Life Sci. Int. J., Vol: 3, Issue-1, January 2009, Page: 981-985 http://www.isphyderabad.com.

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Progeny Estimation of Different Entomopathogenic Nematodes in Full Fed Larvae of Fruit Flies Drosophilla Sp. (Drosophilidae: Diptera) Khalid Abdullah1, Said Mir Khan2, Sumbal Yasmin2, Simon Gowen3, Masood K. Khattak2, 4 5 Amjad R. Kiani and Donald A.Ukeh 1 Agricultural Research Institute, Dera Ismail Khan, Pakistan 2 Department of Entomology, Gomal University, Dera Ismail Khan, Pakistan 3 School of Agriculture, University of Reading, White Knights Campus, Reading, Berkshire, RG6 6AR, UK 4 Government Sarwar Shaheed Degree College, Gujar Khan, Rawalpindi 5 Arthropod Functional Ecology & Behaviour Lab., Zoology Building, University of Aberdeen, AB24 2TZ, UK Email: khalidabdullah99@yahoo.com ABSTRACT The progeny development experiment with three nematode species viz., Steinernema carpocapsie, Steinernema feltiae and Heterorhabditis bacterophora, in the laboratory, showed that J3 started emerging in all nematode after day 11 which increased till day 13 for S. feltaie and ended on day 23. H. bacterophora produced a total of 697 infective juveniles in 16 days, whereas, S. carpocapsae with a total of 847 J3 till post infective day-26. Progeny production as hosts unit body weight for S. carocapsae was recorded as 784 J3/mg followed by H. bacterophora and S. feltiae with 580 and 221 J3/mg., respectively. In another experiment, LC50 for S. feltiase was 516 J3/ml and H. bacterophora was 600 J3. Keywords: Entomolopathogenic nematodes; Progeny; LC50, Fruit fly; Steinernema carpocapsie; Steinernema feltiae; Heterorhabditis bacterophora INTRODUCTION Nematodes of Steinernema and Hetrerorhabditis genera are of special interests for the entomologists because of their infective characteristics to a wide range of insects. Entomopathogenic nematodes (EPN) kill their host by entering into their body either through natural openings (anus, mouth or spiricals) or making a hole by its tooth at a softer cuticular area like intersegmental membrane (Bedding and Molyneux, 1982). The nematodes contain symbiotic bacterium of the genus Xenorhabdus. The bacterium has no free-living stage and is found only either in nematode or in the body of infected host insect. When nematode enters into the body of the host insects, it releases mutualistic bacteria, which produces a toxin; causing septicaemia and the insect die within 24-48 h (Bedding et al., 1993). The entomopathogenic nematodes are reported to infect the insects representing orders Diptera, Lepidoptera, Orthoptera, Coleoptera, Thysonoptera, Hymenoptera and some non-insects species of plant pests like snails and slugs (Poinar, 1989; Grewal et al., 1993). Though entomopathogenic nematodes infect variety of insects and are commercially used in many countries, but still EPNs have never been tested against many insect species (Shapiro-llan et al., 2002). Fruit flies Drosophila sp. are one of the less evaluated insects. Beavers and Calkins, 1984, Lindegren and Vail, 1986, Lindegren et al., 1990, demonstrated infective capability of various EPN species against different fruit flies but none of these studies focus progeny production in the host body. The information is of vital importance for time and does of subsequent application, and could help in reducing the application cost as well. The present studies were, therefore, designed to evaluate the infective juvenile production of different EPNs in the full fed larvae of fruit flies Drosophila sp. MATERIALS AND METHODS Nematode culture The nematodes used in the studies were obtained from the Nematology Research Lab., University of Reading, Reading, UK during 2006 and were cultured by infecting wax moth (Glaria mellonella) larvae supplied by the Live Foods, UK. The 9 cm plastic petri dish was lined with filter paper, moistened by adding 1 ml tap water, and poured 500 nematodes with the help of micropipette. Ten active wax moth larvae were added to each petri dish with different species of nematodes and sealed with a strip of para film to preserve the moisture content, and incubated for 48 h at 20C. The petri dishes were removed from the incubator and dead larvae were transferred to white trap for the extraction of J3. The white trap was made by placing lid of 5 cm plastic petri dish upside down in

