Biochemical Fingerprints of Egg and Salivary Gland Proteins

Characterizing Four Common Tick Genera in Egypt

(Received: 07.11.1999)

Kawther M. EL Kammah* and Makram A. Sayed**

*
Zoology Department, Faculty of Agriculture, Cairo Univ.
**
Plant Protection Dept. Fayoum Fac. of Agric., Cairo Univ.

ABSTRACT

Total protein values and estimated number of bands extracted from eggs and salivary
glands of the Egyptian tick genera; Argas, Hyalomma, Boophilus, and Rhipicephalus were
estimated using polyacrylamide gel electrophoresis technique (PAGE). The silver stain technique
was modified by the second author (Table 1).
Total protein values in eggs were 479.04 ± 8.3, 267.44± 10, 241.44± 8.2 and 750.3± 15.1
mg/g and in salivary glands were 16.5±1.3; 35.8± 2.0 ;29.0±0.4, and 19.6 ± 0.0mg/g isolated from
the genera A., Hy., B., and Rhip., respectively.
A taxonomic key is givin to differentiate between these genera based on the differences of
protein molecular weights, presence or absence of protein bands, and their relative flow values.
The presented biochemical fingerprints indicated that each genus is characterized by:
Eggs:
Argas: the presence of bands 2,6 and the absence of bands 11 to 15
Hyalomma: the presence of band 13; and the absence of 8 to 11
Boophilus: the presence of band 11; and the absence of 7and 13 to 15
Rhipicephalus: the presence of bands 14,15; and the absence of 3 and 4

Salivary gland extract:

Argas: the presence of bands 4,20,22 and the absence of band 7
Hyalomma: the absence of band 12.
Boophilus: the presence of bands 1,8,9,24; and the absence of 6
picephalus: the presence of bands 26,27; and the absence of 3, 10,18, (and as a group
18 to 25)

Rf and molecular weight values are given for each protien band (Tables 2,3 and Figs 1,2)

Key words: Argas, Hyalomma, Boophilus, and Rhipicephalus, Biochemical fingerprints.

Arab J. Biotech., Vol.2, No.(2)Dec. (1999): 127-134.


INTRODUCTION
T
he identification of tick species has
always bean based on
morphological characters of the
mouthparts and adjacent structures.
These parts may become damaged during
removal of a tick from its host. Recently,
protein electrophoresis and molecular genetic
research enable taxonomists to differentiate
between the genera and/or species.Poucher et
al (1999). It was found that the DNA sequence
of the mitochondrially enclosed 16s RNA gene
from the 10 Ixodes species and one
Dermacentor sp. show identifiable differences
between the species (Caporale et. al., 1995)
Image analysis is a system, which facilitates
the analysis so that numerous routine
measurements can be carried out on one
specimen (Heyne and McKay, 1998)
It is our goal to present a key to
identify the most economic important tick
genera in Egypt based on biochemical
fingerprinting of the genera Argas, Hyalomma,
Boophilus and Rhipicephalus.The tick species
used are the chicken tick Argas persicus , the
camel tick Hyalomma excavatum, the cattle
tick Boophilus annulatus, and the dog tick
Rhipicephalus sanguineus.
MATERIALS AND METHODS
Mature stages of Argas and Hyalomma
were obtained from Laboratory colonies in
“Animal Acarin Research Center”, Faculty of
Agriculture, Cairo University, Giza. They
were reared on chicken and rabbits,
respectively, and incubated under a constant
temperature of 28°C and RH 70%. Boophilus
adults were collected from the Experimental
Station, Faculty of Agriculture, Cairo
University. Rhipicephalus adults were
collected from animals in veterinary medical
praxis.
Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.
Kawther M. EL Kammah and Makram A. Sayed
Egg extract
One to two days old eggs from the
above mentioned genera were collected and
homogenized in cooled saline solution
followed by centrifugation at 10000 rpm for
15 min. The supernatants were collected and
stored under -40°C until used.
Salivary gland extract
Salivary glands of fed females were
dissected in cold sodium chloride (0.9%),
homogenized in the same solution, and
centrifuged at 10000 rpm for 15 min. The
supernatant was collected and kept under -
40°C.
Protein content and electrophoretic
technique
The extracted total protein content was
determined by using the Lowery-method
(Lowery et al., 1951). Preparations of 10%
SDS-Polyacrylamide gel were made using the
methods described by (Hames ,1987). Equal
amounts from the eggs and salivary glands
protein were mixed with the sample buffer at a
ratio 1:1 and incubated for 5 min in a water
bath at 100°C. Each sample was loaded in a
separate well of the gel and the electrophoresis
run was carried out at 100V for 1h, followed
by 200V for 3h. After the running time, the
protein bands were visualized by using the
silver stain technique following the protocol
shown in Table (1). (This protocol was the
solitar idea of the seconed author ).
Phoretix 1D image analysis system
(Phoretix International, London) was used to
integrate the data of the protein bands.
Five protein moleculor weight
standards were used as MW markers (Serva,
Catalogue no. 39064), consisted of Aldolase
rabbit (160000D), Albumine egg
(45000D),Myoglobin equine (17800D),
Cytochrome C (12400 D), and DNP-L- alanine
(255D).
 Biochemical Fingerprints of Four Tick Genera in Egypt

