Characterization of Midgut and Salivary Gland Proteins of Hyalomma
dromedarii Females Controlled by Entomopathogenic Nematodes
Hanan A. El-Sadawy,
Biological control of hard ticks, Hyalomma dromedarii
(Acari: Ixodidae) using entomopathogenic nematode of two families;
Heterorhabditidae and Steinernematidae was studied. The protective effect
of controlled ticks including haemolymph and haemocytes against these
biological agents were also investigated. It was found that heterorhabditid
strains cause a higher effect in biological control of engorged female
H. dromedarii than those of stienernematid strains. It induced
mortality rates ranged from 12-92% versus 4-88% for stienernematid strains.
It was also found that these entomopathogenic nematodes can not reproduce
within the exposed ticks. SDS-PAGE of proteins extracted from midguts
and salivary glands infected with 4000 IJs tick-1 separated
21 and 25 protein bands versus 13 and 19 protein bands from non-infected
ones, respectively. It was concluded that entomopathogenic nematodes of
family Heterorhabditidae proved to have a potential acaricidal effect
in the control of hard ticks. Moreover, the controlled ticks released
unknown proteins in their haemolymph that may promote the haemocytes to
phagocyte the nematodes as a type of defense mechanism.
Ticks are important acarines infesting most farm animals
and poultry. Its obligatory blood sucker arthropods causing severe economic
losses through blood feeding and destructing of skin which led to decrease
in milk, meat yields and egg production. Moreover, the ticks are considered
an important vector for transmission of many viral, bacterial, rickettesial
and parasitical pathogens such as Anaplasma sp., Babesia sp.
(Zayed, 2004). Ticks control has been affected primarily by application
of chemicals which often produce undesirable effects. Acaricids require
stringent application regimes where ticks develop resistance to them.
The residual chemicals often cause environmental pollution, which may
cause toxic reactions in domestic animals and the expense of this labor-intensive
control method is enormous (Norval et al., 1992). Biological control
has become a promising alternative for controlling ticks, since it minimizes
the problems caused by chemical control and moreover, solves the problem
of Acaricids resistance.
Entomopathogenic nematodes of the families Steinernematidae
and Heterorhabditidae have been used for biological control of several
important insects of plant (Akhurst, 1983). Their pathogenicity depends
in large part on a mutualistic nematode-bacterial complex (With Xenorhabdus
and Photorhabdus, respectively) (Gaugler, 2002). Recent
studies have shown that engorged females of some tick species are susceptible
to infection with entomopathogenic nematodes (Samish and Glazer, 2001).
The aim of study is to define the nematode`s recommendation
dose which can be used for ticks biocontrol and characterization of midgut
and salivary gland proteins of infected H. dromedarii females with
MATERIALS AND METHODS
Collection of ticks: One thousand
engorged females of Hyalomma dromedarii (Acari: Ixodidae) were
collected from sandy soil of resting house of naturally infested camels
at Berkash market, Giza province, Egypt during summer of 2005. The ticks
were identified according to description of Hoogstraal and Kaiser (1959).
Entomopathogenic nematode culture: Infective
third stage juveniles (IJs) of eight different species of entomopathogenic
nematode belonging to Heterorhabditidae and Steinernematidae families
were maintained and at Parasitology and Animal Diseases Department, National
Research Center which cultured in the last instar larvae of Galleria
mellonella (L.) according to the method of Dutky et al. (1964).
Steinernematid nematodes species were Steinernema carpocapsae DD136
(Sc)., S. agriotes (Sa), S. riobrave (Sr), S. carpocapsae
All. Heterorhabditid nematodes species were Heterorhabditis bacteriophora
HP88, H. bacteriophora (Eg1), H. bacteriophora (TWF)
and H. indicus (RM1). The strains of Eg1, TWF and RM1 are of
Experimental design: One thousand of healthy engorged females were first put
individually in plastic pots (25 cm in diameter 2 cm in height) containing
10 g of clean, moistened and sieved sand (Kocan et al., 1998).