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a 300 ml plastic cup and a Whatman No.1 filter paper was placed over the petri dish in the plastic cup. Twenty ml water was added to it and dead larvae were placed carefully on the raised platform on the filter paper. The nematodes developing in the body of wax moth moved out and swam into the water from the raised platform. The emerged J3 were then collected into 150 ml plastic cup everyday and stored at 10C for future use. Less than 10-days old cultures of EPN were used in all studies. Fruit fly culture The mother cultures of the fruit flies were received from the Global Live foods, West Yorkshire, UK and cultured in the lab, in a 50 x 50 x 100 cm cadges, on an artificial diet (composed of wheat barn, yeast, lysine and preservative). Freshly prepared diet in 150 ml disposable plastic cups were air cooled to room temperature and offered to the fly culture chamber for 24 h. The cup was removed after one day with considerable numbers of fruit fly eggs in it. The cup was then placed in a separate chamber, with no flies, to hatch and with the intention to have uniform-age larvae for further use in experiments. After 8 days, the larvae hatched and were crawling out of food (Lindegran and Vail, 1986), looking for place to pupate. These full fed larvae were used for further experiments. Experiment-1: Daily Progeny Production All the nematode cultures were formulated to a 1000-J3 per ml strength solution for the experiment. The 9-cm petri dish, lined with Whatman No. 1 filter paper in the cover was used for the experiments. 0.5 ml of the nematode solution (with approximately 500 J3) was added with a micropipette to each petri dish and allowed the solution spread evenly all over the filter paper. Ten full fed fruit fly larvae of the apparently uniform size were collected from the culture with fine insect handling forceps (Bioquip, Rancho Dominguez, CA, USA) ensuring the larvae may not get injured, and carefully introduced into each petri dish with different nematode species. The petri dishes were then sealed with para film and incubated at 20C for two days. After 48 h, the larvae/pupae were washed with running tap water for 1 min. Small white traps were prepared in a 5-cm plastic petri dish by placing a detached-cap of the 3 ml micro centrifugal plastic vial in a petri dish and covering it with No. 1 Whatman filter paper. Each infected larvae/pupae was placed on the raised platform individually and 3 ml water was added. The petri dishes were incubated at 20C, in complete darkness. Every day, the water in the white trap was extracted with the help of a pipette and observed in pre-marked petri dish under the stereomicroscope to count the number of nematodes (J3). The procedure was continued until no nematode emerged in the water. The data were then averaged for each nematode species and liner

