Transmission of Different Nucleopolyhedroviruses by Two Ectoparasitoids – Bracon hebetor Say ( Hymenoptera : Braconidae ) and Euplectrus plathypenae ( Howard ) ( Hymenoptera : Eulophidae )

The transmission of nucleopolyhedroviruses (NPVs) of Autographa gamma (AgNPV), Mamestra brassicae (MbNPV), Lacanobia oleraceae (LoNPV), Helicoverpa armigera (HaNPV) and Xantia c-nigrum (XnNPV) to their relevant larvae by the ectoparasitoid Bracon hebetor and the transmission of the multiple-enveloped NPVs of Spodoptera exigua (SeMNPV) and Spodoptera frugiperda (SfMNPV) by the ectoparasitoid Euplectrus plathypenae was examined. Two methods of contamination of the both parasitoids (exposure to infected hosts and total body surface) and two subsequent transmissions of the viruses by Bracon hebetor to healthy hosts were tested. The results showed that both parasitoids were capable to be mechanical vectors of the tested NPVs. Every Bracon hebetor female was able to transmit subsequently twice the virus in 27% to 52.2% of the five Noctuidae species by preliminary exposing to infected larvae. The second method of contamination (applying virus suspension to the total body surface of the parasitoid) was also efficient causing virus infection in between 29.4% and 54.15% of the larvae. The parasitoid E. plathypenae transmited the virus from infected to noninfected larvae in 20% and 25.57% of the S. frugiperda and S. exigua larvae, and 6.43% and 11.10%, respectively of them died from the virus infection. The same observation was established by the second method of contamination - respectively 33.33% and 40% infection and between 13.23% and 16.67% mortality. The mortality of all tested larvae exposed to virus contaminated parasitoids was higher when the parasitoid entire body surface had been artificially contaminated with the virus than when the parasitoid itself was previously allowed to oviposit the larvae.


INTRODUCTION
Certain hymenopterous parasitoids can transmit mechanically entomopathogenic viruses to lepidopterous larvae.Female parasitoids that have become contaminated with virus during oviposition in an infected host may subsequently infect other hosts during oviposition.Another possible route of pathogen transmission is the artificial contamination of the parasitoid body surfaces leading to viral infection of the host (Raimo et al., 1977;Hochberg, 1991;Young and Yearian, 1990).Information on transmission from infected to noninfected hosts would provide insight on the role of parasitoids in dispersal of baculoviruses within or between natural host populations.In addition, dispersal of the viruses by artificially contaminated parasitoids may be a means of an artificial introduction of the viruses into host populations in pest management programs.Dissemination of insect viruses by parasitic insects especially parasitic hymenoptera has been suggested by several authors.For example McCutchen et al. (1996) reported virus transmission by Microplitis croceipess (Cresson) from 3 wild type viruses with 4.0, 10.6, and 8.3% infection of the uninfected larvae.Five percent and three percent of Heliothis virescens Fabricius larvae exposed to female Cotesia marginiventris Cresson that developed in a wild type virus died from virus infection.Raimo et al. (1977) demonstrated intrahaemocoelic inoculation of Lymantria dispar Linnaeus NPV by Apanteles melanoscelus Ratzeburg when the parasitoid oviposited in noninfected larvae after oviposition in NPV-infected larvae.Adults of Campoletis sonorensis Cameron transmitted heliothis NPV to as many as 40% of larvae of H. virescens in which they oviposited after ovipositoin in NPV-infected larvae (Irabagon and Brooks, 1974).Levin et al. (1979) reported a higher frequency (81% to 95%) of transmission of the GV of Pieris rapae Linnaeus by the parasitoid Apanteles glomeratus Linnaeus.
The aims of this study was to determine the potential role of the ectoparasitoids Bracon hebetor Say to transmit the nucleopolyhedroviruses of Autographa gamma Linnaeus, Lacanobia oleraceae Linnaeus, Helicoverpa armigera Hübner and Xantia c-nigrum Linnaeus and Euplectrus plathypenae Howard to be a vector of NPVs of Spodoptera exigua Hübner and Spodoptera frugiperda J.E. Smith.

MATERIAL AND METHODS
Two methods of contaminating female parasitoids with NPVs were tested: (1) exposing the parasitoid to infected larvae (transmission type A) and (2) applying virus suspension to the total body surface of the parasitoid (transmission type B).

Bioassays with E. plathypenae
(1) Twenty newly molted third and four instar S. frugiperda and larvae were fed with an aqueous suspension containing 2.44 x 10 6 and 2.3 x 10 7 OBs ml -1 , in a solution of 10% sucrose and 0.01% Flourella blue dye.These concentrations represented LC 90 values for the third and fourth instar larvae, respectively (Escribiano et al., 1999).S. exigua larvae were fed with a viral suspension with LC 90 -3.32 x 10 7 and 6.83 x 10 8 OBs ml - 1 (for the third and fourth instar, respectively (Muñoz et al.,1999).After virus inoculation larvae were transferred to petri dishes containing fresh diet.When the larvae started molting, they were offered to the parasitoid females.Immediately after they had been parasitized, the parasitoids were removed and offered healthy premolting respectively second to third instar and third to fourth instar S. exigua.The experiment was repeated three times.The larvae were checked for the presence of OBs.
(2) Fifty E. plathypenae parasitoids were put in petri dishes preliminarily smeared with viral suspension for over 30 min.Then the parasitoids were transferred to healthy third instar S. exigua and S. frugiperda.The larvae were checked daily for virus-mortality and the larvae died by the parasitoid were checked for the presence of OBs.
The control group of larvae and parasitoids fed with virus-free solution of 10% sucrose and 0.01% Flourella blue dye.Every bioassay was repeated 3 times.