These pots containing ticks were divided into 8 groups, each containing
125 pots. Each group was subdivided into 5 subgroups, each containing
25 pots. Each subgroup was divided into 5 replicates, each with 5 pots.
The replicates of group 1 to group 2 were infected with infective juveniles
of S. carpocapsae DD136 (Sc)., S. agriotes (Sa),
S. riobrave (Sr), S. carpocapsae All, H. bacteriophora HP88,
H. bacteriophora (Eg1), H. bacteriophora (TWF) and H.
indicus (RM1), respectively at concentrations of 4000, 2000, 1000,
500 and 250 IJs which previously suspended in 1.5 mL tap water. The infection
was applied by introducing the suspended infective juveniles on the ticks
and moistened sand. After the infection, the pots were covered tightly
with plastic lid and incubated at 25°C and 20% RH. A control replicate
was treated with 1.5 mL of tap water only was also incubated at the same
conditions. Mortality rates of tick were recorded at 2, 4 and 6 days post
Collection of tick haemolymph: Fifty engorged female of ticks H. dromedarii were
infected with 4000 IJs tick-1 of H. indicus RM1. The
cuticles of the tested ticks were first cleaned with slightly moistened
cotton. The haemolymph of ticks were collected using capillary tube with
buffered anticoagulant (pH, 7.2) by made an incision of 2-3 mm long in
the dorsum just posterior to the scutum (Roberta, 1970). The collected
haemolymph was immediately examined using phase contrast microscope. Haemolymph
control sample were collected from 20 non infected semi-engorged females
Collection of salivary glands and midguts: Salivary glands and
midguts were separated from semifed H. dromedarii females collected
from naturally infected camels as a method described by Purnell and Jouyner
(1968). Female tick was first fixed at ventral surface in a wax-filled
Petri dish and covered by thin layer of cold normal saline. Dorsal integument
of the tick was then carefully removed under dissecting microscope using
a fine sharp scalpel and finally the salivary gland and the midguts were
Preparation of salivary glands and midguts antigens: Thirty salivary glands and 10 midguts were separately
homogenized in 1 mL of PBS (pH 7.2 in an ice bath). Suspensions of both
salivary glands and midguts were centrifuged at 3000 rpm for 15 min at
4°C. The supernatant of each suspension was harvested as antigen and stored
at -30°C until used.
Sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE):
Haemolymph of infected (2 days PI) and non-infected H. dromedarii
were electrophorsed by SDS-PAGE using a dis continuous gel system of Laemmli
(1970). After staking, separating and simultaneously pouring gel, the
comb was inserted by a slop way. The haemolymph antigens (50-100 µg lane-1)
were treated with the reducing buffer 12% SDS containing 0.7 M 2-mercoptoethanol,
5% glycerol and 0.001% bromophenol bleu) in the ration of 1:2. The treated
antigens were immersed in a boiling water bath for 2 min to ensure protein
denaturation. After polymerization of the gel (about 2 h) and removing
of the comb, unstained protein molecular weight marker (Amersham Company,
USA) and the treated antigens were loaded in the wells. A voltage of 100
v. was applied until the bromophenol blue had reached the bottom of the
gel. The gel was then stained with 0.025% commassie blue (0.25 g L-1)
at room temperature, over night. To visualize the protein bands for each
antigen, the gel was washed several times with destaining solution (45%
methanol, 5% glacial acetic acid and 50% distilled water) until the back
ground become completely clear. Finally, the gel was photographed.
Statistical analysis: The data
were subjected to statistical analysis using t-test and F-test (one way
classification least significant differences LSD) according to (Snedercor
and Cochron, 1967). The significance of the main effects was determined
by analysis of variance (ANOVA). The significance of various treatments
was evaluated by Duncan's multiple range test (p< 0.05, 0.01). All analysis
was made using a software package Costat, a product of Cohort Software
Inc. Berkeley, California.
