cinnamomum malaricum,
Cinnamomum zeylanicum are used as roosting trees
by LTM viz, the height of these plants and the
presence of primary and secondary branches
favored roosting. And the canopy provides an
opportunity to hide to themselves to the predators.
The variations in the canopy spread mean of tree
Density, Girth at Breast Height, and Tree height in
different fragments could be associated with
altitudinal variations and also the variations in the
climate conditions.
Perhaps LTM was not observed in Solayar
Dam, Kavarakkal, Sangarankudi. All these areas
have extensive Cardamom (Elettaria cardamomum)
plantations, human settlements, no water source and
low density of food species, which could be the
reason for the lack of LTM populations in these
areas. Akkamalai fragment was geographically
varied from other fragments since it consists of vast
grasslands and many small island pocket shola
forest which was not habitat for arboreal mammals
such as LTM (Menon and Poirier, 1996).
Furthermore the dynamite explosions in quarry
mined in this area could also be a reason.
We attribute this unusual growth rate in our
study group to its feeding habitats and the nature of
available resources. It is reported that most of the
forest fragments, especially those that are located in
private owned lands such as tea and coffee gardens,
have a much lower resource base than the relatively
undisturbed rainforest complex (Ramachandran and
Joseph, 2000). The major demography changes due
to fragmentation were in birth rate and proportion
of juveniles in the group, and an increase in the
number of adult males and variability in group size
adult sex- ratios with decreasing area and increasing
disturbance levels (Malcom, 1991). Vegetation
status and fragment area are highly correlated
(Umapathy and Kumar, 2000b) which explains
similar variation in the demographic parameters
with different habitat parameters. On comparison
with the demographic pattern of LTM in these
fragments, it was concluded that the group size and
number of troops or the individual population of
LTM in this study area are mainly determined by
the availability of food and other secondary factors
includes disturbance, competition, predation.
Moreover the ecotourism is one of the serious
threats for LTM populations, people knowingly
feeding the plastics and food items to LTMs, which
are existing on the road side. We suggest that
tourists traveling between Azhiyar to Valparai,
should be checked properly with mobile patrol and
spot penalty is to be needed (Fig; 2) for the control
of these activities.
ACKNOWLEDGEMENT
We acknowledged Tamil Nadu Forest
Department and all the Forest staffs of Anamalai
Tiger Reserve. I personally thanks to my Guru Dr.
K. Thiyagesan, Reader in A.V.C College, We
express our sincere thanks to Principle, HOD and
All Staff members of department of Wildlife
Biology, A.V.C. College. Author thanking the field
Asst. Mr. Ravi and Mr. Prakash without whom this
was not possible.

Journal of Research in Biology (2011) 5: 352-362 358

 Narasimmarajan et al.,2011

Table 5: List showing the Plant species and Edible portions of Lion-tailed Macaque
'- indicates that the respective part of that plant was eaten where as ' x ' - indicates that
the parts was not eaten
Plants species Leaf Flower Fruit Mesocarp Seed coat Seed
Aglaia roxburghina x X x X x
Artocarpus heterophyllus x X x X x
Beasa indicum x X x X x
Bischofia javanica x X x X X
Cardia mixa x X x X x
Calophillium sp. x X x X x
Cambendra sp. x X x X x
Carcinia cambogia x x X x
Cassia sp. x x X x
Colikkarna sp. x x x X x
Coffea arabica x x X x x
Cullenia excellrata x x x x x
Dalbergia sissu x x x X x x
Disocyleum sp. x x x X
Divesperous sp. x x x X x
Ficus bengalensis x x X x x
Ficus glomerata x x X x x
Ficus microcarpa x x X x x
Harppulia imbricata x x x x
Kitnocarpus sp. x x X x x
Lechea globerata x x x x
Litsea deccanenis x x x x x
Litsea oleoides x x x x x
Maccaranga peltata x x x x x
Maccaranga roxburghii x x X x x
Mealeosma sp. x X x x
Measa indigum x x x x x
Measua ferrea x x X x x
Mimusops elengii x x x x x
Mimusops eleatum x x x x x
Myristica beddomi x x x X x
Myristica indicum x x x x
Myristica malabaricum x x x x x
Palaquium ellipticum x x x x x
Randia dumetorum x x x X x
Sembcarprus sp. x X x x
Semicarpus anacardium x x x X x
Sheleichera oleosa x x X x
Shorea talura x x X x x
Sterculia guttata x x x X x
Syzygium cumini x x x X x
Syzygium lanceolatum x x x x x
Toona ciliata x x X x x
Tripatacean sp. x X x x
Vateria indica x x x X x
Vernonia sp. x x X x x
Unknown x x
359 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Fig 2. (A). LTM in its natural habitat; (B).LTM consuming fruits from the tree, Cullenia excellrata ; (C&D).
LTM consuming plastics due to effects of ecotourism and loss of their natural habitat & behaviour; (E&F). Due
to ecotourism and habitat loss, LTM is losing its arboreal nature and original feeding behavior.

Journal of Research in Biology (2011) 5: 352-362 360

 Narasimmarajan et al.,2011

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 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Species diversity of water birds in Deepor Beel, Assam

Journal of Research in Biology

Authors: ABSTRACT:
Jyotismita Das1 and
Saikia PK2.
Study has been carried out to assess the water bird diversity in Deepor Beel
Ramsar site of Assam from March 2007 to March 2010. Study revealed the presence
Institution:
1 of 39 species of water birds belonging to 16 families. The family Anatidae represented
Research Scholar,
Department of Zoology, highest of eight species followed by seven species in the family Ardeidae whereas
Gauhati University. other 14 species represented by rest of the family. However, there were marked
2
Associate Professor, variation of species abundance in different years of study, of which, the family
Department of Zoology, Anatidae represented the highest abundant family throughout the study period. The
Gauhati University. analysis of species diversity index using Shannon diversity index showed that the
diversity was highest during 3rd year study period. Study also highlighted the threat
factors prevailing in the Deepor Beel Ramsar Site.
Corresponding author:
Jyotismita

Keywords:
Email: Diversity, water birds, Shannon Diversity index, Threats factor,
deeporbeel@gmail.com anthropogenic, Deepor Beel.

Web Address: Article Citation:

http://jresearchbiology.com/ Jyotismita Das and Saikia PK.
Documents/RA0103.pdf. Species diversity of water birds in Deepor Beel, Assam
Journal of research in Biology (2011) 5: 363-369

Dates:
Received: 12 Sep 2011 /Accepted: 16 Sep 2011 /Published: 20 Sep 2011

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cited.

363-369 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION
Wetlands are widely recognized as fragile
ecosystems with diverse attributes including distinct
avifauna (Burger, 1985). Wetlands have many
ecological roles and are of importance to birds
(Gill, 1994). Wetlands are highly important because
they serve as critical breeding, staging and
wintering grounds for wide array of globally
important bird species (Kristen and Brander, 2004).
The It has been estimated that freshwater wetlands
hold more than 40% of bird species of the entire
world and 12% of all animal species (Zakaria et al.
2009).Wetlands are important because they serve as
critical breeding, staging and wintering grounds for
wide array of globally important bird species
(Kristen and Brander, 2004). The species richness
and relative abundance of birds depend upon
wetland characteristics such as size, water level,
quality of water, availability and distribution of
food resources, presence of suitable roosting and
nursery sites (Wiens,1989).Variation in habitat
condition may also cause changes in relative
abundance of bird species composition (Garcia et
al.2007;Caziani and Derlindati,2000). Birds are bio
-indicators as they notify us of certain changes
occurring in our environment. They are among the
numerous fauna that may be at risk by the use of
Agricultural pesticides (Mineau et al.
1990).Besides, the beauty of birds but also,
particularly, water birds has made bird watching a
very useful way of spending leisure and gathering
revenue from both local and international tourists.
Avian distribution and abundance are expected to
respond seasonal changes in wetlands (Colwell and
Dodd, 1997; Farmer and Parent, 1997).
MATERIAL AND METHODS
Study area
Deepor Beel is a large natural wetland
having great biological and environmental
importance (Deka and Goswami, 1992). This large
water body is a great food source and breeding
ground for a variety of migratory birds, amphibians,
reptiles, insects, micro and macrophytes, terrestrial
weeds and important taxa of ecological and
economic importance (Bera et al., 2008). This is
endowed with both floral and faunal biodiversity.
The Deepor Beel Ramsar site has a total area of 40
Km2 of which 4.14 Km2 had declared as a Bird
Sanctuary. In November 2002, it was listed as a
Ramsar site owing to its rich wetland biodiversity
and sociocultural importance. Again, considering
the varieties of bird species found in the Beel,
364
Jyotismita et al.,2011
Birdlife International has also declared Deepor Beel
as an Important Bird Area (IBA). At maximum
flooding the Beel becomes above 4 meters in deep
and during the dry season the depth drops to about
1-1.5 meter. Deepor Beel (Coordination: 260326
260926N and 903639904125E) is
situated on the Southern bank of the river
Brahmaputra and Village Maj Jalukbari, Pachim
Jalukbari, Dharapur and National Highway No.37
lie on the North; Dakhin Jalukbari, Tetelia and
Pachim Baragoan to the East; Gorbhanga Reserve
Forest, Chakardew Hill and Chilla Hill to the South
West and the Village Azara and Kahikuchi to the
west. Owing to its high biological diversity, this
was included in the Directory of Asian wetlands.
Deepor Beel has a meso-thermal climate,
characterized by high humidity and moderate
temperature. The temperature ranges between 10.6
C to 30C. The pre-monsoon season (March-May)
has a maximum temperature of 27 C and minimum
of 24 C, and relative humidity between 50.5-76.8
%. (Saikia and Kakati, 2010). The monsoon season
(May -September) has a maximum temperature of
32C and minimum of 27.3C. The relative
humidity is 82.5%. The retreating monsoon
(September to October) has maximum and
minimum temperatures of 27 and 25 C
respectively. (Saikia and Kakati, 2010). The
relative humidity is 82% and the rainfall gradually
decreases to average as the season advances. The
winter season begins in November and continues
until January. The average field temperature during
this period remains at 20 2C and the relative
humidity measures about 77.5%. January is the
coldest month, with a lowest temperature of 7 oC
but sometimes the temperature falls to 6 oC. At least
232 species of avian fauna from 72 different
families were recorded in previous studies from this
wetland (Saikia and Kakati, 2010). Apart from
these; the Beel is associated with a rich variety of
other flora and fauna. Deepor Beel appears to be
relatively high with respect to the biodiversity of
free floating, emergent and submerged aquatic
macrophyte (Saikia and Bhattacharjee, 1987).The
free floating plant species are identified as Eicchornia
crassipes, Azolla pinnate, Pistia stratiotes, Lemna
minor, Lemna major. Again the emergent water plants
so far recorded are Trapa bispinosa, Utricularia
flexuosa, Eleocharis pantaginea, Nelumbo nucifera,
Nymphaea alba, Euryale ferox. Ipomea reptans. The
submerged plants which are so far identified are
Potamogeton crispum, Valisnaria spiralis, Hydrilla
verticillata, Najas foveolata.
Journal of Research in Biology (2011) 5: 363-369
Jyotismita et al.,2011

Data Collection
Avian data were collected from March 2007
to March 2010.Data were collected in the early
morning (5a.m-9 p.m) and in the afternoon time
(4p.m-5p.m).For data collection four to five days
were allotted in one month. For watching, counting
and identifying birds Binocular (10X50), telescope
(25-40X), camera (Cannon 110 PS), note book,
guide book, pen, pencil etc were used.
Meteorological data (Temparature, Rainfall, and
Humidity) were collected from the Central
Meteorological Department, Borjhar, Assam.
Photographs and videos were taken to justify the
species type for those species that are difficult to
identify. Birds were identified by seeing their Figure 1. Water bird composition of the study area
characteristics feature in accordance with the During the study period declining trend was
identification keys involved in Ali and Ripley noticed regarding the presence of total numbers of
(1983), Grimmett et al. (1999). Counts were not individuals of the species in the three successive
made on days with fog, rain or strong wind to years. In 2007-08 the total numbers of individuals
lessen the bias caused by the effect of extreme of water birds recorded were 14,104 numbers. In
weather (Verner, 1985). 2008-09 total numbers of individuals of species
Data analysis were recorded as 11,508 numbers and in 2009-10
The diversity of water birds were analyzed the total number of birds were 10,380 (Fig. 2).
using species diversity and richness software (3.02
version).The Shannon diversity index (H) was
calculated to analyze the water bird diversity in the
study area. Bootstrap method was used to calculate
95% confidence intervals. In order to test the
difference in diversity between the three successive
years of study (First year, Second year and third
year) pair-wise randomization test was carried out
based on 10,000 re-sample of species abundance
based on Solow (1993).