Bioassays with Bracon hebetor
The bioassays were similar but the species used were A. gamma, M. brassicae, H. armigera, X. c-nigrum and L. oleraceae and their homologous NPVs (AgNPV,

Emanouela E. Stoianova and Nikolay A. Balevski
The mortality of all tested larvae exposed to virus contaminated parasitoids was higher when the parasitoid entire body surface had been artificially contaminated with the virus than when the parasitoid itself was previously allowed to oviposit the larvae.Keywords: Hymenoptera; Lepidoptera; Nucleopolyhedroviruses; Transmission HaNPV, XcNPV and LoNPV, respectively) with suspensions with lethal concentration LC 90 defined by Величкова-Кожухарова (1984) and in the bioassay transmission type A every parasitoid female was consecutively transferred to two healthy larvae of every mentioned species.

Analysis of data
Data in all tests were transformed to arcsine and analyzed by analysis of variance (ANOVA).

RESULTS
Our study showed that both parasitoids B. hebetor and E. plathypenae transmitted different viruses virus to noninfected larvae.Every female of B. hebetor was able to transmit the virus twice from infected to noninfected larvae between 27% to 52,2% of the five tested species.The second method of contamination (applying virus suspension to the total body surface of the parasitoid) was also efficient causing virus infection from 29.4% to 54.15% of the larvae (Figure 1).
Pestic.Phytomed.(Belgrade), 25(2), 2010, 133-137 The parasitoid E. plathypenae transmits the virus from infected to noninfected larvae in 25.57% of the larvae and 11.10% of the larvae died from the virus infection.The same observation was established in the second method of contamination -40% infection of the noninfected larvae from which 16.67% mortality.Similar results in S. frugiperda larvae were observed (Table 1).

DISCUSSION
The present study indicates the significant role that the ectoparasitoids B. hebetor and E. plathypenae may have in the dissemination of nucleopolyhedroviruses in field populations.The high incidence of mortality of the mentioned Noctuidae larvae by NPVs after oviposition by adult parasitoids that had developed in NPV-deseased larvae indicate the potential role of the parasitoid in the dissemination of NPVs in field populations of Noctuidae larvae.In addition, the finding that the adult parasitoids which oviposited in NPV-infected larvae transmitted the viruses to larvae in which they subsequently oviposited suggested a second mechanism by which parasitism is implicated in the dissemination of this virus.
The both entomophagous E. plathypenae and B. hebetor are ectoparasitoids that means they oviposited their eggs on the host body.Both species prefer ovipositing larvae in last ages.In our study we tested two ages -third and fourth instar which are relatively susceptible to the virus infection and at the same time prefferred by parasitoid females.A special feature of B. hebetor ovipositional behaiviour is that the females stinging the larvae paralize them constantly in the most of the cases.In that meaning our study is aiming not to kill the larvae from viral infection but to use this transmission for subsequent dissemination of the virus.
Our study showed presence of OBs in the larvae-recipients after oviposition with virus-contaminated parasitoid females but not all of them showed virus mortality.The presence of virus infection in the larva's body itself is a potential factor for further dissemination of the virus by vertical or horizontal transmission.
The statistical analysis showed significant differences between the two types of virus transmission.The transmission of the virus by applying the virus suspension to the total body surface of the parasitoid is significantly higher than the transmission from infected to noninfected larvae.That could be explained by the fact the parasitoid could supply additionaly the transmission by parasitizing the larvae and also by contaminating their food with its contaminated body.Our results are analogical of the Young and Yearian (1990) that established that the transmission of the virus by contaminating its body surface M. croiceps was higher than transmission from infected to healthy larvae.
The successful transmission by parasitoids is reported by many authors.Lopes et al. (2002) reported that one female E. viticolee was able to transmist an iridescence virus in 3.7% of S. frugiperda larvae.Irabagon and Brooks (1974) found that the C. sonorensis adults could transmist a NPV in 40% of H. virescens larvae.
This study did not determine the exact mechanism by which B. hebetor and E. plathypenae transmitted the virus to their hosts.The experimental techniques and the behaiviour of the parasitoid during oviposition suggested, however, that the virus was transmitted by the ovipositor or by contact with food.
The role of the parasitoids as vectors of insect baculoviruses is very important and has been frequently mentioned and even demonstrated under laboratory conditions (Brooks, 1993).The finding that E. plathypenae and B. hebetor can transmit NPVs under laboratory conditions suggests a mechanism by which the viruses could be transmitted in field populations of susceptible insects.Pestic. Phytomed. (Belgrade), 25(2), 2010, 133-137 hypervariable genomic region. Virus Research, 59: 61-74, 1999.

Figure 1 .
Figure 1.Transmission (type A and B) of various viruses by the parasitoid B. hebetor to their homologous Noctuidae larvae

Table 1 .
Transmission (type A and B) of the viruses SeMNPV и SfMNPV by the parasitoid E. рlathypenae to healthy Spodoptera exigua and S. frugiperda larvae Donor-larvae -larvae that were artificially infected with virus Donor parasitoids-parasitoid females that were artifically contaminated with virus Larvae-recipients-larvae that were infected by parasitoid ovipositoin