Effect of entomopathologic nematodes in control of engorged
H. dromedarii females ticks (Susceptibility test): The
results indicated that all heterorhabditis and stienernematid strains
were found to be effective in the control of engorged H. dromedarii
females causing variable mortality rates on the exposed ticks (Table
1). These mortality rates were increased as inoculum level and/or
exposure time of applied nematode increased. Heterorhabditis strains showed
to cause higher mortality rates than those of stienernematid strains.
It induced mortality rates varied from 88-92, 52-92, 36-64, 12-52 and
12-36% versus 76-88, 24-68, 8-20, 8-16 and 4-16% for stienernematid strains
at inoculum levels of 4000, 2000, 1000, 500 and 250 IJs tick-1,
respectively. Moreover, LD50 (460-1053) indicated also that
heterorhabditis strains induce the highest effect as biologic agents especially
H. indicus (RM1) in the control of this species of hard tick.
Entomopathologic nematodes infection of haemolymph and the mechanism
of tick protection response against it: Phase contrast microscopy
revealed that H. indicus RM1 larvae were first observed to penetrate
H. dromedarii at 24 h post-exposure and remained inside it throughout
the whole experiment (Fig. 1). The greatest numbers
of nematode were observed in ticks haemolymph collected at 4 days post-exposure.
Aggregations of nematode larvae were showed engulfed and encapsulated
in phagocytic cells of haemolymph as a mean of protection. As a result
of encapsulation, the nematodes can not reproduce inside the infected
ticks as it occur in other insect larvae. Therefore, they did not emerge
Phase contrast micrographs
of Hyalomma dromedarii haemolymph infected with Heterorhabditis
indicus (IJs): A and B; Non infected haemocytes (Pr = Prohemocyte,
Sp = Spherul cell, Pl = Plasmatocyte), C; The Capsule of haemocytes
(He) around the nematode (Ne), D; The encapsulated bacteria,
Photorhabdus lummences (Ba) within haemocytes; Dc, dividing
Effect of various nematode
strains at different concentrations upon the lethal dose of
50 (LD50) of engorged H. dromedarii females
after 6 days of exposure
indicus (native nematode), HP88= H. bacteriophora
(imported nematode), TWF = H. bacteriophora (native nematode),
Eg1 = H. bacteriophora (native nematode), All = Steinernema
carpocapsae All (imported nematode), Sa = S. agriotes
(imported nematode), Sc = S. carpocapsae DD136 (imported
nematode), Sr = S. riobravae (imported nematode), A,
B, C, D: Means significant between different doses for eight
strains, a,b,c,d: Means significant between different doses
for each strain, *: Highly significant p>0.05 non significant
Electrophoretic salivary gland
profiles of female H. dromedarii infected with H.
bacteriophora HP88 by using Gel-Pro-Analyzer
MW: Molecular weight, kDa: Kilo Dalton Conc.:
Concentration, *: Shared protein bands
nematode exposed ticks. Moreover, Symbiotic bacteria of
nematodes were observed in tick haemolymph at 24 h post-exposure. The
bacteria were first observed in the haemolymph and later in the degenerated
tissues of all nematode exposed ticks. Cell phagocytosis and encapsulation
were also observed around the symbiotic bacteria in tick haemolymphs.
A clear space was often shown separating the nematodes from the surrounding
tissues and bacteria. Nematode guts were filled with these bacteria that
presumably served as a source of food for the nematodes.