RESULTS Figure 2. Total numbers of individuals recorded in

Water bird composition of the study area
Altogether 35,555 individuals from 39
species of 16 different families were found during
the study period (Fig.1). The highest number of
species (8) was found to be in the bird family
Anatidae which is followed by the family Ardeidae
containing seven numbers of species. The families
Ciconiidae, Alcidinidae and Rallidae were
represented by three numbers of species
respectively. The families Accipitridae, Jacanidae
and Passeridae are represented by two numbers of
species respectively. Again one species of each of
the families Phalacrocoracidae, Corvidae,
Charadridae, Apodidae, Hirundinidae, Upopidae,
Meropidae and Laridae were reported during the
study period.
three successive years
Percent occurrence of water birds in three
successive years
While calculating the percentage of water
birds in the study area in three successive years it
was seen that in the first year the percent
occurrence of the Anatidae family was found to be
highest (67.6%) and the same value was found to be
lowest (0.13%) in Passeridae family (Fig.3). Again
in the second year the percent occurrence of the
Anatidae family was highest (69.2%) as compared
to the other families and the same was calculated to
be lowest (0.15%) in Passeridae family (Fig.4). In
the third year also the percent occurrence of
Anatidae family was highest (69.2%) than other
families (Fig.5). The percent occurrence of the

Journal of Research in Biology (2011) 5: 363-369 365


Figure 3. Percent occurrence of water birds in first year
Figure 4. Percent occurrence of water birds in second year
Figure 5. Percent occurrence of water birds in third year
Passeridae family was reported to be lowest
(0.15%) during the study period. Overall it was
seen that the percent occurrence of Anatidae family
was reported to be highest in all the three
successive years.
Comparison of diversity indices between first,
second and third year
In these section Shannon diversity index (H)
of the three successive years had been worked out
to analyze the species diversity of water birds and it
was presented in Table 1.
365
Jyotismita et al.,2011
Table 1. The values of Shannon diversity indices (H) of
three successive years
Variance Lower Upper
Sample H
H 95% 95%
First year 1.269 0.0001381 1.247 1.293
second year 1.228 0.0001703 1.2 1.253
Third year 1.335 0.0001902 1.308 1.36
Comparing Shannon index (H) between first
year and second year showed that the first year was
more diverse than the second year, at 5% level (pair
wise randomized test based on 10,000 random
samples; SI: First year: H=1.3; second year: H=1.2;
^=0.04). Again the comparison between second and
third year had showed that in the third year of study
diversity was found to be higher as compared to the
second year, at 5% level (pair wise randomized test
based on 10,000 random samples; SI: second year:
H=1.2,third year: 1.3; ^=0.10). Again while
comparing the diversity between first year and third
year it was seen that the third year was more diverse
than the first year, at 5% level (pair wise
randomized test based on 10,000 random samples;
SI: first year: H=1.2, third year: H=1.3;
^=0.06).Overall, the study showed that Shannon
Diversity Index was reported to be highest (H=1.3)
in third year of study period (Fig.6).
Figure 6. Shannon diversity indices (H) of avian fauna
of three successive years
DISCUSSION
The results clearly showed the presence of
39 species of water birds from 16 different families
in the study area. The present findings contradict
the views of Saikia (2005) who had reported the
presence of 232 species of aquatic avifauna from
different families. Likewise, A. U. Choudhury
(2000) had also recorded 150 species of birds in and
around the Sanctuary. Barman (1995) had reported
62 species of water birds of which 16 species were
of Anatidae family. Interestingly, they had also
Journal of Research in Biology (2011) 5: 363-369
Jyotismita et al.,2011
mentioned the presence of 1,018 individuals of
Aythya baeri in their survey in 1988-89, but
subsequently the population declined to 250 in 1989
- 90, 3 in 1990-91, 135 in 1991-92, and none in
1992-93. The decline in species content as reveled
from the present study might be due to degradation
and habitat fragmentation due to anthropogenic
activities in and around the Beel ecosystem. Four
factors impact on migratory birds population at
their stop-over sites and winter quarters: restriction
of habitats, hunting and trapping, disturbance, and
effects of biocides (Berthold, 1994). Similarly the
major threats to the water birds of Deepor Beel
were encroachment, habitat fragmentation, hunting
and trapping of water birds, Soil digging, over
fishing, Application of pesticides in the agricultural
field. Likewise in Yunnan province habitat
destruction and over-hunting were the major threats
to the wetland species (Wen et al. 1995). Bharatha
Lakhmi (2006) had also revealed in his study that
anthropogenic pressure affects the habitat of water
birds. Apart from this hunting and trapping of water
birds can also play a major role in declining of the
water birds number in an alarming level. Poaching
of water birds also greatly contributed in declining
water bird species. This fact was also supported by
Barman (1995) in his work by mentioning the
highest threat factors in Deepor Beel as compared
to other Beel. Saikia and Kakati (2010) had also
mentioned about the heavy destruction process
which was prevailing within the Beel periphery
since last few years. Saikia (2005) had mentioned
about the recent soil digging of the Beel bed in
number of locations in northern boundaries and
heavy encroachments for settlements caused
tremendous loss of wetland area in their works.
Apart from these he also mentioned different
anthropogenic factors which promotes in
degradation of the Beel in a quite high rate. Apart
from this the Expert Team constituted by the
Planning Commission, Government of India, to
Review the status of implementation of the National
Wetland Conservation and Management
Programme (NWCMP) of the Ministry of
Environment & Forests in 2008 had also reported
that the past two decades had witnessed a lots of
transformation in the ecological and social character
of Deepor Beel.
Similar findings for the declining trend in
various waterfowls in many regions of the world
were observed by Korschgen and Dahlgren (1992),
Vaisanen and Solonen (1996), Lebedeva and
Journal of Research in Biology (2011) 5: 363-369
Markitan (2001), Houdkova (2003), Horn et al,
(2008), Phillips (2008). Likewise year wise
declining trend in population also might be
attributed to different threat factors within Beel
ecosystem. The percent occurrence of Anatidae
family was reported to be highest in three
successive years which was also supported by
Saikia (2005). Among the members of Anatidae
family the population of both lesser whistling teal
(19,661) and large whistling teal (3,540) were
reported to be highest in three successive years.
Saikia (2005) also had reported the highest
occurrence of large whistling teal in Deepor Beel.
The high density of aquatic vegetation might be
contributed in the high abundance of whistling teal
in the study area as makhana act as a special food
component of both the species of whistling teal.
Saikia and Bhattacharjee (1987) had also reported
high density of free floating, emergent and
submerged aquatic macrophyte in Deepor Beel.
Shannon diversity index was reported to be
highest in third year of study (H=1.3).This may be
due to the fact that the diversity of wetland
component and the adaption of different aquatic
avian fauna to exploit the resources of wetland
ecosystem might be the cause of supporting high
diversity of water birds in the third year. Again
different vegetation types as well as abundant food
resources also might be playing a greater role in
difference in habitat preference by bird species. The
rich and high vegetation might be providing
heterogeneous and suitable site for foraging,
roosting and nesting of birds. Similar findings of
high species diversity were reported by Macdonald
(1977), Weller (1978) and Puttick (1984) in their
work. Similar findings regarding the affect of
vegetation diversity and richness on the bird
population richness were studied by Karr and Roth
(1971), Cody (1981), Canterbury et al. (1999),
Soderstrom and Part (1999), Paracuellos (2006) had
mentioned in his work that the bird assemblage is
affected by various factors like food availability, the
size of the wetland etc. Zakaria et al. (2009) had
reported that diversity of flora subsequently
affected the abundance and diversity of birds,
insects, amphibians, fishes, reptiles and small
mammals. Again, he mentioned that the abundance
of insects, amphibians, reptiles and small mammals
had also attracted waders and raptors. Similar
findings were documented by the work of Lee &
Rotenberry (2005) where they had reported that the
distribution and abundance of many bird species are
366

determined by the composition of the vegetation
that forms a major element of their habitats. As
vegetation changes along complex geographical and
environmental gradients, a particular bird species
may appear, increase or decrease in number, and
disappear as the habitat changes. Smith (1992) had
also worked out that differences in feeding habits
and habitats could also increase diversity, evenness
and species richness.
CONCLUSION
Deepor Beel which is the lone Ramsar site
in Assam is very rich in biodiversity. Apart from
the extensive destruction processes which are
continuously prevailing within the Beel, the water
birds are still visiting the wetland body for its rich
biodiversity. So, proper management practices
should be taken for the proper conservation of the
wetland ecosystem as well as the water birds.
ACKNOWLEDGEMENT
Authors are very much helpful to Mr.
S.Upadhaya, Assistant Professor, T.H.B College,
Sonitpur for providing necessary information.
Again the authors are grateful to Dr. Hemen Deka,
Assistant Professor, B.B.K College and some local
peoples for providing the immediate helps in proper
completion of the work. The Authors express proud
thanks to UGC for providing financial help as well
as Head of the Department Zoology, Gauhati
University for providing help for proper completion
of the work.
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369 Journal of Research in Biology (2011) 5: 363-369

 Journal of Research in Biology

Original Research Paper An International Online Open Access

Publication group

Feasible use of rock oyster ( Crassostrea commercialis) and seaweeds (Gracilaria salicornia
and Caulerpa lentillife) as biofilter in a laboratory - scale closed recirculating system for
juveniles spotted babylon (Babylonia areolata)
Journal of Research in Biology

Authors: ABSTRACT:
Nilnaj Chaitanawisuti1,
Wannanee Santhaweesuk1, This study was conducted to assess the feasible use of rock oyster
Sirusa Kritsanapuntu 2 (Crassostrea commercialis) in biofiltration and two seaweeds (Gracilaria salicornia and
Caulerpa lentillife) as nutrient absorbant in a laboratory - scale recirculating system for
growing of juveniles spotted babylon (Babylonia areolata). The experiment was
Institution:
1. Aquatic Resources carried out in triplicates over a period of 90 days. The experiment was a complete
Research Institute, randomized design with three growth trials: Treatments 1: without oyster and
Chulalongkorn University, seaweed biofilter used as a control; Treatment 2: Oyster biofiltration (1,500 g per
Phya Thai Road, Bangkok, tank) and seaweed (G. salicornia) absorption (250 g per tank); and Treatment 3:
Thailand 10330. Oyster biofiltration (1,500 g per tank) and seaweed (C. lentillife) absorption (250 g per
2. Faculty of Science and tank). No significant differences (P>0.05) in final shell length, final body weight, body
Industrial Technology, weight gains, shell length increment and growth rate among all treatments. Growth
Prince of Songkla rate in shell length and body weight of spotted babylon ranged from 0.33 0.34 cm
University, Surattani mo-1 and 0.62 0.67 g mo-1, respectively. Significant differences (P<0.05) in final
province, Thailand 84100. survival rate of spotted babylon were found among treatments, ranging from 86.72 to
86.98 % compared with those of the control (84.27%). There were no significant
differences (P>0.05) in water temperature, salinity, pH, dissolved oxygen, ammonia-
Corresponding author: nitrogen, nitrite-nitrogen, nitrate-nitrogen and phosphate-phosphorus among the growth
Nilnaj Chaitanawisuti trials but not for alkalinity. This study can conclude that G. salicorniand C. lentillifera can
be used as nutrient biofilter for regulating of water quality in a closed recirculating
system for growing juveniles spotted babylon but not suitable on using oyster.
Email:
nilnajc1@hotmail.com; Keywords:
saenisa479@hotmail.com; Seaweed, biofilter, recirculating culture, water quality, Gracilaria salicornia,
wannanee295@hotmail.com Caulerpa lentillifera, Babylonia areolata.

