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BIOLOGICAL ACTIVITY OF THE TOXIC PEPTIDES FROM VENOM OF 

BRACON HEBETOR (SAY.) (HYMENOPTERA: BRACONIDAE) 

Atif Manzoor, Zain-ul-Abdin,* Muhammad Arshad, Muhammad Dildar Gogi, Hoor Shaina, Erum Mubarik, 

Saqi Kosar Abbas and M.Ahsan Khan 

1 

Department of Agricultural Entomology, University of Agriculture, 38040 Faisalabad Pakistan 

ARTICLE INFORMATION 

Received: June 13, 2011 

Received in revised form: August 20, 2011 

Accepted: September 30, 2011 

Corresponding Author: 

Zain-ul-Abdin 

Email: zainentomology@yahoo.com 

ABSTRACT 

The physiological effects of the venom of ectophagous larval parasitoid, Bracon hebetor 

(Say.) (Hymenoptera; Braconidae) were studied by performing bioassay of microinjections 

th 

of both heat and protinase K treated venom extract in non-parasitized and synchronized 5 

instar larvae of the host greater wax moth Galleria mellonella (L.) (Lepidoptera: Pyralidae), 

where as Pringle's saline solution injected larvae of the host were acted as control. The major 

effects for artificially envenomated host larvae were observed for their survival and 

mortality. The biological activity of the venom was evaluated by heat and protinase K 

treatments. Results showed a high level of mortality (70%) of the host larvae by the injections 

method of crude venom whereas, heat and protease treated venom injections showed non 

significant mortality of the host, due to protein nature of venom which lost its activity upon 

treatment with heat and protinase K. This basic information serves as a starting point for 

assessing in a more comprehensive way the role that the venom of B.hebetor has in the host 

regulation process. 

Keywords: Bracon hebetor, Galleria melonella , parasitization, venom, microinjections, bioactive 

components, biological activity. 

INTRODUCTION 

term biopesticides describes a large number of control 

techniques, including the application of microbial organisms, 

Reduction in the use of chemical insecticide is one of the key entomophagous nematodes, plants-derived pesticides, 

factors in safe food production which has encouraged secondary metabolites from micro-organisms, insect 

considerable efforts in research for developing pioneering pheromones applied for mating disruption. Genes also are 

technologies based on the use of bio-control agents (Bale et used to increase the resistance of crops to insect, fungal, viral 

al., 2008), toxins of protein or peptides, deriving from insects or herbicide damage (Copping and Menn, 2000). A variety of 

and plant natural antagonists (Whetstone and Hammock, peptides and proteins have been used to produce biopesticides 

2007) and natural insecticides comprising small organic and a list of these compounds, derived from many different 

molecules (Dayan et al., 2009). Since the introduction of sources, has been recently reviewed by Whetstone and 

DDT in the 1940s, insect pests have been controlled almost Hammock (2007). The study of the physiological and 

exclusively with chemical insecticides (Casida and Quistad, molecular mechanisms underlying the antagonistic host- 

1998). Fast acting, cheap to produce, relatively easy to deliver parasitoid associations in insects provides a very interesting, 

and highly potent, chemical insecticides have been viewed and nearly unexploited, opportunity to isolate genes and 

with extreme optimism. Some decades after their molecules with potential insecticide activity. Parasitoids in 

introduction, these compounds showed all the problems the order Hymenoptera show an astonishing variety of 

associated with their use, including poor species specificity regulation strategies of their hosts, characterized by a number 

and development of resistance in target organisms. In of serious pathologies, ranging from severe immune 

response to the environmental hazards that these compounds suppression (Schmidt et al., 2001; Lavine and Strand, 2002) 

cause, in 1970s in many countries DDT and many other to neuroendocrine disorders associated with developmental 

chlorinated insecticides were banned from agricultural use. In and reproductive disruptions (Lawrence and Lanzrein, 1993; ; 

parallel, chemical alternatives were developed and research Pennacchio et al., 2001). Most of these host syndromes are 

was focused on biopesticides (Copping and Menn, 2000). The induced by regulation factors injected by the ovipositing 

Cite this article as: 

Manzoor, A., Zain-ul-Abdin, M. Arshad, M.D. Gogi, H. Shaina, E. Mubarik, S.K. Abbas and M.A. Khan, 2011. Biological activity 

of the toxic peptides from venom of Bracon hebetor (Say.) (Hymenoptera: Braconidae). Pak. Entomol., 33(2): 125-130. 