Characterization of midgut and salivary gland proteins of H. dromedarii
Salivary glands: SDS-PAGE of proteins extracted from
salivary gland homogenates infected with 4000 IJs tick-1 of
H. dromedarii females (Fig. 2) detected 25 protein
bands (Table 2). The molecular weights of these bands
ranged from 3-241 kDa. However, it was detected only 19 protein bands
in non-infected salivary glands with molecular weights ranged also from
3-241 kDa. Twelve shared common protein bands were demonstrated between
infected and non-infected salivary glands. The molecular weights of these
shared bands were of 241, 78, 61, 55, 52, 46, 35, 32, 17, 5, 4 and 3 kDa
of female H. dromedarii
salivary glands proteins; Lane
M: Molecular weight markers, Lane 1: Non infected salivary glands,
Lane 2: Infected salivary glands with entomopathogenic nematodes
of female H. dromedarii
midguts proteins; Lane M: Molecular
weight markers, Lane 1: Non infected midguts, Lane 2: Infected
midguts with entomopathogenic nematodes
Midgut: SDS-PAGE of proteins extracted from midgut homogenates
infected with 4000 Ijs tick-1 of H. dromedarii females
(Fig. 3) detected 21 protein bands versus 13 protein
bands in the non-infected guts (Table 3). These protein
bands of infected midguts exhibited great variations in their molecular
weights which ranged from 4-233 kDa versus 3-250 kDa in non-infected
Electrophoretic midgut protein profiles of female
H. dromedarii infected with H. bacteriophora HP88
(4000 IJs tick-1) by using Gel-Pro-Analyzer
kDa: Kilo Dalton, MW: Molecular
weight, *: Shared Protein bands, Conc.: Concentration
ones. Five shared common protein bands with molecular weights
of 48, 41, 35, 33 and 26 kDa were detected between the infected and non-infected
guts (Table 3).
This study found that the engorged females of H. dromedarii
were highly susceptible to infection by heterorhabditid nematodes
than those belonging to steinernematid. Mortality rates of ticks were
proportional to either the dose or exposure time of applied nematodes.
The four tested heterorhabditis strains; H. bacteriophora HP88,
H. indicus RM1, H. bacteriophora Eg1 and H. bacteriophora
TWF were more virulent than Steinernematid strains; S. carpocapsae
DD136, S. riobravae, S. carpocapsae all and S. agriotes.
These results were evident in the higher mortality rates recorded after
increasing exposure times and dosages of nematodes. These findings were
in agreement with other studies reported by Hassanin et al. (1997),
Kocan et al. (1998), El Sadawy and Habeeb (1998) and Glazer et
al. (2001). The death of ticks was attributed to the fact that infective
juveniles of nematodes carry a specific bacteria in their intestine. After
the invasion of these juveniles into the tick body, they release its bacteria
(Photorhabdus sp. for heterorhabditid and Xenorhabdus sp.
for steinernematids) which multiply rapidly releasing large amount of
protolytic enzymes that hydrolyses the tick protein and subsequently decreases
the resistance of the tick to the bacteria causing death (Kocan et
al., 1998; Samish et al., 2000; Vasconcelos et al.,
As reported by Samish and Glazer (1992), Glazer et al.
(1991), Hassanin et al. (1997), Kocan et al. (1998) and
present study found that the applied nematodes did not multiply in ticks
and consequently not complete their life cycle inside the infected ticks.
Therefore, there were no subsequent generations of infective juveniles
releasing from the cadaver searching to infect new hosts. The most rapid
mortality of engorged females H. dromedarii was obtained with infective
juveniles of H. bacteriophora HP88, H. indicus RM1, H.
bacteriophora TWF and H. bacteriophora Eg1. This finding might
be attributed to the ability of heterorhabditid infective juveniles to
penetrate through soft cuticles and thin membranes with the help of a
cuticular tooth in their head region. These results were in accordance
with the previous study of different tick models by Georgis (1990), Samish
and Glazer (1992) and El Sadawy and Habeeb (1998).
Previous studies on ticks were also demonstrated that there
were marked differences in the virulence of various applied nematode species
when used as a biological mean against A. americanum, I. scapularis,
B. annulatus, H. excavatum and R. bursa (Samish and Glazer,
1992; Glazer and Samish, 1993; Mauleon et al., 1993; Zhioua et
al., 1995; Kocan et al., 1998; Samish et al., 1999,
2000). It was found that the strains of nematodes species were also varied
greatly in their effectiveness against insects (Coroli et al.,
1996; Ricci et al., 1996). Therefore, the factors influencing the
mortality rates of ticks were probably included; dose and penetration
rate of nematodes in the host haemocoel (Glazer, 1992; Coroli et al.,
1996; Ricci et al., 1996), proliferation rate of nematodes within
the host (Samish et al., 2000) and finally efficiency of the ticks
to protect itself against the nematode symbiotic bacteria and their deleterious
secretions (Wang and Bedding, 1996).