Web Address: Article Citation:

http://jresearchbiology.com/
Documents/RA0078.pdf.
Nilnaj Chaitanawisuti, Wannanee Santhaweesuk, Sirusa Kritsanapuntu.
Feasible use of rock oyster (Crassostrea commercialis) and seaweeds (Gracilaria
salicornia and Caulerpa lentillife) as biofilter in a laboratory - scale closed recirculating
system for juveniles spotted babylon (Babylonia areolata).
Journal of research in Biology (2011) 5: 370-375

Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 23 Sep 2011

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

370-375 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION:
Commercial growing of juveniles spotted
babylon, Babylonia areolata, in Thailand are
cultivated under the flow-through seawater system.
This type of culture needs about 100% water
exchange per day to maintain water quality and also
had some criticism regarding its environmental
impact due to its tendency to release waste effluents
containing elevated levels of nitrogen or
phosphorus rich compounds which may be
considered as water pollutants. One of the main
drawbacks of spotted babylon culture lies in the
wastes derived from feed and their metabolic
products. The main products are uneaten food,
faeces and excreted dissolved inorganic nutrients
which are transported in the water at various
concentrations (Chaitanawisuti, Krisanapuntu and
Natsukari 2005). Many valuable marine species can
be successfully grown and have high growth and
survival rates in recirculating aquaculture system
(RAS) due to the high-quality culture water. Water
reuse methods require maintaining good water
quality with a system that easily operated at a low
cost. RAS have attractive much attention because
they consume less water than flow-through systems
and also reduce water exchange rates. Therefore,
RAS is biologically and technically feasible and
secondly that it will produce worthwhile income to
the producers. It has been used extensively for
rearing and maintaining adult and juvenile marine
species. Pagand et al. (2000) reported that the use of
recirculating water systems is one approach used to
limit the impact of aquaculture on the aquatic
environment. Although the total quantity of
nutrients released is similar in open and
recirculating systems, the small volumes of
concentrated effluents that are produced should be
easier to deal with, although proper technical and
economical solutions have to be implemented. The
use of seaweeds for the treatment of marine
aquaculture wastes has been proposed by a number
of authors since marine seaweeds have high
capacities for absorption and metabolism of N and
P rich compound excreted by marine animals. In
addition to improving the water quality, seaweeds
can provide an additional source of income from a
valuable by-product in the process. Jones, Dennison
& Preston (2001) suggested that nutrient removal
efficiency of macroalgae may be improved with
increased light, particularly for nitrate uptake,
increased water flow to reduce the boundary layer,
and higher stocking densities. The special interest is
the use of seaweeds for recycling some dissolved
371
Chaitanawisuti et al.,2011
nutrients, in particularly nitrogen in the term of
ammonia (Buschmann 1996, Neori et al. 1996,
Chow et al. 2001, Hernandez et al. 2002, Matinez-
Aragon et al. 2002, Msuya et al. 2006). Thus, we
have developed a simpler recirculating system for
the production of juvenile spotted babylon available
use for commercial applications. This study was
conducted to assess the capable use of rock oyster
(Crassostrea commercialis) as biofiltration and two
seaweeds (Gracilaria salicornia and Caulerpa
lentillife) as nutrient absorption in a laboratory -
scale closed recirculating system for growing of
juveniles spotted babylon (Babylonia areolata)
MATERIALS AND METHODS
This study was conducted at the Spotted
Babylon Aquaculture Research Unit, Aquatic
Resources Research Institute, Chulalongkorn
University, Petchaburi province, Thailand. The
laboratory experiment was designed to test the use
of rock oyster (C. commercialis) and seaweeds (G.
salicornia and C. lentillife) as biofilter in a
laboratory - scale closed recirculating system for
growing juveniles spotted babylon (B. areolata).
The experiment was a complete randomized design
with three replications as following: Treatment 1:
Without oyster and seaweed biofilter used as a
control; Treatment 2: Oyster biofiltration (1,500 g
per tank) and seaweed (G. salicornia) absorption
(250 g per tank); and Treatment 3: Oyster
biofiltration (1,500 g per tank) and seaweed (C.
lentillife) absorption (250 g per tank). Each
experimental unit was designed as an independent
closed - recirculating system consisting of a rearing
tank, an oyster filtration tank and a seaweed
absorption tank. Cylindrical plastic tanks of 300 l
capacity were used for each tank of the recirculating
unit. The bottom area of the rearing tank was 0.78
m2 and was covered with a 5 cm layer of coarse
sand (0.5 1.0 mean grain size) to serve as a
substrate. The seaweed absorption tank contained
one of the marine seaweeds as a biofilter. All
experimental units were located in an indoor
hatchery, but the roof over all seaweed absorption
tanks was transparent and permitted about 80% of
sunlight to pass thus enabling seaweed
photosynthesis. Seawater was pumped from an
earthen pond, filtered mechanically, and circulated
through the experimental unit. The water depth in
all tanks was 30 cm. Water flowed from the
biofilter tank through the rearing tank and was
maintained in a small submersible pump at a
Journal of Research in Biology (2011) 5: 370-375
Chaitanawisuti et al.,2011
constant flow rate of 530 l h-1 before it was returned
to the rearing tank. No seawater was exchanged
throughout the experimental period of 90 days. The
tanks were moderately aerated by air diffusers
placed at the bottom of each tank. Water
temperature was maintained at room temperature
1.5oC. Salinity was monitored daily, as necessary,
to keep the variation within 1.0 ppt by addition of
fresh water to correct for any increased salinity due
to water evaporation. Photoperiod was a natural 12
l:12 day:night.
Juvenile spotted babylons, B. areolata, used
in growth and survival experiments were produced
at a private hatchery in Rayong province, located on
the eastern coast of the Gulf of Thailand.
Individuals from the same cohort were sorted by
size to prevent possible growth retardation of small
spotted babylons when cultured with larger ones.
Their initial shell length and whole body weight
averaged 1.23 0.005 cm and 0.37 0.01 g, n = 30,
respectively. Mean shell lengths and body weights
used in each treatment were not statistically
different, and hence, treatments could be compared
statistically. Juveniles were held in the experimental
rearing tanks at a stocking density of 300 individual
m-2 or 192 snails per tank. The rock oyster
Crassostrea commercialis with average body
weight of 4.0 - 10.0 g were collected from the
oyster farm at Ang-sila, Cholburi province. Two
seaweeds Gracilaria salicornia and Caulerpa
lentillifera were used as biofilters. The first was
collected from intertidal pools along the coastal
water of Samui Island, Surattani province, and the
latter was collected from broodstock-rearing ponds
of an intensive shrimp farm. Oysters and seaweeds
were placed in a plastic basket of 25.0 x 35.0 x 25.0
cm which contained numerous pores of 1.5 cm2 (4
holes cm-2) at each side and were suspended 30 cm
above the bottom of biofilter tanks. Oyster and
seaweeds in all tanks were not harvested throughout
the experiments.
The snails were fed ad libitum with fresh
trash fish once daily at 10:00 h. The amount of food
consumed was recorded daily, and uneaten food
was removed immediately after the animals stopped
feeding and air dried for a period of 10 min before
weighing. Size grading of snails in all treatments
was not done throughout the grow-out period. No
chemical and antibiotic agents were used
throughout the entire experimental period. To
determine growth performance, 50% of snails were
sampled randomly from each treatment at 15 days
Journal of Research in Biology (2011) 5: 370-375
intervals, and shell length and whole body weight
were determined. Shell length was measured with
calipers to the nearest millimetre from the
maximum anterior to posterior distance of a shell,
and the whole weight was measured after air drying
for a period of 10 min before weighing and then
returned to the tank. The number of dead
individuals was recorded at 15-day intervals.
Seaweeds from each tank were also weighed to
determine the increase in biomass, and specific
growth rates were calculated at 15-day intervals.
Seaweeds were placed in a plastic basket to drain
off excess water. Visible epiphytes were carefully
removed. The experiment was terminated after a 90
days during May to June, 2009. Average shell
length increments, body weight gains and growth
rates were calculated after the method of
Chaitanawisuti and Kritsanapuntu 1999). Body
weight gains (BWf - BWi), and monthly growth
rates for body weight (BWf - BWi)/T) were
calculated, in which BWf = mean final body
weight, BWi = initial body weight, and T = time in
months. The specific growth rate (SGR, % day-1) =
100 9 [ln (final weight, g) - ln (initial weight, g)/
(culture period, days). Mortality, expressed as the
percentage of the initial stocking density, was
calculated from the difference between the number
of snails stocked and harvested. The following
seawater quality parameters in each rearing tank
were analysed weekly: water temperature (Hg
thermometer), salinity (portable multiparameter
metre model YSI#63), conductivity (portable
multiparameter metre model YSI#63), pH (pH
metre), total alkalinity (phenolphthalein methyl
orange indicator), dissolved oxygen (DO metre
model YSI#52), total ammonianitrogen (phenate
method), nitritenitrogen (colorimetry method),
nitratenitrogen (cadmium reduction) and
orthophosphate (ascorbic acid method) (APHA et
al. 1998).
Data on growth performance and water
quality were analyzed using the SPSS statistical
software package (version 10). Analysis of variance
(ANOVA) was used to test the interaction of
seaweeds and stocking density at = 0.05, and
differences between means were compared using
Tukeys test at = 0.05.
RESULTS
Growth in shell length and body weight of
juvenile spotted babylon (Babylonia areolata) in a
laboratory - scale closed recirculating system using
372

oyster and two seaweeds as biofilters over 90 days
is shown in Figure 1. One-way ANOVA showed no
significant differences (P>0.05). in final shell
length, final body weight, body weight gains, shell
length increment and growth rate among all
treatments (Table 1). Shell length increments of
spotted Babylon ranged from 0.98 to 1.00 cm and
1.88 to 2.03 g for those of body weight gains.
Growth rate in shell length and body weight of
spotted babylon ranged from 0.33 0.34 cm mo-1
and 0.62 0.67 g mo-1, respectively. Significant
differences (P<0.05). in final survival rate of
spotted babylon were found among rearing
treatments (P<0.05), ranging from 86.72 to 86.98
% compared with those of the control (84.27%).
Growth of seaweeds (G. salicornia and C.
lentillifera) were used as biofilters in a laboratory
scale, closed recirculating system for juveniles
spotted babylon (B. areolata) for 90 days is shown
in Figure 2. There were significant differences
(P<0.05) in absolute growth rates between the two
seaweed biofilter treatments. Weight gain of C.
lentillifera (716.0 g) was significantly higher than
those of G. salicornia (353.0 g mo -1). Growth rate
of C. lentillifera (238.6 g mo -1) was significantly
higher than those of G. salicornia (117.6 g mo -1).
However, growth of rock oyster (C. commercialis)
is very low with less than 0.05 g mo-1 for all
treatments but final survival was high (100%) for
Treatment 1 and 2.
Water quality parameters in a closed
recirculating system using rock oyster (C.
-
373
Chaitanawisuti et al.,2011
commercialis) and seaweeds (G. salicornia and C.
lentillifera) as biofilters over 90 days is shown in
Table 2. There were no significant differences
(P>0.05) in water temperature (24.2 24.60C),
salinity (30.5 30.7 psu), pH (8.35 8.42), dissolved
oxygen (6.5 6.7 mg/l), ammonia - nitrogen (0.0766
0.0922 mg/l), nitrite -nitrogen (0.1555 0.1728 mg/
l), nitrate - nitrogen (0.8584 0.9073 mg/l) and
phosphate - phosphorus (0.4863 0.5185 mg/l)
among the growth trials but not for total alkalinity
(77.0 81.0 mg/l) (P<0.05).
DISCUSSION
This study showed that no significant
differences in final shell length, final body weight,
body weight gains, shell length increment and
growth rate among all treatments. Growth rate in
shell length and body weight of spotted babylon.
Significant differences in final survival rate of
spotted babylon were found among treatments,
ranging from 86.72 to 86.98 % compared with
those of the control (84.27%). There were no
significant differences in water temperature, salinity,
pH, dissolved oxygen, ammonia-nitrogen, nitrite-
nitrogen, nitrate-nitrogen and phosphate-phosphorus
among the growth trials but not for alkalinity. This
study can conclude that G. salicorni and C. lentillifera
can be used as nutrient biofilter for regulating of
water quality in a closed recirculating system for
growing juveniles spotted babylon but not suitable
use of oyster. As compared to the study of
Chaitanawisuti, Kritsanapuntu and Natsukari
-
Journal of Research in Biology (2011) 5: 370-375
Chaitanawisuti et al.,2011

Table 1. Growth of juveniles spotted babylon (B. areolata) in a laboratory - scale closed recirculating system
using oyster and two seaweeds as biofilters for 90 days
Parameters Treatment 1 Treatment Treatment
1.230.02 1.230.01 1.230.001
0.300.02 0.290.01 0.300.004
2.230.04 2.230.16 2.210.10
2.320.11 2.300.57 2.170.27
1.00+0.03 1.00+0.15 0.98+0.10
2.03+0.11 2.01+0.58 1.88+0.27
0.33+0.01 0.34+0.05 0.33+0.03
0.67+0.04 0.67+0.19 0.62+0.09
84.27b 86.72a 86.98a
Values are means +standard deviation. Means with different superscript in the same row are significantly
different (P<0.05).