Table (1): Silver stain protocol, used to visualize the protein bands.
Solution Incub. Period
FIX Solution I: 1 x 30 min
150 ml Ethanol, 17.1 g Sulfosalysilic acid, 285 ml TCA and 285
ml, then add 500 ml deionised water
Washing Solution: 2 x 10 min
60 ml Ethanol, add 1000 ml water
FIX Solution II: 2 x 10 min
40 ml Glutraldhyde 25%, 60 ml deionizedwater
Washing Solution 2 x 10 min
Rinsing with deionized water 3 x 10 min
Staining Solution: 1 x 30 min
1 g Silver nitrate, 3.75 ml Ammonium hydroxide, 31.5 ml 0.1 N
Sodium hydroxide, add 250 ml water.
The components are added in the same sequence
Rinsing with deionized water 1 x 5 min
Developing solution: Until
10 ml citric acid 0.05%, 100 µl Formaldhyde 37 – 40%, add. 100 visualized
ml deionized water good bands
Stop solution: 1 x 5 min
10 ml acetic acid, add. 100 ml deionized water
Preservative solution:
250 ml ethanol, 80 ml acetic acid, 80 ml glycrin, add 1000ml
deionized water
Immerse the gel and a piece of cellophane sheet in the 1 x 15 min
preservative solution
Keep the gel in the cellophane sheet and let it to dry in the air or
by using gel dryer.

RESULTS their molecular weights (Table 2). The protein

Eggs:
The protein content value in eggs was
479.04+8.30, 267.44+ 10.00, 241.44 + 8.20
and 750.3+15.10 mg/g eggs for Argas,
Boophilus, Hyalomma and Rhipicephalus,
respectively.
The electrophoretic pattern of the
proteins extracted from the newly oviposited
eggs from the genera under investigation
showed significant differences in the number
of the protein bands, their relative flows, and
band number was 10, 7, 8, and 6 for A., Hy., B.
and Rhip., respectively. The presence of the
bands with Rf values 0.11 and 0.25 (Molecular
weights 104811 and 82501Dalton) and the
absence of the bands with relative flow Rf
0.46, 0.48, 0.50, 0.76 and 0.84 are
characteristic for the genus Argas. The
absence of the band Rf 0.27 (MW 78051D)
and the presence of the band Rf 0.5 (MW
43462D) are distinguishable for the genus
Hyalomma. The disappearance of the protein
band Rf 0.26 (81094) and the presence of the

Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.

 Kawther M. EL Kammah and Makram A. Sayed

protein band Rf 0.46 (MW 48754) are the Rhipicephalus eggs, but it is the only one
characteristic for the Boophilus. The protein which has the protein bands with Rf 0.76 and
bands with Rf 0.16 and 0.19 (MW 99085 and 0.84 and molecular weights 5356 and 1200,
92524D) did not exist in the protein pattern of respectively,(Table,2,Fig1.).

Table (2): Relative flow and approximate molecular weights of the protein bands of Argas,
Hyalomma, Boophilus and Rhipicephalus (eggs).
Band No. Relative flow Molecular weight
Rf Dalton
Marker Argas Hyalomma Boophilus Rhipicephalus
Protein
1 0.08 ----- 110216 110216 ----- -----
2 0.11 ----- 104811 ----- ----- -----
3 0.16 ----- 99085 99085 99085 -----
4 0.19 ----- 92524 92524 92524 -----
5 0.21 ----- 88228 88228 88228 88228
6 0.25 ----- 82501 ----- ----- -----
7 0.26 ----- 81094 81094 ----- 81094
8 0.27 ----- 78051 ----- 78051 78051
9 0.30 ----- 72701 ----- 72701 -----
10 0.44 160000 52440 ----- 52440 -----
11 0.46 ----- ----- ----- 48754 -----
12 0.48 ----- ----- 45487 45487 45487
13 0.50 45000 ----- 43462 ----- -----
14 0.76 ----- ----- ----- ----- 5356
15 0.84 ----- ----- ----- ----- 1200

Electrophoretic protien pattern of the genera Argas, Boophilus, Hyalomma and

Rhipicephalus (eggs).

Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.