125

Manzoor et al. / Pakistan Entomologist 2011, 33(2): 125-130 

parasitoid female, such as: venom and polydnaviruses needs, ecological adaptation and developmental 

(PDVs) (Pennacchio and Strand, 2006). 

characteristics. It is used as a host in favor of many 

hymenoptera species (Burges, 1978; Smith, 1965; Haewoon 

The parasitic form of life is very prolific and has evolved in 

et al., 1995; Coskun et al., 2006; Dweck et al., 2010;) The 

copious orders of arthropods. In parasitic insects adults are 

present study was carried out with the aim to evaluate the 

free living and a few numbers of progeny develop per host. 

biological activity of the venom of the wasp. . 

The hosts are not survived during the encounters so many 

parasitic insects are different from other parasitic organisms 

(Vinson, 1975). 

MATERIALS AND METHODS 

Regulation of host by insect parasitoids is a multifaceted 

procedure which consequences in a common physiological 

condition, ultimately increasing the suitability of the host 

Rearing of host parasitoid culture 

(Vinson and Iwantsch, 1981). According to their origin and 

The ectophagous larval parasitic wasp Bracon hebetor (Say.) 

function, there are different categories of host regulation 

(Hymenoptera: Braconidae) was reared under laboratory 

factors. Adult parasitoid females produce maternal origin 

th 

conditions on the mature larvae (5 instar) of greater wax 

factors that are injected at the time of oviposition, while 

moth Galleria mellonella (Lepidoptera: Pyralidae) by 

factors belonging to embryonic origin are obtained from the 

following a slightly modified approach as described by Alserosal 

membrane or from the other tissues associated with 

Tememi, 2005; Khan et al., 2005; Ahmed and Ahmad, 2006. 

embryo. Host physiology is also regulated by the parasitoid 

The adults of the parasitoid, B. hebetor were collected directly 

larvae. 

from fodder Trifolim alexandrium L., located at the campus of 

The host-parasitoid association that was considered in the the University of Agriculture, Faisalabad, Pakistan. The 

present study is Bracon hebetor (Say.) (Hymenoptera: collected parasitoids were identified on the basis of their 

Braconiodae) and Galleria mellonella (L.) (Lepidoptera: morphological characters. This parasitoid has two 

Pyralidae). Bracon hebetor (Say.) is a gregarious, phenotypes, depending on the temperature. During summer 

arrhenotokous, idiobiont, ectoparasitoid wasp, attacking the season, the adults have brownish colour while, during winter 

larvae of lepidopterons (Tuncyurek, 1972; Cline et al., 1984; season, the adults have blackish colour (Ahmed et al., 1985). 

Gul and Gulel 1995; Heimpel et al., 1997; Darwish et al., The larvae, pupae and adults of the host, G. mellonella were 

2003). It has been widely used in studies of host parasitoid collected from the infested bee hives located at the campus of 

interactions because of its high reproductive rate, short University of Agriculture, Faisalabad, Pakistan. The host and 

generation time and significant range of host species parasitoid cultures were maintained in two separate glass jars, 

o 

(Tuncyurek, 1972; Gul and Gulel, 1995). B. hebetor (Say) both were kept at 27-30 C±1, 65±5% temperature, relative 

completes its development on different species of humidity (RH) and 18 h light /6 h dark photoperiod. 

Lepidopteran larvae by using generally Pyralidae The culture of the parasitoid was reared on the larvae of the 

(Lepidoptera) larvae as host . Most of the species of Pyralidae greater wax moth G. mellonela as a host by using glass vials of 

(Lep.) are agricultural pests on some field and storage crops. (2×10 cm) in the insect molecular biology laboratory, 

Toxic bio-active peptides are present in the venom of the Department of Agri. Entomology, University of Agriculture, 

th 

B.hebetor which blocks the neuromuscular transmissions in Faisalabad. Each vial contains 2 to 3 of to 5 instar larvae of 

larvae of lepidopteran species by inhibiting exocytosis of pre the host and one female of the parasitoid, provided with cotton 

synaptic vesicles (Piek, 1966; Walther and Reinecke, 1983). swabs/pads soaked in 50% honey and water as food source for 