(2005), growth rate of B. areolata in this study taxifolia almost doubled internal nitrogen in
(0.62 to 0.67 g mo-1) is the same as those cultured nutrient rich water), Caulerpa species have
in the recirculating system used oyster shells as application in bioremediation of intensive tank-
biofilter (0.62 to 0.68 g mo-1) but lower than those based aquaculture and perhaps treated pond
cultured in flow-through system (1.05 to 1.25 g mo- aquaculture effluent. As compared to other
1
) and This study agreed with the study of Chow et seaweeds commonly used for water treatment in
al. (2001) who indicated that Gracilaria chilensis mariculture purposes, Msuya et al. (2006) reported
culture was highly efficient at biofiltration of the that seaweed, Ulva reticulate, grew at an average of
soluble nutrients but had little effect on particulate 4.0% day-1 at the fishpond outflow with a biomass
emission. The best growth of G. chilensis occurred yield averaging 46 g m-2 day-1, compared with
in the ammonium rich effluent from the fish averaging 2.5% and 2.7 g m-2 day-1at the fishpond
culture. Jones, Dennison & Preston (2001) also inflow. Martinez-Aragon et al. (2002) indicated that
recommended that nutrient removal efficiency of seaweeds (Ulva rotundata and Enteromorpa
macroalgae may be improved with increased light, intestinalis and Gracilaria gracilis removed
particularly for nitrate uptake, increased water flow efficiently the phosphate dissolved in the waste
to reduce the boundary layer, and higher stocking water from the fish culture tanks. Removal
densities. Paul & Nys (2008) reported that seaweed efficiency was highest in U. rotundata (99.6%) and
from the genus Caulerpa culture will not be easily lowest in G. gracilis (62.2%). In addition, the
integrated into settlement ponds in tropical maximum uptake rate of phosphate occurred in U.
aquaculture. However, because some species of rotundata, slightly greater than that for E.
Caulerpa grew well in tank-based system (C. intestinalis, while G. gracilis showed the lowest
racemosa grew at > 7% day-1) and others are uptake rate. Hernandez et al. (2002) also
capable of luxury uptake (C. serrulata and C. indicated that U. rotundata and E. intestinalis and
Table 2. Seawater quality in a laboratory - scale closed recirculating system for juveniles spotted babylon (B.
areolata) using oyster and two seaweeds as biofilters for 90 days
Parameters Treatment 1 Treatment Treatment
Water temperature ( C) 24.22.4 24.32.4 24.62.3
Salinity psu) 30.61.3 30.71.7 30.51.2
pH 8.420.11 8.370.12 8.350.15
Alkalinity (mg/L) 819 a 748c 779b
Dissolved oxygen (mg/L) 6.50.7 6.70.6 6.50.5
Ammonia - nitrogen (mg-N/L) 0.07660.0590 0.09000.0538 0.09220.0604
- 0.17280.0404 0.16380.0809 0.15550.0802
- 0.90730.3112 0.85840.3036 0.88270.3230
- 0.48630.2109 0.51850.2516 0.49220.2643
Values are means +standard deviation. Means with different superscript in the same row are significantly
different (P<0.05).

Journal of Research in Biology (2011) 5: 370-375 374


G. gracilis removed efficiently the ammonium
dissolved in the waste water from the fish culture
tanks. U. rotundata and E. intestinalis showed the
highest ammonium disappearance (97.7%), whereas
G. gracilis removed 93.2% of the nutrient during
the incubation. In addition, the minimum uptake
rate of ammonium occurred in G. gracilis. Wang et
al. (2007) showed that growth rate of Ulva pertusa
was 3.3% day-1 in a indoor recirculating system for
production of juvenile sea cucumber (Apostichopus
japonicas). U. pertusa was efficient in removing
toxic ammonia and in maintaining the water quality
within acceptable levels for sea cucumber culture.
U. pertusa removed 68% of the total ammonia
nitrogen and 26% of the orthophosphate from the
sea cucumber culture effluent; the macroalgae
biofilter removed ammonia at an average rate of
0.459 g N m-2 day-1. This study can conclude that G.
salicorniand C. lentillifera can be used as nutrient
biofilter for regulating of water quality in a closed
recirculating system for growing juveniles spotted
babylon but not suitable use of oyster. However, the
stocking density and production of G. salicorniand
C. lentillif should be considered for optimal use in
the recirculating system for the spotted Babylon.
ACKNOWLEDGES
This research was funded by Chulalongkorn
University in the fiscal year of 2009. The authors
thank to Aquatic Resources Research Institute,
Chulalongkorn University, in particular Mr.
Yongyudth Chankaew for his assistances in the
hatchery and collecting of oysters and seaweeds.
The authors also thank to Associated Professor Dr.
Somkiat Piyatiratitiworakul for valuable comments
during the experiments and preparation of this
manuscript.
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APHA, AWWA, WPCF. 1998. Standard methods
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Buschmann AH. 1996. An introduction to Paul NA, and de Nys R. 2008. Promise and pitfalls of
integrated farming and the use of seaweeds as locally abundance seaweeds as biofilters for integrated
biofilters. Hydrobiologia 326(327):59-60. aquaculture. Aquaculture 281:49-55.
Chaitanawisuti N, Krisanapuntu S, Natsukari Y.
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marketable size at four stocking densities in flow-
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 Journal of Research in Biology

Original Research Paper An International Online Open Access

Publication group

The combined effects of temperature and salinity on hatching success and survival of
early life stages in the economically candidate marine mollusks : Spotted babylon
Babylonia areolata
Journal of Research in Biology

Authors: ABSTRACT:
Nilnaj Chaitanawisuti,
Sirinun Nunim and The combined effects of temperature and salinity on the hatching success
Wannanee Santhaweesuk. and larval and juvenile survival of the spotted babylon Babylonia areolata were
investigated in a 3x3 complete factorial experimental design employing three
temperatures (25, 30 and 350C) and three salinities (27, 30 and 33). The results
showed that hatching of egg capsules B. areolata was only significantly affected by
Institution: salinity but not for temperature. No significant interaction between both factors
Aquatic Resources Research occurred. However, survival of larvae and early juvenile were significantly affected by
Institute, Chulalongkorn temperature and salinity. A significant interaction between both factors occurred in
University, Phya Thai Road, this experiment. The highest survival of early juveniles was founded at the lowest
Bangkok, Thailand 10330. temperature (250C) with the highest salinity (33) and the highest temperature
(250C) with the highest salinity (33). From an aquaculture point of view, high
hatching of egg capsules B. areolata was obtained at temperature of 29 -350C and
salinity above 32 to 33. As well as the high survival of larvae was obtained at
temperature of 29 -30.50C and salinity above 32 to 33 and 29 -350C and 32 - 33
Corresponding author: for early juveniles.
Nilnaj Chaitanawisuti

Email:
nilnajc1@hotmail.com,
Keywords:
saenisa479@hotmail.com, Spotted babylon, Babylonia areolata, temperature, salinity, hatching,
wannanee295@hotmail.com. survival.

Web Address: Article Citation:

http://jresearchbiology.com/
Documents/RA0079.pdf.
Nilnaj Chaitanawisuti, Sirinun Nunim and Wannanee Santhaweesuk.
The combined effects of temperature and salinity on hatching success and survival of
early life stages in the economically candidate marine mollusks : Spotted babylon
Babylonia areolata
Journal of research in Biology (2011) 5: 376-384

Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 23 Sep 2011

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
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cited.

376-384 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION:
Temperature and salinity are considered to
be the most important physical factors influencing
marine organisms, and the biological effects of
these factors are complex and wide ranging.
Temperature is one of the most critical external
factors of ontogeny in the early life stages of fish.
There are two ways in which temperature affects
ontogeny. Firstly, temperature, if within a viable
range, strongly affects the rate of ontogeny. A
temperature beyond this range is lethal for the
species. Secondly, temperature affects the hatch
rate, incubation period, the size of the newly
hatched larvae, larval yolk absorption and
utilization, larval feeding behavior, larval survival
and larval growth (Shi et al. 2010). Tolerance and
resistance tests can be used to determine the
survival capacity of various life stages of a species
in relation to certain factors. The combined effects
of temperature and salinity on the survival of
marine animals have been demonstrated in many
marine organisms, mainly fish (Shi et al. 2010,
Berlinsky et al. 2004, Cook et al. 2005), crustaceans
(Dawirs 1979, Ponce-Palafox et al. 1997, Mene et
al. 1991, Nagaraj 1988, Albuquerque et al. 2009),
echinoderms (Asha & Muthiah 2005, Dong & Dong
2006, Ji et al. 2008, Lavitra et al. 2010), bivalves
(Taylor et al. 2004, Robert et al. 1988, Tettelbach et
al. 1981), and gastropods (Lu et al. 2004, Davis
2000, Chen & Chen 2004, Zheng et al. 2000).
However, there is scant information on the effects
of environmental fluctuation, such as temperature,
salinity on growth and survival of Babylonia
areolata particularly the combined effects of
temperature and salinity. This is the case for early
life stage of Babylonia areolata that may be more
sensitive to environmental change. Albuquerque et
al. (2009) stated that salinity could modify the
effects of temperature and alter the temperature
range of many biological processes. In turn,
temperature can also modify the effects of salinity.
The objective of this study is to investigate the
combined effect of temperature and salinity on the
eggs, larvae and early juvenile of spotted Babylon
Babylonia areolata to determine the optimal
temperature and salinity for incubation and larval
culture of this species. The results of this study will
be useful in increasing the production of this
species through incubation and larval culture.
MATERIALS AND METHODS
Experimental design
The study was conducted from February to
377
Chaitanawisuti et al.,2011
April 2011 in indoor air condition room at Sichang
Marine Science Research and Training Station,
Chulalongkorn University, located on Sichang
Island Cholburi province, Thailand. To verify the
combined effects of temperature and salinity on
various life stages of the spotted babylon resistance,
a 3x3 complete factorial design was used for the
experiments including three temperatures (25, 30
and 350C) and three salinities (27, 30 and 33).
Each of the nine different temperaturesalinity
combinations was conducted in three replicates. A
total of 27 experimental units were set up initially.
The experiment followed a completely randomized
design, using the egg capsule, larvae and juveniles
from a single spawn to minimize variability
between experimental units. This experiment was
conducted in the air condition room with normal
temperature control at 25 0C. Seawater used in all
treatments was filtered (10 m) and dechlorinated
for two days before use to ensure no chlorine
residue. Seawater with low salinities was made up
daily by diluting a source of natural seawater with
de-chlorinated freshwater and high salinities water
was made by evaporating seawater. Temperature
were maintained within +0.2 0C using 3 identical
22.0 L thermostatically controlled water baths (L x
W x H = 50 x 30 x 15 cm). Each water bath
contained nine experimental units (1000 mL glass
beaker). Gently aeration was provided during larval
culture, a normal photoperiod of 12L:12D was
adopted throughout the experiment. Water
exchange was not done during the experiment for
all aquaria. Salinity and temperature in all
experimental units were measured every six hours
using a portable refractosalinometer and a mercury
thermometer, respectively. All experimental units
were covered with aluminum foil to prevent
evaporation. The experiments lasted after 96 h.
Experiment 1: Effect of temperature and salinity
on the hatching success
After spawning, the egg capsules were
transferred to experimental units maintained at
different temperature salinity combinations. The
mean size (+SD) of egg capsules at the start of the
experiment was 1.2+0.03 cm in length. For each
temperature and salinity combination, each
experimental unit consisted of 100 egg capsules
acclimated in a 1000 mL glass beaker filled with
700 mL seawater. Each unit was stocked with low
stocking density of 10 egg capsules per unit so that
stocking density was not a limiting factor for
survival. Hatching out of veliger larvae from egg
capsule usually was observed by naked eyes and
Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011
considered as hatched or unhatch egg capsule.
Hatched egg capsules expressed by no fertilized
eggs inside or partial retain of fertilized eggs. The
number of hatched capsules and duration of
incubating period in each experimental unit were
recorded. The mean percentage of hatching rate was
calculated by combining the data from three
replicates at the end of the experiment.
Experiment 2: Effect of temperature and salinity
on the survival rate of veliger larvae
Within 6 h after hatching, aeration was
stopped, and the veliger larvae were allowed to
concentrate at the water surface; they were then
collected from the hatching tanks for use in the
experiments. The mean size (+SD) of larvae at the
start of the experiment was 428+0.01 m in shell
length. For each temperature and salinity
combination, each experimental unit consisted of
100 larvae reared in a 1000 mL glass beaker filled
with 700 mL seawater. Each unit was stocked with
low stocking density of 100 larvae per unit so that
stocking density was not a limiting factor for
survival. Larvae were fed with a single algal diet
(Chaetoceros calcitrans) once daily during the
experiment. Optimal concentrations of algal cells
(6x104 cell ml-1) were fed so that food availability
was not a limiting factor for survival and was
adjusted for each experiment according to the size
of larvae (Chaitanawisuti and Kritsanapuntu 1997).
Each experimental unit was initially examined the
dead larvae after 6 h, and every 12 h thereafter. The
number of larvae which sink down to bottom of the
aquaria / empty shell or no movement of velum
were observed microscopically and considered as
dead. The mean percentage of survival rate was
calculated by combining the data from three
replicates at the end of the experiment.
Experiment 3: Effect of temperature and salinity
on the survival rate of early juveniles
Within 48 h after settlement, the mean size
(+SD) of juveniles was 1393.33+8.18 m in shell
length and are used at the start of the experiment.
For each temperature and salinity combination,
Table 1. Percentage hatching of egg capsules B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature
(0C) 27
25 63.33+25.20
30 76.67+11.50
35 76.67+20.80
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)
Journal of Research in Biology (2011) 5: 376-384
each experimental unit (1000 mL glass beaker filled
with 700 mL seawater) was stocked with low
stocking density of 50 larvae per unit so that
stocking density was not a limiting factor for
survival. Juveniles were fed with fresh meat of trash
fish at once daily during the experiment. Uneaten
food was removed immediately after they stopped
eating to prevent water degradation. Then tested
juveniles were fed so that food availability was not
a limiting factor for survival. Each experimental
unit was initially examined for the dead juveniles
after 6 h, and every 12 h thereafter. The number of
juveniles which did not response to the touch of a
needle were observed by naked eyes and considered
as dead. The mean percentage of survival rate was
calculated by combining the data from three
replicates at the end of the experiment.
Data analysis
To investigate the effect of temperature and
salinity on hatching of egg capsule, and survival of
larvae and juveniles, a two-way analysis of variance
(ANOVA) (fixed factors: temperature and salinity)
with a 95% confidence interval was used. All data
were tested for normality and homoscedasticity. On
significant difference indication, Tukey test was
used to verify the difference among the treatments.
The correlation between the survival and the
temperature and salinity was estimated by multiple
regression analysis.
RESULTS
Experiment 1: Effect of temperature and salinity
on the hatching success
Hatching of egg capsules B. areolata over
the three temperatures and three salinity
combinations for 96 h was presented in Table1.
The highest hatching of 86.67% and 100% were
founded at the lowest temperature (25 0C) with the
highest salinity (33) and the highest temperature
(350C) with the highest salinity (33),
respectively, while the lowest hatching 63.33% was
founded at the lowest temperature (25 0C) with the
lowest salinity (27).
Salinity ()
30 33
83.33+15.30 86.67+5.70
86.67+15.30 96.67+20.80
83.33+15.30 100
378