 Biochemical Fingerprints of Four Tick Genera in Egypt

Salivary glands: absence of the band Rf 0.17 is remarkable for

The absence of the band of the relative
flow 0.19 (MW 92524D) and the presence of
the band of Rf 0.11 (MW 104811D) are
characteristic for the genus Argas. The
Hyalomma genus can be distinguished by the
absence of the band of Rf 0.26 (MW 81094D).
Four kinds of proteins, which have molecular
weights 118111, 90930, 88228 and 30051D
and Rf 0.03, 0.2, 0.21 and 0.57, respectively,
are characteristic for Boophilus., and the
this genus. The electrophoretic pattern of
salivary gland proteins isolated from
Rhipicephalus did not include the bands of Rf
0.08, 0.22 and 0.41 (MW 110216, 86401 and
57239D, respectively). In addition, the
Rhipicephalus protein profile is the only one
which has the bands with molecular weights
16036 and 2356D (Rf 0.66 and 0.79,
respectively),(Table,3andFig.2).

Table (3): Relative flow and approximate molecular weight of the protein bands isolated
from of the genera Argas, Hyalomma, Boophilus and Rhipicephalus (salivary
glands).

Band No. Relative flow Molecular weight

Rf Dalton
Marker Argas Hyalomma Boophilus Rhipicephalus
Protein
1 0.03 ----- ----- ----- 11811 -----
2 0.05 ----- ----- 113887 113887 -----
3 0.08 ----- 110216 110216 110216 -----
4 0.11 ----- 104811 ----- ----- -----
5 0.13 ----- 101951 ----- ----- 101951
6 0.17 ----- 95874 95874 ----- 95874
7 0.19 ----- ----- 92524 92524 92524
8 0.2 ----- ----- ----- 90930 -----
9 0.21 ----- ----- ----- 88228 -----
10 0.22 160000 86401 86401 86401 -----
11 0.24 ----- 83400 ----- 83400 -----
12 0.26 ----- 81094 ----- 81904 81094
13 0.27 45000 78051 ----- 78051 -----
14 0.32 ----- 71350 71350 71350 71350
15 0.34 ----- 67601 ----- 67601 -----
16 0.36 ----- 64077 64077 ----- -----
17 0.38 ----- 61453 ----- 61453 -----
18 0.41 ----- 57239 57239 57239 -----
19 0.44 17800 52440 ----- 52440 -----
20 0.45 ----- 50168 ----- ----- -----
21 0.48 ----- 45487 45487 45487 -----
22 0.51 12400 40072 ----- ----- -----
23 0.55 ----- 34440 34440 34440 -----
24 0.57 ----- ----- ----- 30051 -----
25 0.61 ----- 24342 ----- 24342 -----
26 0.66 ----- ----- ----- ----- 16036
27 0.79 ----- ----- ----- ----- 2356
28 255 ----- ----- ----- -----

Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.

 Kawther M. EL Kammah and Makram A. Sayed

Fig. (2): Electrophoretic protien pattern of the genera Rhipicephalus

Boophilus, Argas, and Hyalomma, (Salivary glands).

Protien bands and molecular weight taxonomic key for the tick genera; Argas, Hyalomma,
Boophilus, and Rhipicephalus Egg and salivery gland.

Eggs:
Presence of bands:
1 2 (Rf 0.11) and 6 (Rf 0.25); MW.104811, 82501------------------------ Argas
2 13 (Rf 0.50); MW 43462---------------------------------------------------- Hyalomma
3 11 (Rf 0.46); MW 48754---------------------------------------------------- Boophilus
4 14 (Rf 0.76), 15 (Rf 0.84); MW 5356, 1200-------------------------------------- Rhipicephalus
---------
Absence of bands:
1a 11 to 15 (Rf 0.46, 0.48, 0.50, 0.76, 0.84, respectively)------------------ Argas
2a. 8 to 11 (Rf 0.27, .030, 0.44, 0.46) ----------------------------------------- Hyalomma
3a. 7 (Rf 0.26) and 13 to 15 (Rf 0.50, 0.76, 0.84, repectively)-------------- Boophilus
4a 3 (Rf 0.16) and 4 (Rf 0.19);--------------------------------------------------------- Rhipicephalus
----------
Salivary gland extract:
Presence of bands:
1b. 4,20,22 (Rf.011, 0.45, 0.51, respectevily); MW 104811,50168, 40072------ Argas
3b. 1,8,9,24 (Rf 0.03, 0.2, 0.21, 0.57, respectively); MW 118111, 90930, 88228, 30051
-------------------------------------------------------------------------------------- Boophilus
4b. 26,27 (Rf 0.66 and 0.79) ; MW 16036, 2356---------------------------------- Rhipicephalus
Absence of bands:
1c. 7 (Rf 0.19) ------------------------------------------------------------------------- Argas
2c. 12 (Rf 0.7) ------------------------------------------------------------------------- Hyalomma
3c. 6 (Rf 0.17) ------------------------------------------------------------------------- Boophilus
4c. 3,10,18 (Rf 0.08, 0.22, 0.44, respectively) ------------------------------------- Rhipicephalus

Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.