model was applied to see the progeny production differences among the nematode species. The nematode species tested were S. carpocapsie, S. feltiae and H. bacterophora. The experiment was replicated 10 times. Experiment-2: Total Progeny Production In the second experiment, the full fed fruit fly larvae were offered to nematodes contained in 9-cm petri dishes to get infected following the previously described procedure. After 2 days, larvae/pupae were carefully washed with running tap water for 1 minute and allowed to air dry. The larvae/pupae were weighed and placed each one in a separate 5 ml centrifugal test tube (a modified white trap). A small piece of No. 1 Whatman filter paper (0.75 x 5 cm) was placed in a test tube and 4 ml tap water was added. The larvae/ pupae were carefully placed on the upper end of the filter paper to prevent direct contact with water. The tubes were sealed with para film and incubated at 20C. After three weeks the filter paper was carefully removed and the water along with the emerged J3 was pipetted in the petri dish and counted under the stereoscope. The nematode species evaluated were H. bacterophora, S. feltiae, S. krausseri, S. glasarie, H. indica and S. scapterisci. There were 10 replications for each EPN species. Experiment-3: Lethal Concentration Studies To calculate the LC50 and perform probit analysis of two promising EPN species i.e. S. feltiae and H. bacterophora, five concentrations of each EPN were prepared by assessing the stock culture for its strength and diluting it accordingly. The concentrations tested were 1600, 800, 400, 200 rd and 100 J3/ml (3 juvenile stage of nematodes per ml of water) and tap water (control). The bioassay was performed in lab in 9-cm petri dish lined with Watman 1 filter paper. One ml of each concentration was poured in each petri dish with the help of pipette and allowed for 5 minutes to equally distribute the nematodes in petri dishes. Ten full fed active fruit fly larvae collected from the stock culture were released in each petri dish and sealed with Para film to secure moisture therein. Each treatment was replicated five times. The petri dishes were placed in incubator at 20C for 5 days. Data were recorded on fly emergence, nonemerging flies were considered infected and expressed in percent infection (Lindegren et al., 1990). Statistical analysis Probit analysis was performed to the data generated in the experiment where appropriate. All statistical analysis was done using MSTATC (Michigan State University, MI, USA) computer software.

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RESULTS Experiment-1: Daily Progeny Production All nematodes started producing J3 from day-11 (Figs. 13). Maximum (12) J3 were produced by S. carpocapsae followed by S. feltiae (8) and H. bacteriophora (3) on day-1. The increase in J3 was quite rapid until day-13 for S. feltiae, reaching 96 J3 per day; and then dropped afterward. The last J3 produced by S. feltiae was on day-23. Total J3s produced by S. feltiae in the host of fruit fly larvae were 384 in 12 days (Fig. 1). In case of H. bacteriophora, the progeny build-up gained its two peaks, first on day-14 where it produced 107 J3s, and second peak was attained on day-18 with 125 J3s per day. The nematode th was kept emerging up to 26 post infective day and produced 697 J3s in 16 days (Fig. 2). The nematode S. carpocapsae produced its first batch of J3 on 11th post infective day and had its peak on day-15 (post infective) 1(15 J3). The J3 production dropped gradually and reached zero on 26 post infective day. The total J3 produced by the S. carpocapsae were 847 in 15 days (Fig. 3). Experiment-2: Total Progeny Production Progeny development of three species of entomopathogenic nematodes are presented in Fig. 4. The data revealed that S. carpocapsae produced maximum number of J3 (847) from the fruit fly larvae followed by H. bacterophora (697). S. feltaie with 384 J3 proved to be the least producer among the studied nematodes with a maximum and minimum range of 383 to 1039 per larvae. Maximum and minimum progeny production range for S. carpocapsae and H. bactrophora was from 456 to 3622 and 1108 to 1635 nematodes from single fruit fly larvae. Looking at the progeny production from a unit body weight of fruit fly larvae (mg), it was noted that S. carpocapsae produced the maximum number of J3 (784), followed by H. bacterophora with J3 580 per milligram of fruit fly larvae. S. feltiae with 221 J3/mg of body weight proved to be the least J3 producer among the studied EPN. Experiment-3: Lethal Concentration Studies The fruit fly mortality data plotted against the nematode concentrations showed a significant and positive correlation between two factors. The concentration explained 87% of the mortality in the current studies, whereas, the calculated LC50 for S. feltiae was 516 J3 per ml (Fig. 5). The percent infection of fruit fly larvae by six different concentrations of H. bacterophora J3 is presented in Fig. 6. The coefficient of correlation (r2) revealed a positive and significant relation of percent infection and rises in EPN concentration and proved that 93% of variation in fruit fly larvae was due to EPN concentration. Probit analysis depict 600 J3 per ml is the lethal concentration for 50% mortality of fruit flies population.