Two-way ANOVA showed that hatching of
egg capsules B. areolata was only significantly
affected by salinity (F = 5.102; P = 0.018) but not
for temperature (F = 1.085; P = 0.359). No
significant interaction between both factors were
occurred in this experiment (F = 0.220; P = 0.924).
Tukey test showed that hatching of egg capsules
were significantly different between the salinity of
27 and 33 (Table 2).
Multiple regression analysis showed that
only salinity (P = 0.002) were statistically
significant, showing a negative correlation with the
percentage hatching of egg capsules B. areolata
(Table 3). In additional, standard coefficient (Beta)
of salinity (0.570) was higher than that of
temperature (0.228), indicating a higher correlation
between salinity and hatching. The higher salinity
provided the higher hatching. The multiple
regression equation on hatching of egg capsules B.
areolata over the combined effects of temperatures
and salinity were estimated as follows:
Hatching = -54.074 + 0.889 Temperature + 3.704
Salinity
The response surface plots summarizing
percentage hatching under nine different
combinations of temperature and salinity over 96 h
showed that high hatching (95%) was obtained at
temperature of 29 -350C and salinity above 32 to
33. Lower temperatures and salinities reduced
hatching markedly and higher salinities had a
marked positive effect at lower temperatures (Fig
1).
Fig 1. Response surface plots with estimating the mean
percentage hatching of egg capsules B. babylonia after
96 h under 9 different combinations of temperature
and salinity.
379
Chaitanawisuti et al.,2011
Experiment 2: Effect of temperature and salinity
on the survival rate of veliger larvae
Survival of veliger larvae B. areolata over
the three temperatures and three salinity
combinations for 96 h was presented in Table4.
The highest survival of veliger larvae of 78.67%
and 47.00% were founded at the lowest temperature
(250C) with the highest salinity (33), and the
highest temperature (350C) with the highest salinity
(33), respectively, while the lowest survival of
veliger larvae of 23.33% and 17.67% were founded
at the lowest temperature (25 0C) with the lowest
salinity (27), and the highest temperature (35 0C)
with the lowest salinity (27), respectively.
Two-way ANOVA showed that survival of
veliger larvae B. areolata was significantly affected
by temperature (F = 1662.01; P = 0.000) and
salinity (F = 488.96; P = 0.000). A significant
interaction between both factors were occurred in
this experiment are observed (F = 152.69; P =
0.000). Tukey test showed that survival of veliger
larvae were significantly different one another
among temperature and salinity treatments (Table
5).
Multiple regression analysis showed that
both temperature (P = 0.020) and salinity (P =
0.012) were statistically significant, showing a
negative correlation with the percentage survival of
veliger larvae B. areolata (Table 6). In additional,
standard coefficient (Beta) of salinity (0.445) was
higher than that of temperature (0.406), indicating a
higher correlation between salinity and survival. It
also indicated that the higher salinity provided the
higher survival. The multiple regression equation
on survival of veliger larvae B. areolata over the
combined effects of temperatures and salinity was
estimated as follows:
Survival = -13.037 - 2.789 Temperature + 5.093 Salinity
The response surface plots summarizing
percentage hatching under nine different
combinations of temperature and salinity over 96 h
showed that high hatching (90%) was obtained at
temperature of 29 -30.50C and salinity above 32
to 33. Lower temperatures and salinity reduced
hatching markedly but higher temperature and
salinities had a marked positive effect (Fig 2).
Experiment 3: Effect of temperature and salinity
on the survival rate of early juveniles
Survival of early juvenile B. areolata over
the three temperatures and three salinity
combinations for 96 h was presented in Table 7.
Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

salinity (F = 49.060; P = 0.000). A significant

Fig 2. Response surface plots with estimating the mean
percentage survival of veliger larvae B. babylonia after
96 h under 9 different combinations of temperature
and salinity.
The highest survival of early juveniles of
100% and 65.56% was founded at the lowest
temperature (250C) with the highest salinity (33),
and the highest temperature (25 0C) with the highest
salinity (33 ), respectively, while the lowest
survival of early juveniles (15.56%) was founded at
the highest temperature (35 0C) with the lowest
salinity (27).
Two-way ANOVA showed that survival of
early juvenile B. areolata was significantly affected
by temperature (F = 605.192; P = 0.000) and
interaction between both factors occurred in this
experiment are observed (F = 36.376; P = 0.000).
Tukey test showed that survival of early juveniles
were significantly different one another among
temperature and salinity treatments (Table 8).
Multiple regression analysis showed that
both temperature (P = 0.002) and salinity (P =
0.026) were statistically significant, showing a
negative correlation with the percentage survival of
early juvenile B. areolata (Table 9). Standard
coefficient (Beta) of temperature (0.818) was higher
than that of salinity (0.251), indicating a higher
correlation between temperature and survival. The
multiple regression equation on survival of early
juvenile B. areolata over the combined effects of
temperatures and salinity was estimated as follows:
Survival = 165.176 6.148 Temperature + 3.148
Salinity
The response surface plots summarizing
percentage survival under nine different
combinations of temperature and salinity over 96 h
showed that high survival (100%) was obtained at
29 -350C and 32 - 33. Higher temperatures at all
salinities reduced hatching markedly (Fig 3).
DISCUSSION
Our results showed that hatching of egg
capsules B. areolata was only significantly affected
by salinity but not for temperature. No significant
interaction between both factors occurred in this
experiment. The highest hatching was founded at
the lowest temperature (25 0C) with the highest

Table 2. ANOVA comparing the effects of temperature and salinity on percentage hatching of egg capsules B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 189170.37 1 189170.37 865.69 0.000
Temperature 474.07 2 237.03 1.08 0.359
Salinity 2229.63 2 1114.81 5.10 0.018
Temperature x salinity 192.59 4 48.14 0.22 0.924
Error 3933.33 18 218.51

Table 3. Multiple regression analysis of data on hatching of egg capsules B. areolata through 96 h under 9
different combinations of temperature and salinity on (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept -54.074 36.676 -1.474 0.153
Temperature 0.889 0.627 0.228 1.417 0.169
Salinity 3.704 1.046 0.570 3.542 0.002
Standard error of estimation = 13.310
R = 0.614 R2 = 0.377 Adjusted R2 = 0.326
F = 7.275 P = 0.003

Journal of Research in Biology (2011) 5: 376-384 380

 Chaitanawisuti et al.,2011

Table 4. Percentage survival rate of veliger larvae B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature Salinity ()
(0C) 27 30 33
25 23.33+1.53 65.67+2.08 78.67+1.53
30 81.33+3.51 84.00+3.00 87.67+1.53
35 17.67+1.15 18.67+1.53 47.00+2.65
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)

salinity (33) and the highest temperature (25 0C)

Fig 3. Response surface plots with estimating the mean
percentage survival of new settled juvenile B.
babylonia after 96 h under 9 different combinations of
temperature and salinity.
salinity (33) and the highest temperature (35 0C)
with the highest salinity (33). Multiple
regressions showed a higher correlation between
salinity and hatching. High hatching was obtained
at temperature of 29 -350C and salinity above 32
to 33. Survival of larvae and early juvenile B.
areolata were significantly affected by temperature
and salinity. A significant interaction between both
factors occurred in this experiment was observed.
The highest survival of early juveniles was founded
at the lowest temperature (25 0C) with the highest
with the highest salinity (33 ). Multiple
regression analysis showed a negative correlation
with the percentage survival of early juvenile B.
areolata as well as indicating a higher correlation
between temperature and larval survival, as well as
salinity and early juvenile survival. The response
surface plots showed that high survival of larvae
was obtained at temperature of 29 -30.50C and
salinity above 32 to 33 and 29 -350C and 32 -
33 for early juveniles. The results of this study
agreed with various studies that salinity had
strongly effect on larvae of various mollusks
(Doroudi et al. 1999, Tettelbach & Rhode 1981,
Davis 2000 and Dove & OOconner 2007. Doroudi
et al. (1999) reported that optimal conditions for
maximum larval survival of the black-lip pearl
oyster Pinctada margaritifera were 26-290C and 28
-32. Temperature of 350C or greater were lethal
for larvae and at all temperature tested, larval
survival were lowest at a salinity of 40.
Tettelbach & Rhode (1981) reported that optimum
combination of temperature and salinity for survival
of the Northern Bay scallop Argopecten irradians
larvae from 2 to 5 day after fertilization, as
estimated from the response surface plot, was
18.70C and 28.1. Temperatures of 35 0C or
greater and / or salinities of 10 or less were lethal
for all life stages of this species. Davis (2000)
showed that at the end of 0 to 7 day interval,
percent mortality of tropical gastropod veligers
Strombus gigas was highest for veligers grown at
20 and 240C and at salinity of 45, while percent
mortality was low and not different for veligers