DISCUSSION
It is well known that proteins are
synthesized in the cell ribosomes through the
transcription and translation processes.
However, the synthesis and maturation of
some specific proteins may occur in a post
transitional event. In all cases, these processes
are still very specific and generic closely
related to the genetic information. On the other
hand, the translation process and protein
induction express the already stored genetic
information. Thus, we can utilize the
electrophoretic pattern of any subject as a
specialized fingerprint useful in the
classification and evolutionary sudies. Keys
depending on molecular genetic markers were
also used to identify 17 Ixodes, tick species by
Paucher et al (1999) and 10 Ixodes and one
Dermacentor species, by Caporale et. al.,
(1995).
An additional tool for the solution of
the taxonomic problems has been provided by
the detection of some enzymes using
electrophoresis on starch or polyacrylamid gel.
It was also used in the taxonomic studies of
cereal aphids (Loxdale et. al, 1983). Different
Meloidogyne species were also identified on
the basis of isozyme phenotypes (Esbenshade,
1985 & 1990). Although these ticks should be
sacrificed for the identification with these
methods, the numerous oviposted eggs or
offspring could be used with the same methods
for this purpose. It is confirmed that all these
genera had unique specific protein fingerprints
shown by either eggs or salivary galnds
extract. The electrophoretic pattern will form
an anglestone in identifying species and strains
of ticks and mites explained by using the
image analysis system.
Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.
Biochemical Fingerprints of Four Tick Genera in Egypt
ACKNOWLEDGMENT
Research Project (416-B) supported by
the Ministry of Economy and International
Cooperation (Departement of Economic
Cooperation with USA)
REFERENCES
Caporale, D.A., Rich, S.M., Speilman, A.,
Telford, S.R., and Kocher, T.D. (1995).
Discriminating between Ixodes ticks by means of
mitochondrial DNA sequeinces. Molecular-
Phylogenetic and Evolution 4(4): 361-365.
Esbeshade, R.P., and Triantaphyllou, A.C.
(1985). Use of enzyme phenotypes for
identification of Meliodogyne species. Journal of
Nematology 17: 6-20.
Esbeshade, R.P., and Triantaphyllou, A.C.
(1990). Isosyyme phenotypes for the
identification of Meliodogyne species. Journal of
Nematology 22: 10-15.
Hames, B.D. (1987). Gel electrophoresis (eds, B.
D. Hames & D. Rickwood) 6th edition, IRL Press
: 1-86.
Heyne, H. and Mckay, I. J. (1998). Tick
taxmomy : beyond 2000 (From naked eye to
nucleotide). First African Acarology Symposium.
Nov. 1998 :26.
Lowery, O. H.; Rosebrough, N. J.; Farr, A. L.
and Randall, R. J. (1951). Protien measurment
with the Folin Phenol reagent. J. Biol. Chem. 193
: 265-275.
Loxdale, H. D.;Castaner, P. and Brookes, C.
(1983). Electrophoretic study of enzymes from
cereal aphid population. I.
Electrophoretechniques and staining system for
characterising isoenzymes from six species of
cereal aphis (Hemiptera: Aphididae). Bull.
Ent.Res. 73 : 645-657.
Poucher, K. L.; Hutcheson, H. J.; Keirans, J.
E.; Durden, L. A. and Black ,Iv. W. C (1999).
Molecular genetic key for the identification of 17
Ixodes species of the United States (Acari:
Ixodidae) : A methods model. Journal of Parasit.
85 (4) : 623-629.
 Kawther M. EL Kammah and Makram A. Sayed

! ! "#$

– –
– –

# $ % & ' ( ! " !

# # 1 PAGE 0 ) %* ! * +- , ./ - , -, %, %
{ -* 5 " / + } 2 3' 4
#' %( # # : !. +; 5 ! -6 7 89
A * < , & => ? ! * , @
A(
, % ………………………………………………… B C D B +;
…………………………………………………… !E C D +;
, ./ ……………………………………….. !E B C D +;
, ……………………………………………… B C D B +;
A '
, ………………………………………………… C D B B +;
……… ……………………………………………………… C D
, ./ …………………………………………… C D B B B +;
- - C D - +;
, …………………………………………………… 1 !E C DF)
1 - H% 0 Rf 9 & G 2

Arab J. Biotech., Vol.2, No. (2) Dec. (1999): 127-134.