Its females have a preference to attack last (fifth) instar of host adult of B. hebetor. The females of the parasitoid start to 

larvae (Benson, 1973). The host is first injected with venom parasitize the larvae of the host by first injecting a small 

th 

that induces entire and everlasting paralysis within 15 quantity of paralyzing venom into mature (5 instar) larvae of 

minutes (Piek, 1966; Piek et al., 1974). After host-feeding, a the host before egg laying on them, which induces partial or 

female lays between 5 and 25 eggs per host depending upon complete paralysis and then deposit between 3 to 20 eggs on 

the size of the host, host-encounter pace, and physiological the outside of the host. After parasitism, the larvae of the host 

state of the ovipositing female (Benson, 1973; Hagstrum and become sluggish and later on, the females start egg laying on 

Smittle, 1977) . Females lay eggs on the host body and after these larvae. After 24 hours, the parasitized larvae of the host 

hatching the eggs, the larvae of the parasitoid produce silken were shifted from the vials to new sterilized glass jars (9×5 

cocoons and pupate outside the body of the host. Adult wasps cm) by using brush, provided with wax, pollen and honey as 

emerge from pupae ~16 days after oviposition. 

food for developing host larvae by keeping at the same 

The greater wax moth, Galleria mellonella (L.) (Lepidoptera: environmental conditions stated as above and the females of 

th 

Pyralidae) is an economically vital pest of the honeybee and is the parasitoid were shifted to new vials containing 2-3 (4-5 

one of the most devastating pests of wax in the world. Moth's instar) larvae of the host for further parasitization and this 

larval stages feed on wax, honey and pollen thus causing process remained continue until the last moment of the female 

serious economic damage as they tunnel through the comb. life. The eggs were hatched within 2-3 days into transparent 

The larvae of the wax moth, cause considerable damage to larvae, which were directly bored into the body of the host 

combs left unattended by bees (Caron, 1992). G. mellonella is larvae for feeding. These larvae, after completing their 

preferred in entomological studies, because of its nutritional feeding/development came out the host body for pupation. 




126

Life cycle of the parasitoid ranged from 9 to 12 days, through micro injection bioassay. Injections of venom (Heat 

depending on the rearing conditions (Temperature and and protease treated) were performed on non-parasitized 

humidity). Finally, these pupae were collected singly in hosts, on day 1 fourth and fifth instars, which were 

glass vials plugged with cotton swab/pad soaked with 50% synchronized by following the approach as described by 

honey and water as food source upon emergence of Pennacchio et al. (1995). Experimental insects were 

naive/virgin parasitoid adults which were separated by anaesthetized by placing in refrigerator or on ice and 

sexes: morphologically the females were distinguished injections containing 0.5-µl aliquots of treated venom were 

from the males by their pointed/conical abdomen performed into the pronotum of the larvae as stated by Quistad 

(ovipositor). The collected parasitoids were frozen at et al., 1996 and by following a slightly modified protocol of 

−80°C. 

Digilio et al. (1998, 2000) by using a glass microcapillary 

Venom extraction 

tube with a very fine tip/Micro-sampling syringe, 0.5 µl 

BioBasics (Cat.No. SR.031). Host Larvae receiving saline 

Females of B. hebetor of mixed ages were collected directly injections were acted as control. After injection, larvae were 

from the rearing cages singly in glass vials and were 

o 

kept at 30 C±1, 65±5% relative humidity (RH) and 18 h light 

anaesthetized by putting vials at 4°C in refrigerator for 30 /6 h dark photoperiod and data was scored for mortality at 24 

2 

min. The whole reproductive tract of adult females, hrs. A χ test was used to compare the recorded frequencies. 

previously anaesthetized was pulled out by grasping the 

ovipositor tip with a fine forceps, while holding and keeping 

the abdomen of the female with another forceps in a drop of 

Pringle's saline solution (Pringle, 1938) placed on a Petri dish 

RESULTS 

under microscope. The venom glands and reservoirs obtained Biological activities of the venom of B. hebetor and 

from each female were subsequently separated from the determination of its biochemical nature 

ovaries and other unnecessary tissues by using fine micro 

dissecting needles and transferred to a 20-µl drop of ice-cold The venom of B. hebetor showed considerable biological 

Pringle's solution. The venom reservoirs were gently opened activity against Galleria mellonella (Table 1). 

with fine needles to allow the diffusion of venom in a drop of 

Pringle's solution. Up to 20 venom reservoirs were pooled per 

Table 1 

each 20-µl drop of Pringle's solution. At the end all drops of 

Mortality of the G. mellonella caused by the venom of B. 