Table 5. ANOVA comparing the effects of temperature and salinity on percentage survival of veliger larvae B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 84896.14 1 84896.14 19760.31 0.000
Temperature 14280.96 2 7140.48 1662.00 0.000
Salinity 4201.40 2 2100.70 488.95 0.000
Temperature x salinity 2624.14 4 656.03 152.69 0.000
Error 77.33 18 4.29
381 Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

Table 6. Multiple regression analysis of data on survival of veliger larvae B. areolata through 96 h under 9
different combinations of temperature and salinity on (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept -13.037 65.309 -.0200 0.843
Temperature -2.789 1.117 -0.406 -2.496 0.020
Salinity 5.093 4.862 0.445 2.735 0.012
Standard error of estimation = 23.701
R = 0.603 R2 = 0.364 Adjusted R2 = 0.311
F = 6.855 P = 0.004

Table 7. Percentage survival rate of early juvenile B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature Salinity ()
(0C) 27 30 33
25 93.33+1 98.89+1.92 100
30 92.23+1.93 92.23+3.05 92.23+1.93
35 15.56+5.09 26.67+5.77 65.56+7.70
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)

grown at 240C and at salinity of 30, 35 and 40.
Dove & OOconner (2007) showed that salinity had
a significant effect on D-veliger larval survival of
Sydney rock oysters Saccostrea glomerata whereas
temperature significantly affected survival of both
D-veliger and pediveliger larvae. There was an
interaction between salinity and temperature for D-
veliger larval survival. While spat survival was
significantly affected by salinity only and no
interaction was detected between salinity and
temperature for spat survival.
In addition, results of this study also agreed
with the combined effects of temperature and
salinity on larvae and juveniles of various
organisms; crab (Paula et al. 2003, Nurdiani &
Zeng 2007) and shrimp (Palafox et al. 1997,
Jackson & Burford 2003 and Zacharia & Kakati
2004). Palafox et al. (1997) indicated that good
survival (80 to 90%) of Penaeus vannamei
postlarvae was obtained below 30 0C and below
40. Higher temperatures reduced survival
markedly. Higher salinities only had a marked
negative effect at higher temperatures. The
optimum temperature and salinity conditions for
survival of P. vannamei postlarvae coincides the
best at 28 to 300C and 33 to 40. Jackson &
Burford (2003) showed that salinity did not have a
significant effect on survival of larval shrimp
Penaeus semisulcatus above 28. At 28,
survival rate decreased, while temperature had a
substantial and regular influence on growth rate.
Zacharia & Kakati (2004) showed that salinity
exerted a greater influence than temperature on the
survival and development of larvae Penaeus
merguiensis. The best temperature salinity
combination for hatching and larval survival was
obtained at 330C and 35, and a salinity range of
30-35 is ideal for larval development. Paula et al.
(2003) indicated that for all zoeal stages of
mangrove crab Parasesarma catenaata, the highest
survival was obtained at temperature of 25 0C and
salinity of 35, but survival decreased towards the
lower and higher salinity. Nurdiani & Zeng (2007)
showed that temperature and salinity as well as the
interaction of the two parameters significantly
affected the survival of zoeal larvae of mud crab

Table 8. ANOVA comparing the effects of temperature and salinity on percentage survival of early juvenile B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 152616.90 1 152616.90 8826.82 0.000
Temperature 20927.67 2 10463.84 605.19 0.000
Salinity 1696.51 2 848.25 49.06 0.000
Temperature x salinity 2515.77 4 628.94 36.37 0.000
Error 311.22 18 17.29
Journal of Research in Biology (2011) 5: 376-384 382
 Chaitanawisuti et al.,2011

Table 9. Multiple regression analysis of data on survival of early juvenile B. areolata through 96 h under 9
different combinations of temperature and salinity (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept 165.176 46.503 3.552 0.002
Temperature -6.148 0.796 -0.818 -7.728 0.000
Salinity 3.148 1.326 0.251 2.347 0.026
Standard error of estimation = 16.876
R = 0.855 R2 = 0.731 Adjusted R2 = 0.709
F = 32.679 P = 0.000

Scylla serrate. At low salinity, both high and low Berlinsky DL, Taylor JC, Howell RA, Bradley
temperature led to mass mortality of newly hatched TM and Smith TIJ. 2004. The effects of
larvae, while the low temperature and high salinity temperature and salinity on early life stages of black
combination of 250C and 35 gL-1 resulted in the sea bass Centropristis striata. Journal of the World
highest survival to the megalopal stage. Aquaculture Society 35:335-344.
This study is the first study to investigate
salinity and temperature effects on hatching Chaitanawisuti N and Kritsanapuntu A. 1997.
success, and survival of larvae and early juvenile B. Laboratory spawning and juvenile rearing of the
areolata. Although the results suggest the marine gastropod: Spotted babylon, Babylonia
possibility that the culture and natural population of areolata Link, 1807 (Neogastropoda: Buccinidae)
this species in Gulf of Thailand may suffer in Thailand. Journal of Shellfish Research 16:31-
mortality from higher water temperature due to 37.
climate change and lower salinity from heavy
rainfall. Cook MA, Guthriel KM, Rust MB and Plesha
PD. 2005. Effects of temperature and salinity
ACKNOWLEDGEMENT during incubation on hatching and development of
This research was a part of the Research lingcod Ophiodon elongates Girard, embryos.
University Program funded by Chulalongkorn Aquaculture Research 36:1298-1303.
University (CC103A). The authors thank Sichang
Marine Science Research and Training Station, Chen JC and Chen WC. 2000. Salinity tolerance
Aquatic Resource s Re searc h Institute, of Haliotis diversicolor supertexta at different
Chulalongkorn University, in particular Mr. salinity and temperature levels. Aquaculture
Soontorn Thepmoon for help in the hatchery and 181:191-203.
providing of algae. The authors also thank to
Associated Professor Dr. Gullaya Wattayakorn for Doroudi MS, Southgate PC and Mayer RJ. 1999.
valuable comments and coordinations during the The combined effects of temperature and salinity on
preparation of this research project. embryos and larvae of the black-lip pearl oyster
Pinctada margaritifera. Aquaculture Research
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 Journal of Research in Biology

Original Research Paper An International Online Open Access

Publication group

Biorestraining potentials of marine macroalgae collected from

Rameshwaram, Tamil nadu
Journal of Research in Biology

Authors: ABSTRACT:
Anandhan S and
Sorna kumari H.
The marine ecosystem is the treasure place for many natural resources. In
this study, about five different marine macroalgae were chosen to study the
antibacterial activity and larvicidal activity of Aedes sp. About five different stranded
strains of bacteria have been selected to detect the antibacterial activity of collected
Institution:
Sri Sankara Arts & algae. Among the five algae collected, Gracilaria crassa and Hypnea valentia have
Science College -Enathur, shown maximum antibacterial activity in methanolic extraction by antibiosis of Kirby-
Tamilnadu. Bauers method. The active biocompound from methanol was determined by using
different solvent systems in TLC. The partially purified antibacterial compound was
identified as saponins by phytochemical tests. Larvicidal bioassay was carried out with
the two algae Gracilaria crassa and Hypnea valentia. The LC50 determined by the
Gracilaria crassa and Hypnea valentia was noted as 52.2 and 53.4 respectively.
Corresponding author:
Sorna kumari H

Email: Keywords:
sornask@gmail.com Larvicidal bioassay, LC50, antibiosis.

Web Address: Article Citation:

http://jresearchbiology.com/
Anandhan S and Sorna kumari H.
Documents/RA0100.pdf.
Biorestraining potentials of marine macroalgae ollected from Rameshwaram,
Tamil nadu.
Journal of research in Biology (2011) 5: 385-392