DISCUSSION Many authors have assessed the progeny production of EPNs with varying host range and other factors, Elwad et al. (2001) reported 234,000 dauer juveniles (DJs) of Steinernema abbasi emerged from wax moth, 220000 from Helicoverpa virescens and 166000 from Spodoptera exiga. The higher number of J3 was most likely due to the comparative difference in host species than the size of host insect used in two studies. It was supported by the data presented in the studies of Elwad et al. (2001) where larger sized or heavier insects host could not produce as many DJs as produced by the wax moth larvae. It is presumed that not only just the food source, which inhibit the progeny development in the insect host body, but, probably, the nutritional status of the hoemocle that arrested the development and production of nematode and vice versa. The IJs emergence started on 4th day reaching to its peak on 5th or 6th post infection day, which was quite a shorter period than what observed in the present studies for the same genus. Unlu and Ozer (2003) reported that Steinernema feltiae took 9 days after death to emergence from the cadaver (wax moth) and continued for 10 days. This was quite close to observations in the present experiment (11 days for S. feltiae), in case of H. bacteriophora, emergence started on day-7 after the death of the insect while contradictory to that it was 11 days in present studies. Average number of J3 produced by host insect (wax moth larvae) were 13829 for S. feltiae and 141562 for H. bacteriophora, of 200 mg of host, comparing to that of 284, 887 and 697 for S. feltiae, S. carpocapsae and H. bacteriophora, respectively, from a fruit fly larvae with an average weight of 1.3 mg. Though Unlu and Ozer (2003) reported number of J3 produce is a function of average body weight of host insect, however, other factors like host species, number of nematodes entered into the host body and the potential of the nematode species were indicated as well. The overall reproduction potential of the EPN greatly influenced by the isolates, species, host susceptibility, number of bacteria present per infective juvenile, invasion rate and other environmental factors (Unlu and Ozer, 2003). The lethal concentration of 516 and 600 J3 /ml is very close to what other workers reported for varying fruit flies species. (Attalla et al., 2002, Koppler et al., 2003, Koppler et al., 2004 and Gazit et al., 2000) With the background data of the soil persistence of EPN, such bioassay might give a grey idea how effective the bio control program would be and interval between two EPN applications can be estimated. This ensures sustainability with varying host insects available in different time interval and population in the specific agro eco system (Yee and Lacey, 2003).

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Acknowledgments Higher Education Commission of Pakistan is cordially acknowledged for providing funds to conduct these studies at University of Reading, Reading, UK. Fig. 1: Daily progeny emergence of S. feltiae from the fruit fly larvae

Fig. 4: Progeny development of different EPN from the fruit fly larvae

Fig. 5: Probit analysis for LC50 of S. feltaiae for fruit fly larvae

Fig. 2: Daily progeny emergence of H. actrophora from the fruit fly larvae

Fig. 6: Probit analysis for LC50 of H. bacterophora for fruit fly larvae Fig. 3: Daily progeny emergence of S. arpocapsai from the fruit fly larvae

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Steinernema feltiae in field tests in Hawaii. Environ. Entomol. 19 (2): 383-386. Poinar, G. O. Jr. 1989. Non-insect hosts for entomogenous rhabditoides Neoaplectana (Steinernematidae) and Heterorhabditts (Heterhabdittdae) Revue. Nematol. 12 (4): 423428. Shapiro-Ilan, D. I., R. Gaugler, W. L. Tedders, I. Brown and E. E. Lewis. 2002. Optimization of inoculation for in vivo production of Entomopathogenic Nematodes. J. Nematl. 34 (4): 343 350. Unlu, I. O. and N. Ozer. 2003. Evaluation of the reproduction potential and competition between two entomopathogenic nematodes Steinernema feltiae Filipjev. 1934 (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora, Poinar 1976 (Rhabditida: Heterorhabditidae). Turk J. Biol. 27: 149-155. Yee, W. L. and L. A. Lacey. 2003. Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes. Biological Control. 27: 349-356

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