Pringle's solution containing gently opened/ruptured venom 

hebetor 

reservoirs were transferred to 1.5 ml sterile eppendorf tube by 

Mortality 

Treatment 

Replications 

sucking only the liquid part of the drop with micropipette and 

% 

leaving behind the ruptured tissues of the reservoirs in the 

Petri dish, in order to ensure the maximum purity of the 

Crude venom 50 70 

venom. The resulted crude extract was centrifuged at 5000 g Control ( Saline injections) 50 2 

for 5 min, at 4°C. to remove any fragments of tissue and the 

supernatant was recovered and transferred to a sterile 

eppendorf tube which was finally be diluted with appropriate 

Micro injections of crude venom in the host species G. 

saline solution in order to obtain the needed concentration of 

mellonella, caused immediate paralysis, feeding disruptions 

female equivalents (number of females processed as 

and death occurs within 72 hours, but in some cases the venom 

described above)/µl. Extracted venom was stored at −80°C. 

has an immediate paralysis leading to death within 24 hours, 

the death rates recorded in the host species were high, 

Venom inactivation treatments 

reaching 70% of mature larvae of G. mellonella. Bioassays 

Heat treatment 

were performed to evaluate the bioactivity of the venom after 

digestion with protease and heat treatment, it was deduced 

To assess the sensitivity of the venom extract to heat, an that the active components of the venom were proteins, as 

inactivation protocol was adopted as described by Digilio et they lost their effectiveness after heat and protease treatments 

al. (2000). The heat inactivating treatment was performed by (Table 2). 

boiling the extract for 5 min. After a 5 min centrifugation at 

12000g, the supernatant was recovered and stored at−80°C. 

Table 2 

Activities of the venom of B. hebetor after treatment with 

Protease treatment 

protease and heat 

The enzymatic digestion treatment was carried out by using 

active proteases Protinase K (21.4mg/ml) (Fermentas Life 

Sciences, catalog # E00492), which was incubated with the 

venom extract, at 37°C, for 1.5 h. Untreated venom extract 

acted as control. 

Biological activity of venom 

Biological activity of total venom of B. hebetor was assessed 



Treatment 

Venom treated with heat 

Venom treated with 

proteases (Protinase K) 

Control ( Saline injections) 

Replications 

Mortality (%) of 

G. 

mellonella 

50 6 

50 4 

50 2 



127

The host mortality caused by the heat and protease treated 

venom was almost similar to that mortality observed in 

control, which suggest that heat and protease treatments of 

venom have inactivated the proteins found in the venom 

blend of the wasp, confirming that the active components of 

the venom are proteins which are thermolabile and protease 

sensitive. 

Table 3 

Crude venom vs saline injections 

Total No. 

of injected 

host larvae 


Dead Alive % age of 

Mortality 

Mortality (%) 

Venom injected larvae 50 35 

15 

70 

Saline injected larvae 50 1 49 2 

(χ 2 = 50.173, P ≥ 0.05) df=1 

Table 4 

Venom treated with Heat vs saline injections 

Total No. 

of injected 

host larvae 

Dead Alive % age of 

Mortality 

Fig. 2 

Mortality of the host with heat treated venom and saline 

injected larvae of G. mellonella 

Venom treated with Heat 50 3 47 6 

Saline injected larvae 50 1 49 2 

χ 2 

=1.041 (χ 2 

=1.041, P ≤ 0.05) df=1 

Table 5 

Venom treated with proteases vs Saline injections 

Total No. 

of injected 

host larvae 

Venom treated with Heat 50 

Dead 

2 

Alive 

48 

% age of 

Mortality 

4 

Mortality (%) 

Saline injected larvae 

50 

χ 2 = 0.343 (χ 2 =0.034, P ≤ 0.05)df=1 

1 

49 

2 

Mortality (%) 

Fig. 1 

Mortality of the host with crude venom and saline injected 

larvae of G. mellonella 


Fig. 3 

Mortality of the host with protease treated venom and saline 

injected larvae of G. mellonella 

DISCUSSION 

This preliminary study of functional analysis of the maternal 

secretions (venom) injected by ectoparasitoid B. hebetor in its 

host G. mellonella at the time of oviposition has allowed us to 

understand the mechanism of parasitization in host regulation 

process along with the chemical nature of the female secretion 

like venom to be involved in successful parasitization of the 

host. The venom of B. hebetor, is produced by the venom 

glands attached to the reproductive system of the female of the 

parasitoid which is responsible for immediate paralysis and 

death of the host, or at least stop the development of the host. 