Dates:
Received: 09 Sep 2011 /Accepted: 16 Sep 2011 /Published: 23 Sep 2011

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

385-392 | JRB | 2011 | Vol 1 | No 5

Journal of Research in biology
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INTRODUCTION
Man relied on natural products in general
and plants in particular to promote and maintain
good health and fight sickness, pain and disease
since in time immemorial. India is an important
country in world where ambient system of medicine
such as ayurveda, siddha and unani has been in
practice for many years. In common all the above
mentioned system of medicine are directly
dependent upon resources such as plants with the
advances in experimental methods in
phytochemistry and pharmacology, several
medicinal plants were screened for active principles
and biological activites (Prashant Kumar et al.,
2006). Marine environment is a rich source of
biological and chemical diversity. The diversity has
been a unique source of chemical compounds of
potential for pharmaceuticals, cosmetics, dietary
supplements and agrochemicals (Chau Van Minh et
al., 2005). In recent years, a significant number of
novel metabolites with potent pharmacological
properties have been discovered from the marine
organisms.
(Narisnh et al., 2005).Marine algae were
also reported to have some antioxidant properties
(Faten et al., 2009) Commercially available
varieties of marine macro algae are commonly
referred to as seaweeds. Seaweeds have some of
valuable medicinal value compounds such as
antibiotics, laxatives, anticoagulants, antiulcer
products and suspending agents in radiological
preparation (Rajasulochana et al., 2009).Fresh and
dry seaweeds are extensively consumed by people
especially living in coastal areas. Seaweeds are
classified as rhodophyta (red algae) or phaeophyta
(brown algae) or chlorophyta (green algae)
depending on their nutrient and chemical
composition (Cox et al ., 2010).As a consequence
of an increasing demand in screening for new
therapeutic drugs from natural products, there is a
greater interest towards marine organisms. Several
marine organisms produce bioactive metabolites in
response to ecological pressure such as competition
for space maintenance of unfolded surface
deterrence of predation and the ability to
successfully reproduce (Fangming Kong et al.,
1994). The antibacterial agent found in the algae
include terpenoid, phlorotannins, acryl acid,
phenolic compounds, steroid, halogenated ketone
and alkaline, cylic polysulphides and fatty acid. In a
number of marine algae antimicrobial activites are
attributed to the presence of acryl acid (James et al.,
1975). Seaweeds provide a rich source of
386
Anandhan et al.,2011
structurally diverse secondary metabolites. The
secondary metabolites offer a defense against
herbivores, fouling organism and pathogens. They
also play a role in reproduction, protection from UV
radiation and as allelopathic agent. There is an
urgent need to search for alternatives to synthetic
antibiotics. The revalution of the discovery of new
groups antimicrobial peptides make natural
antibiotics the basic elements of novel generation
drugs for the treatment of bacterial and fungal
infection. Marine algae have become recognized as
potential source of antibiotic substances (Zheng Yi
et al., 2001). The antibacterial activity of six marine
algae belonging Rhodophyceae and phaeophyceae
were studied against pathogenic microbes
(Staphylococcus aureus, E.coli, Micrococcus,
Enterobacter aerogens, Enterococcus faecalis were
studied (Taskin et al., 2007). Mosquito transmits
serious human diseases like Malaria, Filariasis,
Japanese encephalitis, dengue, hemorrhagic fever
and yellow fever causing millions of death every
year. Extensive use of chemical insecticides for
control of vector borne diseases has created
problems related to physiological resistance to
vectors, adverse environmental effects, high
operational cost and community acceptance,
numerous plant products have been reported either
as insecticides for killing larva or adult mosquit oes
or as repellent for mosquito biting and are one the
best alternatives for mosquito control (Rajkumar et
al., 2009). The revolution therapy of infection
disease by the use of antibacterial drugs has certain
limitation due to changing pattern of resistance in
pathogens and side effects they produced. These
limitation demands for search of new antimicrobial
compounds for development of drugs. Seaweeds are
used alternative and traditional remedies in many
parts of the world.
The production on inhibitor substance by
seaweeds has antibacterial actions and some of their
substances have potential use in mosquito control
(Nagi et al., 2010).Use of synthetic insecticides to
control vector mosquitoes has caused physiological
resistance and adverse environmental effects in
addition to high operational cost (Virendra et al.,
2009). The treatment of this disease becoming more
difficult since Plasmodium falciparum parasite has
developed resistance to wide range of anti malarial
drugs (Anne Platt Mc Ginn et al., 2002). Secondary
metabolites of many marine algae species isolated
from Canaria Island were shown to have a wide
antimicrobial activity (Antonio Gonzalez et al.,
2001). Thus the screening of marine organism for a
Journal of Research in Biology (2011) 5: 385-392
Anandhan et al.,2011
variety of biological activities with in aim
identifying novel with interesting and potentially
therapeutic activities continues till this date.
(Sreenivasan Sasidharan et al., 2010).
MATERIALS AND METHODS
Collection of algae samples
Marine algae samples were collected from
the coastal region of Rameshwaram. Algae were
washed with sea water to remove extraneous
materials and brought to the laboratory in plastic
bag containing sea water to prevent evaporation.
Sample preparation
After collection of sample, it was brought to
the laboratory. Algal samples were washed in
running tap water to remove any associated debris
and then with the distilled water. After washing, the
samples were dried in a blotting paper for two
weeks. After drying, the samples was grinded in to
powder form, which was then stored in 4C for
further studies. (Rajasulochana et al., 2009).
Preparation of different solvent extracts
One gram of each algal sample was
extracted with different solvents systems 10ml of
methanol, Hexane, and ethyl acetate in a beaker for
24 hours at room temperature. Then the solvent
portion was centrifuge at 5000rpm for 10minutes.
The supernatant was collected from the centrifuge
tube and the solvent were evaporated. Finally crude
extract was obtained. The extracts were collected in
plastic vials and stored in the refrigerator for further
studies. (Aseer et al., 2009)
Disc preparation
The solvent extracts were used for disc
diffusion assay to test for antibacterial activity. The
discs were prepared by using Whatman filter paper
approximately 5mm in diameter and then it was
soaked in extracts and then dried. Then the discs
were stored and used for further works.
Antibacterial assay
The stranded strains of Bacillus subtilis,
Staphylococcus aureus, Klebsiella pneumoniae,
Pseudomonas aeruginosa and E.coli cultures were
collected from the Department of Microbiology, Sri
Sankara Arts and Science College, Enathur,
Kanchipuram. The antibacterial activity of algae
extracts were studied by disc diffusion methods by
using Muller Hinton Agar (MHA). 18hours old
broth culture was prepared and inoculated on
Muller Hinton agar plates by using sterile cotton
swab. After the swabbing, 20l of crude extract disc
were added in to sterile filter paper disc
Journal of Research in Biology (2011) 5: 385-392
approximately 5mm in diameter and allowed at
room temperature for few minutes. After that the
disc were placed on the Muller Hinton agar plates
by using sterile forceps. All the plates were
incubated at 37C for 24 hours. After incubation of
24 hours at 37C a clean zone around the disc was
evidence of antimicrobial activity (Rajasulochana et
al., 2009)
Partial purification of selected algae extract
Based on the antibacterial activity of potential algae
extracts further investigations like partial
purification by thin layer chromatography were
done.
Partial purification of crude extract by thin
layer chromatography (TLC)
Analytical TLC
The crude extract was purified by using thin layer
chromatography. For determining the best solvent
system for good separation of crude compound,
solvents such as methanol, chloroform, n-hexane,
diethyl ether, n-butanol, ethyl acetate and acetic
acid were used in the following ratio.
B u t a n o l : A c e t i c a c i d : w a t e r
[30:45:25,45:30:25,50:35:15],hexane:ethylacetate
[90:10,80:20,70:30,60:40,50:5
0],hexane:diethylether:aceticacid
[80:10:10,70:20:10,60:30:10], hexane:methanol
[70:30,60:40,50:50], meth anol:ethylacetate:hexane
[70:20:10,60:30:10,75:20:5].The crude extract was
dissolved in 200l of methanol. With the help of
capillary tube the sample was spotted on the silica
gel coated slide and placed in the developing
chamber which contain solvent mobile phase,
covered with the watch glass in order to prevent the
evaporation of solvents. The solvent was allowed to
run till it reaches about half a cm below the top of
the plate. After running the slide was kept in room
temperature for the complete drying of the plate.
Then the slide was kept closed in iodine chamber to
visualize the separated compound as clear spots
(Jebakumar Solomon et al., 2008). The RF value
were determine by RF=Movement of the solute
from the origin /movement of solvent from the
origin.
Preparative TLC
Preparative TLC was performed to get
partially purified compound.TLC plate was
prepared by spreading the slurry of silica gel evenly
on the plate. The plate was activated at 100C for
15 minutes. Crude extract was applied on the plate
as a single line and the chromatogram was
performed with the solvent system Hexane:
387

ethylacetate solvent system (50:50).After
separation, the active spot band was scrapped,
mixed with methanol and centrifuged at 3000rpm
for 15 minutes. Supernatant was collected in a pre
weighed vial and kept for evaporation. The partially
purified compound obtained from preparative TLC
was tested for antibacterial activity against stranded
organisms by disc diffusion method.
Antimicrobial Activity of TLC fractions
After the swabbing the stranded organism, 20l of
TLC extracts disc were added on to the sterile filter
paper disc approximately 5mm in diameter and
allowed at room temperature for few minutes. After
that the disc were placed on the Muller Hinton agar
plates by using sterile forceps. All the plates were
incubated at 37C for 24 hours. After incubation of
24 hours at 37C a clean zone around the disc was
evidence of antimicrobial activity.
Phytochemical test
The partially purified compound obtained
from preparative TLC was subjected to qualitative
phytochemical analysis for the identification of
compound present in the purified extract. All the
tests were carried out using standard methods
(Aliyu et al., 2008).
Larvicidal bioassay
Mosquito larva was collected from ditch
area near Enathur, in Kanchipuram district and it
was examined by experts for the confirmation of
mosquito larva of Aedes sp. The algal extracts of
Gracilaria crassa and Hypnea valentia were
volumetrically diluted to obtain the test
concentrations of 25, 50 and 75 mg/10ml. Control
was set up by 1 ml of methanol in 10 ml of water.
Twenty five late third instar larvae were introduced
to each of the test concentration as well as control.
The larval mortality was recorded after 24 h of
exposure, during which no food was given to the
larvae. The lethal concentrations (LC50) were
calculated by probit analysis (Rajkumar et al.,
2009).
RESULTS AND DISCUSSION
Screening of algae extract for antibacterial
activity
The macroalgae from the Moroccan coast
are potential sources of bioactive compounds for
investigating natural antibiotics(Chiheb et al.,
2009).Different extracts of the brown algae
Sargassum cinereum have different anti bioactive
properties such as antibacterial and antifungal
activity (Divya et al., 2011). Antibacterial activity
of selected algae extracts were given in table 1.In
388
Anandhan et al.,2011
the present study, among the five different algae
with varying extracts tested by disc diffusion
method, the methanolic extract of all algae have
satisfactory inhibition properties. Stranded strain of
Bacillus subtilis had shown maximum sensitivity to
the algae Gracilaria crassa and Hypnea valentia
with a maximum zone of inhibition of about
14mm.Similarly, in a study conducted on
a nti mi crobial acti vitie s of mic roal gae
Trichodesmium erythraeum, the hexane extracts
have shown good inhibitory activities (Kasinathan
thillairajasekar et al., 2009). The crude methanolic
extract of different algae like Asparagopsis,
Laurencia, and Hypnea showed significant
antimicrobial activity(Nagi et al., 2010). It was
observed that kappaphycus, a red sea weed algae
showed maximum activity against Pseudomonas
flouresences, Staphylococcus aureus and less
inhibition on Vibrio cholera and Proteus mirabilis
(Rajasulochana et al., 2009). In a study it was
observed that the crude butanol extract of Isochrysis
galbana, marine algae had shown a satisfactory
inhibitory effect against the selected bacterial
pathogens (Srinivasakumar et al., 2009).Among the
sea weeds highest inhibitory activities was
documented among the members of red, green and
brown algae against both the gram positive and
gram negative (Vallinayagam et al., 2009).In this
study, the second highest sensitivity is shown in
Enteromorpha intestinalis against Pseudomonas of
an inhibition zone about 13mm as shown is table 1.
Based on this result, Gracilaria crassa and Hypnea
valentia were selected for further studies.
Partial purification of antibacterial compound
by TLC
Among the various solvent systems analyzed
in TLC, three well separated spots were observed in
Hexane: ethylacetate solvent system for Gracilaria
crassa and two well separated spots were seen for
Hypnea valentia. RF values for Gracilaria crassa
was calculated as 0.64, 0.78, and 0.84 and for
Hypnea valentia it was calculated as 0.62 and 0.67
respectively. The antimicrobial effect of bioactive
compound present in Dictyota acutiloba has been
purified by TLC to examine the compound for
further analysis.
Antimicrobial activity of partially purified TLC
fractions
Among the three different spots that were
observed in Gracilaria crassa the third band named
G3 showed maximum inhibitory of 16mm while the
Hypnea valentia showed inhibitory effects of 15
and 18mm.the results were presented in table-2.
Journal of Research in Biology (2011) 5: 385-392
Anandhan et al.,2011

Table .1.Antimicrobial activity of crude algae extract against selected pathogens

Table.2.Effect of partially purified compound against

Bacillus subtilis
S. No Spots
1 G1
2 G2
3 G3
4 G4
5 G5
Phytochemical analysis of partially purified
antibacterial compound
Results of phytochemical analysis of
Zone on inhibition partially purifies antibacterial compound were
(mm) in diameter given in table- 3.Based on the results it was found
- that the partially purified compound consists of
saponins. Similarly in a study, flavonoids and
- tannins extracted from the Acacia albida and
16 Pavetta crassipes which possess the antimicrobial
activity against MRSA (Aliyu et al.,
15 2008).Phytochemical screening of C.decorticatum
18 in methanolic and petroleum ether extracts revealed
the presence of saponins, phytosterols and

Journal of Research in Biology (2011) 5: 385-392 389


Table.3. Phytochemical properties of partially purified active compound
glycosides which showed a well documented
antimicrobial activity (Anbu Jeba Sunilson et al.,
2009). The phytochemical analysis of Gracilaria
fergusonii revealed the presence of coumerins,
phenols, quinines and steroids, which ultimately
may inhibit the microorganisms (Renuka Bai.,
2010).
Larvicidal bioassay
Insecticides of botanical origin may serve as
suitable alternative biocontrol techniques in the
future (Kamaraj et al., 2009). An insecticide
containing azadirachtin, a neem tree (Azadirachta
indica) extract, was tested against mosquito larvae
in the Islamic Republic of Iran under laboratory and
field conditions. (Vatandoost et al., 2004). Certain
species of green algae in the order Chlorococcales
kill larvae primarily because they are indigestible
(Gerald., 2007). The extracts of J. curcas and E.
tirucalli were highly effective against the larvae of
A. aegypti (LC= 8.79 and 4.25 ppm) and against C.
quinquefasciatus (LC =11.34 and5.52 ppm). The
LC values were 35.39, 256.77, 384.19, 703.76, and
13.14 ppm against A. aegypti (Abdul Rahuman et
al., 2008). In this study also, Gracilaria crassa and
Hypnea valentia in methanolic extract have shown
good larvicidal activity with a LC50 of about 52.2
and 53.4 respectively.
Thus, the present study discussed about the
marine algae as potential compound reservoirs
which not only act as antibacterial agents but also
used to control the mosquito larval population
which is a biggest growing threat.
390
Anandhan et al.,2011
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 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Foliar spray of Earthworm urine to improve Amaranthus growth

performance and yield
Journal of Research in Biology

Authors: ABSTRACT:
1
Owa Stephen
Olugbemiga,
2
Ojediran John O and
3
Fadeyi Oluwatosin.
The effects of foliar spray of earthworm urine on plant growth parameters
and greengrocery values were studied on the leaf vegetable Amaranthus. The
Institution:
1. Department of Biological application caused 28% increase in plant height, 166% increase in plant girth taken at
Sciences, Landmark the ground level, 20% increase in the number of leaves per plant, 17% increase in
University, Omu-Aran, length of leaves, 15% in leaf breadth and about 49% in leaf area. It was also observed
Kwara State, Nigeria, that the stomatal diameters increased by about 19%. These results of foliar
2. Department of application indicate that earthworm urine contains some plant growth hormone that
Agricultural Engineering, can be gainfully used to increase not only the yield of the vegetable crop, but also
Landmark University, increase its market visual appeal with respect to the length of an Amaranthus stick,
Omu-Aran, Kwara State, the leaf-density per stick and the length and area of a leaf. On a commercial scale
Nigeria, earthworm urine application can be mechanized using a sprinkler.
3. Formerly, Department
of Plant Science and Applied
Zoology, Olabisi Onabanjo
University, Ogun State,
Nigeria.