The difference in nematode virulence is often attributed
to the forgoing strategy of Lewis et al. (1992) and Campbell and
Gauglar (1993). However, in the present study, both nematodes and ticks
were placed in a close proximity so that the need to seek the target host
was eliminated. Therefore, it may be speculated that penetration rate
of nematodes and perhaps both rate of development and virulence of the
symbiotic bacteria within the ticks played a major role in the nematode-tick
interaction. These observations similar to that reported for Egyptian
cotton leaf worm by Glazer et al. (1991).
The present study found that a high tick mortality at comparatively
low concentration of heterorhabditid strains indicate high susceptibility
to H. dromedarii and their developmental stages to a particular
nematode strain. These findings further imply that the high mortality
rates may be related not only to the concentration of nematodes, but also
to other factors. Therefore, it was noteworthy that a complete mortality
was recorded in the susceptible insects with 24-48 h of exposure at the
same concentrations used in this study (Coroli et al., 1996). Moreover,
our results revealed complete mortality of H. dromadarii females
after 6 days. This delay may be attributed to that the ticks exhibited
suboptimal conditions for the nematodes or symbiotic bacteria or both.
This conclusion may similar to the fact that all studied tick species
have not supported a full nematode life cycle (Glazer and Samish, 1993;
Zhioua et al., 1995; Samish et al., 2000).
In general, the susceptibility of the different tick species
studied here was far less affected by their particular species than by
their developmental stages. However, very pronounced differences were
demonstrated between engorged females of several other tick species in
terms of susceptibility to nematodes (Mauleon et al., 1993; Samish
et al., 1996; Kocan et al., 1998). The preimaginal stages
of ticks are evidently far less susceptible to nematodes than are adults.
Ixodes scapularis larvae and nymphs are totally resistant to nematode
strains which killed the former's engorged females (Zhioua et al.,
1995; Hill, 1998). These differences may be attributed to anatomical differences.
Nematodes are known to enter insects via natural openings; anus, spiracles,
mouth and genital apertures (Peters and Ehlers, 1994). Moreover, nematodes
also are able to penetrate the ticks via thin areas of insect cuticle
(Bedding and Molyneux, 1982). The relation between the penetration sites
of the various nematodes and their efficiency in killing insects has been
demonstrated (Glazer et al., 1991; Glazer, 1992). It was found
that when the nematodes placed near ticks, it concentrate in the vicinity
around their mouth, spiracles, anus and genital apertures (Kocan et
al., 1998; Samish et al., 1999).
Results obtained by phase contrast microscopy clearly demonstrated
that the nematodes penetrated engorged female H. dromedarii. Moreover,
the released bacteria, Photorhabdus or Xenorhabdus were
proliferated within the tick cadaver which showed to be the cause of tick
mortality. Similar observations confirming these results were obtained
by Samish and Glazer (1991), Kocan et al. (1998) and Samish et
al. (2000). Therefore, it should be noted that the symbiotic bacterium
that was released upon invasion into the haemolymph had a vital role in
the pathogenic process.
Penetration route of entomopathogenic nematodes into ticks
is still controversial. Samish and Glazer (1991) reported that nematodes
entered B. annulatus via the mouth parts. However, for the same
tick species, B. annulatus, genital opening was also suggested
as another route of penetration (Mauleon et al., 1993). Similarly
in insects, stienernematids IJs used oral and anal openings as penetration
routes (Poinar, 1979). Furthermore, spiracles were considered as an alternative
routes using dorsal labial tooth (Triggiani and Poinar, 1976). Heterorhabditis
nematodes were suggested to penetrate the insect hemocoel through soft
intersegmental areas of the cuticle (Bedding and Molyneux, 1982).