Venom is a complex mixture of protinaceous compounds 

which usually cause particularly in Microbracon species 



128

flaccid paralysis. Our experimental evidence showed that the (Parkinson et al., 2002), Asobara Tabida (Moreau and 

active components of the venom are proteins, which usually Guillot, 2005) and Cotesia rubecola (Asgari et al., 2003, 

lost their activities upon treatment with heat and protinase K. Zhang et al., 2004). Many of these molecules would be to 

The venom, as well as the toxins, of Microbracon hebetor have neurotoxic and cytolytic function (Moreau and Guillot, 

causes a blockage of presynaptic glutamatergic excitatory 2005). 

transmission. The venom has no effect on the inhibitory As mentioned previously, paralysis was found in the larvae of 

transmission. The venom of Microbracon is slow acting G. mellonella after injection of the venom of B.hebetor could 

(Beard, 1952). The paralysis persists over a long period of be due to a dose of venom injected for this host. Our results are 

time without any apparent change in the ultrastructure of the in accordance with the dose dependent effect of the venom 

nerve endings being caused (Piek et al., 1974; Rathmayer and which was also observed in other parasitoids as Euplectrus 

Walther, 1976). A dose of venom equivalent to one venom plathypenae (Nakamatsu and Tanaka, 2004) and Euplectrus 

gland has the same effect as the mass-collected venom of 100 kuwanae (Uematsu and Sakanoshita, 1987). 

wasps. The 'gland-extract venom' as well as the 'mass- The next step was to fractionate venom components, to isolate 

collected venom' have a paralysing effect on G. mellonella cDNA encoding major proteins of the venom and then 

larvae which is apparently identical to the paralysis observed expression of the isolated cDNA in eukaryotic cells for more 

after a G. mellonella larva has been stung by Microbracon. rapid and abundant production of these proteins, expression in 

Only a few representatives of the family Braconidae behave eukaryotic cells should ensure the maintenance of postas 

ectoparassitoid (koinobiont), regulating the physiology of translational modifications that often can have an important 

the host without interrupting its growth (Pennacchio and role for biological activity associated with the protein. In this 

Strand 2006). Also in Bracon hebetor, venom is responsible way, we can make more easily the necessary functional 

for the paralysis and death of parasitized hosts, these effects studies. 

have been attributed to the active protein components in the One of the advantages of biotechnology to control insect pests 

venom confirmed by the bioassays of heat and protease is just to expand the potential use of parasitoids, not only as 

treatment of the venom and this is in similar with our findings living organisms, but also using genes and molecules derived 

The physiological effects of the venom of B. hebetor were from parasitoid which are involved in the process of 

assessed on the mature larvae of host species G. mellonella increasing their success in programs to control pests. 

through microinjections. The effects of the venom on the host In conclusion, the possibilities offered by biotechnology for 

species were quite significant. The larvae of G. mellonella, the application of the genes and molecules derived from 

immediately after injection, at least at the doses we used, parasitoids in the control of insect pests are increasing, the 

interrupting the feeding activity and there were actual signs of biotechnology industry looks forward to growing this type of 

paralysis. Thus death, takes over 24-48 hours, has always molecules, as shown in some places of patents components of 

been associated with paralysis. We can not conclude at this the venom of the parasitoid Bracon hebetor (Quistad and 

stage that the observed effects of venom on the host are the Leisy, 1996; Windass et al., 1996, Johnson et al., 1996, 1999). 

result of a synergistic effect of proteins occurring in the It is imperative to undertake a solid programme of research 

venom of the wasp because sometimes a single protein found which will address such issues, namely the identification of 

in the venom of certain parasitoids is not sufficient to induce new molecules with insecticidal activity from the indigenous 

paralysis and death of the host and it requires many toxic fauna of insect parasitoids for development of sustainable 

proteins for successful parasitization. Further molecular pest insect control strategies. 

studies are required to fractionate the venom blend of the 

wasp through HPLC and SDS-PAGE and then bioassays ACKNOWLEDGEMENT 

would be performed with chromatographic fractions. 

Concerning the function of the proteins occurring in the Thanks to Higher Education Commission (HEC), 

venom of the wasp, we can only make assumptions because Government of Pakistan and Pakistan Science Foundation) 

we did not isolate the proteins yet and its N-terminal (PSF), for providing financial support for this work, Vide 

sequence, therefore, we can make assumption only based on notification No. HEC/20-1455/R&D/09/223, PSF/Res/Pthe 

previous studies that the similarity of the proteins of the AU/Agr (405). 

venom may be associated with “trypsin-like serine proteases” 

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