Corresponding author: Keywords:

Owa Stephen Earthworm urine, horticulture, greengrocery, plant growth hormone,
Olugbemiga sprinkler application.

Web Address: Article Citation:

http://jresearchbiology.com/ Owa Stephen Olugbemiga, Ojediran John O and Fadeyi Oluwatosin.
Documents/RA0046.pdf. Foliar spray of Earthworm urine to improve Amaranthus growth performance and
yield.
Journal of research in Biology (2011) 5: 393-398

Dates:
Received: 08 Jun 2011 /Accepted: 21 Jun 2011 /Published: 28 Sep 2011

Ficus Publishers.
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393-398 | JRB | 2011 | Vol 1 | No 5

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INTRODUCTION:
The importance of earthworms to the general
health of crops and soil has been well documented.
They affect the soil in many ways. Bayon and Binet
2001 reported that earthworm surface casts affect
soil erosion by runoff water and phosphorus
transfer in a temperate maize crop. Their casts
improve the stabilization of organic matter in fine
soil fractions (McInerney, Little and Bolger 2001).
Their surface movement improves soil porosity and
aeration (Mather and Christensen 1988). According
to Sharpley, Syers and Springett (1979) their
surface casting improves transport of phosphorus
and nitrogen in surface runoff from pasture.
One mechanism of nitrogen efficiency in
agricultural soils is due to release of nitrogen from
herbage residues by earthworms (Ruz Jerez, Ball
and Tillman 1988). According to Mackay and
Kladivko (1985) earthworms improve the rate of
breakdown of soybean and maize residues in the
soil. By competitive feeding, earthworms depress
soil nematode populations (Yeates 1981).
Earthworm burrowing activity reduces soil
compaction (Bostrom 1986). Soil damage due to
opencast mining is sometimes rehabilitated by
earthworm transplant. (Stewart, Scullion, Salih and
Al Bakri 1988).
Owa, Moreyibi and Dedeke (2002)
demonstrated that wormcasting initiates thermal
and nutrient convection current in the soil. By that
earthworms raise soil temperature and thereby
improve the physicochemical activities of crops
(Owa et al 2004a).
When located at the base of a plant crop
their effect could amount to creating an isolating
environment (Owa et al. 2004b). Another positive
effect on plant is that earthworms participate in
dispersal of seeds (Decaens et al. 2003). According
to Ayanlaja et al. (2001) leachate from earthworm
cast can break seed dormancy and promote radicle
growth.
Earthworm affect soil microbial population.
Their casts and compost improve soil microbial
activities and plant nutrient availability (Brown
1995; Chaoni et al. 2003). Microbial biomass and
activities increase after the soil microbes have
passed through the gut of earthworms (Daniel and
Anderson 1992).
Studying earthworm populations and
distribution in Nigerian soils Owa et al. (2003)
showed that earthworms make significant
contribution to the soil nitrogen, and that
application of earthworms in a farm, or their
394
Olugbemiga et al.,2011
protection from farming activity damages can lead
to reduction of the quantities of inorganic fertilizers
needed.
Studying lowland rice agroecosystems Owa
et al. (2003b) showed that rice stands that have
earthworm casts associated with their bases grew
more tillers, grew taller, had more leaves, more
grains and bigger grain size, than others stands
lacking earthworm casts associated with their bases.
Ojediran 1990 reported that increasing irrigation
frequency induced an increase in the shoot length
and root growth of wheat. Both root mass and
length were reported to decrease significantly with
water stress (Ojediran 1990, Snyman 2004).
Two major challenges confront expansion of
earthworm urine irrigated farming: one is
availability of water resource. Irrigation of
vegetables requires significant quantities of water
of suitable quality. The second is water lifting and
distribution device. One traditional method is the
rope and bucket. An alternative traditional method
is the treadle pump which originated in Bangladesh
or motorized pumps.
The present study is aimed at checking if
foliar application of earthworm urine can produce
enhancement in the growth parameters and market
grocery values of the leaf vegetable crop
Amaranthus. An argument is also provided that the
foliar application can be mechanized by use of a
sprinkler. Such a mechanization can significantly
improve the production and availability of this very
important leaf vegetable.
MATERIALS AND METHODS
Earthworm urine was prepared by placing 1
kg live earthworms in 1 L earthworm saline for 1
hr. The decant was funnel filtered under suction.
The filtrate was labeled as earthworm urine.
Ninety (90) plastic plant pots were loaded
each with 1 kg top soil, set in an open space; and
planted with viable seeds of Amaranthus. After
germination, the plants were thinned down to one
per pot. 30 pots randomly selected were reserved
and properly labeled for earthworm urine treatment,
another 30 for earthworm saline treatment and the
last 30 for water treatment.
Each pot was watered with 500 ml water
between 6.30 am and 7 pm daily. Using a handheld
plastic sprayer, 30 cm3 of earthworm urine,
earthworm saline and distilled water were daily
sprayed onto the leaves of each plant in the
respective pots. The application was every morning.
Journal of Research in Biology (2011) 5: 393-398
Olugbemiga et al.,2011

On a regular weekly basis, the height of the

DISCUSSION
plant in each pot was measured from the ground Hitherto, the effects of earthworms on plants
level to the apical bud, using a meter rule. The have been viewed via their impacts on the soil. The
widest stem girth was measured at the ground level,present work shows that earthworm urine can affect
using a thread and plastic ruler. The lengths and plants more directly.
widest diameters of the lowest (and largest) three From agronomic considerations, the gain in
leaves on a plant were measured using a plastic height or length of a crop is a desirable effect
ruler. The total number of leaves on each plant wassimilar to the effect of organic and inorganic
recorded. At the end of the fifth week an additional
fertilizers. For a leaf vegetable, it shortens the time
factor, stomatal diameter, was measured and the taken for the crop to reach table size. It means
experiment was terminated. To measure the savings in time and cost of operations before the
stomatal diameter, nail vanish was applied to the crop is sold for a monetary gain. From the buyers
lowest green leaf on a plant, in order to take an angle, the lengthening of the vegetable stick
impression of the stomata pores. The vanish was produces a visual advantage. In local
allowed to dry, carefully peeled off, placed on a greengroceries, buyers examine the length of the
labeled microslide and taken to the laboratory. sticks of the vegetable, and prefer those that are
Using a stage micrometer and eyepiece graticule, long.
the stomatal diameters were measured. From research angle, this result is similar to
The resulting data were analyzed using the the earlier results of Owa et al. (2002), Owa et al.
Statistical Package for Social Sciences (SPSS) (2003), Owa et al. (2004a, b) And it indicates that
Version 15. Analysis of variance, LSD and Duncan beyond just providing nitrogen and ammonia to
multiple range tests were performed on the data, inplants through the soil, earthworm urine provides
order to test the significance of the differences at p
some growth hormone.
= 0.05. That plant girth is greatly increased by
earthworm urine is beneficial in many ways. In the
RESULTS current drive to minimize artherogenesis and other
Compared to the controls, application of diet-related diseases, there is a growing emphasis
earthworm urine produces about 28% increase in on vegetarianism, especially the use of intake fibers
plant height, 20% in number of leaves per plant, to reduce dietary cholesterol absorption at the villi
166% in plant girth (Table 1), 17% in leaf length level. Among the local community, not only the
and about 40% in leaf area (Table 2). It also leaves of Amaranthus are shredded for cooking, but
produces an increase of about 19% in stomatal pore also the soft succulent stem. Thus, the application
diameter (Table 3). of earthworm urine could be indirectly contributing
Table 1: Effects of foliar application of earthworm urine on the height, number of leaves and stem girth of
Amaranthus
% Gain in
parameter
N Mean Std. Dev Minimum Maximum
relative to
control
Height of plant (cm) Earthworm urine 30 40.302a 6.5102 28.4 56.2 28.0502
Saline water 30 33.411b 5.7132 23.4 48.6 6.157594
Water (control) 30 31.473b 4.9797 21.8 41.1
Average 90 35.062 6.8585 21.8 56.2
No of leaves Earthworm urine 30 12.10a 2.845 7 16 20.19868
Saline water 30 10.70b 2.054 1 13 6.291391
Water (control) 30 10.07b 1.780 4 12
Average 90 10.96 2.403 1 16
Plant girth at ground Earthworm urine 30 3.627a .5836 2.5 5.1 166.4055
level (cm)
Saline water 30 1.660b .3001 1.3 2.2 21.93928
Water (control) 30 1.361c .0687 1.3 1.5
Average 90 2.216 1.0785 1.3 5.1
Note: Values labeled with same letter are not significant different, others with different letters are significantly different.

Journal of Research in Biology (2011) 5: 393-398 395

 Olugbemiga et al.,2011

Table 2: Effects of foliar application of earthworm urine on leaf length, leaf width and leaf area of Amaranthus
% Gain
Std. Std. relative
N Mean Minimum Maximum
Deviation Error to
control
Leaf length
Earthworm urine 30 10.1150a 1.47177 .26871 7.08 14.08 17.11694
(cm)
Saline 30 9.5917a 1.53517 .28028 6.38 13.28 11.05751
Water 30 8.6367b 1.27177 .23219 6.20 11.98
Average 90 9.4478 1.54258 .16260 6.20 14.08
Leaf breath
Earthworm urine 30 5.9350a 3.04315 .55560 2.95 21.35 15.09373
(cm)
Saline 30 5.0192a .85872 .15678 3.30 6.78 -2.66645
Water 30 5.1567a 2.21831 .40501 3.50 16.33
Average 90 5.3703 2.24181 .23631 2.95 21.35
Leaf Area
Earthworm urine 30 20.4851a 12.08548 2.20650 6.95 78.03 39.83027
(cm**2)
Saline 30 16.3841a 5.24115 .95690 8.09 29.95 11.83741
Water 30 14.6499b 5.23739 .95621 8.25 37.37
Average 90 17.1730 8.45779 .89153 6.95 78.03
Note: Values labeled with same letter are not significant different, others with different letters are significantly
different.

significantly to higher fiber intake and thus
contributing to the battle against artherogenic
disease. Similarly amaranth oil is used in the
treatment of ischemic and other artherogenic
diseases (Czerwinski et al., 2004; Gonor et al.
2006; Martirosyan et al. 2007).
Earthworm urine increases the number of
leaves on a plant. To the local consumers the
density of leaves on an Amaranthus stick is a major
buyers consideration. From dietary point of view
many of the nutrients that humans benefit from
Amaranthus are derived from the leaves. These
include, among others, vegetable oils, protein,
carbohydrates, macronutrients and micronutrients.
Vegetable leaf is one of the most important sources
of dietary fiber. Thus, application of earthworm
urine can improve the nutritional value of
Amaranthus for human consumption.
But in addition, there is an esthetic premium
attached to the length of a vegetable leaf. There is a
social and psychological preference for Amaranthus
sticks with longer leaves than for those with shorter
leaves, even when there is no known data that the
one is richer in nutrients than the other. Thus,
application of earthworm urine could significantly
improve the premium value placed on Amaranthus
in a greengrocery.
The widening of stomatal diameter by
earthworm urine may be a factor to the higher
growth performance and productivity of the plants.
It increases the rate of in- and out-diffusion
processes, thus increasing the rate of foliar and
photosynthetic metabolism.
Beyond Amaranthus, the implication of the
effect of earthworm urine on plant growth and yield
performances is that the food basket can be

Table 3. Effects of foliar application of earthworm urine on leaf stomatal diameter of Amaranthus
Diameter of stomata (um)
% Gain
Std.
N Mean Std. Error Minimum Maximum relative to
Deviation
control
Earthworm urine 100 .006500a 0.00188 0.000188 0.0025 0.01 19.26606
Saline water 104 .005962b 0.001608 0.000158 0.0025 0.01 9.386027
Ordinary water 100 .005450c 0.00125 0.000125 0.0025 0.01
Average 304 0.00597 0.001651 9.47E-05 0.0025 0.01
Note: Values labeled with same letter are not significant different, others with different letters are significantly
different.

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