Present results demonstrated that the integumental thickness
of the ticks were suggested to be another factor affecting the entrance
of applied nematodes. The integument is thicker in late instars (adult
males and females) than early ones (larvae and nymphs) where it increases
after each molt. Therefore, size of natural openings was subsequently
affected. It becomes narrower in late instars which hinders the entrance
of applied nematodes. Moreover, the mechanical stretch of the cuticle
in engorged ticks may affect the size of some natural openings. Therefore,
it should be taken into account that large meals cause dramatic internal
changes, integument thickness and cuticle plasticity (Triggiani and Poinar,
1976; Bedding and Molyneux, 1982; Kocan et al., 1998).
The present study focused on the time elapsed between application
of nematodes and death of ticks as an important factor not only in terms
of host-parasite interaction but also it indicates whether, or/and how
long engorged female tick still could lay eggs after they drop off onto
nematode infested soil. Moreover, it could also determine if, or how long,
or both, various unfed ticks could serve as a vector of medical disease
agents in nematode infested soil.
SDS-PAGE results of midguts and salivary glands of applied
ticks showed a remarkable increase in protein bands if compared with non-infected
control ticks. It reached 25 and 21 bands in infected salivary glands
and midguts versus 19 and 13 bands in non-infected ones, respectively.
These findings agreed with those reported by Saad and Kamal (1994), Hernandez
et al. (1995) and Abdel Wahab et al. (1999). They showed
that the gut and salivary gland SDS- dissociated proteins of B. annulatus
and R. sanguineus were separated into 15 and 16 bands respectively.
The protein bands post infection with nematode showed presence of additional
bands. This may be attributed to the capability of the ticks to release
a protein as a type of defense against applied nematodes. Similar changes
were described post-injection of Parasarcophage surcoufi with H.
bacteriophora HP88. All of the isolated protein bands were replaced
by new ones referred as an immune protein (Ayaad et al., 2001;
El-Kady et al., 2005).
The obtained results demonstrated also absence of some bands
in infected midguts and salivary glands if compared with non-infected
ticks. This disappearance of some bands may be interpreted to that the
applied nematodes secrete toxic materials and proteolytic enzymes which
hydrolyse protein of the tick. This run in full agreement with Poinar
(1979), El Sadawy (1994) and Boemare (2002). They reported that the infective
juvenile penetrates insect host through natural body openings such as
the mouth and spiracles. Once inside the host, species-specific, symbiotic
bacteria (Xenorhabdus sp. for Steinernema sp. and Photorhabdus
sp. for Heterorhabditis sp.) are released from the gut into the
insect body cavity causing a bacterial septicemia. In response, the combination
of nematode/bacterial virulence factors kills the host within 24-48 h.
Furthermore, the findings of the present study demonstrated
the presence of 5 common shared protein bands between infected and non-infected
midgut. Also, there were 10 common shared protein bands between infected
and non-infected salivary glands. These shared bands can be considered
as the specific protein bands of midgut and salivary gland. This is in
agreement with that reported by Hernandez et al. (1995) who studied
SDS-PAGE proteins of eggs, larvae, nymphs, male and female salivary glands
and midgut extracts of R. sanguineus. A common band of molecular
weight higher than 250 KDa was observed, although with different intensity
(Hernandez et al., 1995).
In conclusion, entomopathogenic nematodes, belonging to
the families Steinernematidae and Heterorhabditidae, are complex nematode-bacteria
with a broad insect-host range. The effectiveness of these biological
agents in killing the parasites was mainly ascribed to the septicemia
induced by massive release of symbiotic bacteria, in the latter stage
of the infection (Samish and Glazer, 2001; Brivio et al., 2004).
The insect-nematode system studied by Brivio et al. (2004) provided
unique opportunities for studying the interactions of insect immunity
with immune-suppressive nematodes. In particular, the immunoevasion mechanisms
involving parasite body surface through which they are able to escape
host immune recognition (You, 2005; Imamura et al., 2005; Dinglasan
et al., 2005).
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