Using Gamma Irradiated Galleria mellonella L. and Plodia interpunctella (Hübner) Larvae to Optimize Mass Rearing of Parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae)

We evaluated possible improvements to the mass rearing of the larval parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) on irradiated host wax moth Galleria mellonella L. and Indian meal moth Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) larvae. The use of irradiated G. mellonella and P. interpunctella larvae at the dose of 150 Gy proved useful for enhancing the parasitism and adult emergence of H. hebetor due to the absence of negative repercussions on parasitoid development. Overall, parasitism was increased as the host larvae was irradiated with higher doses, while significantly higher parasitism was recorded at 150 and 300 Gy compared to lower doses. The female parasitoids preferred the irradiated larvae and significantly higher numbers of larvae were parasitized compared with non-irradiated larvae. The results also showed that irradiated larvae of G. mellonella served better as hosts for H. hebetor as compared with irradiated larvae of P. interpunctella. The implementation of these findings would be helpful for improving the mass production of parasitoids and the effectiveness of releases of biocontrol agents for the control of stored product pests.


Introduction
Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is a gregarious ectoparasitoid that attacks the larvae of several species of Lepidoptera, including members of the family Pyralidae that infest stored products [1]. In this regard, there are various successful paradigms of control of stored product moths using H. hebetor [2,3]. At the same time, naturally-occurring populations of this species have been frequently found in several parts of the world [4], while it is able to develop easily high population densities, provided there is sufficient number of large host groups [5]. Nevertheless, apart from quantity, which has an apparent influence of population growth, host quality is probably the key element that determines the reproductive success of a given parasitoid. The use of irradiated hosts has improved the rearing efficacy of different parasitoids and perhaps their quality as biological control agents [6,7]. Irradiation has been used to enhance production of insects' natural enemies in mass-rearing units [8]. Nuclear techniques also can play a prime role in augmentative biological

Experimental Procedures
Fifth Instar larvae of G. mellonella and P. interpunctella were selected for irradiation prior to the evaluation of the performance of H. hebetor. Larvae of both species were irradiated with different doses including 0 (control), 50, 75, 150, 300 and 500 Gy. After irradiation, the larvae of both species were transferred separately to plastic jars containing a pair of H. hebetor for parasitization under laboratory conditions at 27 ± 1 • C and 50-60% RH. There were three replications (25 larvae per replicate) for each species and gamma radiation dose. The number of fully grown H. hebetor mature larvae developed in irradiated host larvae was counted. After emergence of adult parasitoid, the parasitism percentage, sex ratio and longevity were recorded carefully. The size of H. hebetor adults including body length, head width and wing span was also measured using the ocular and stage micrometer.

Statistical Analysis
Levene's [21] test was used to test the assumptions of normality and homogeneity of variance prior to statistical analysis and then the data were subjected to analysis of variance (ANOVA) using the PROC GLMMIX [22]. Percent parasitism and emergence data were arcsin-transformed for statistical analysis. Means were separated by Tukey's HSD test when the F-test of the ANOVA was significant at the 5% level. Untransformed means and standard errors are reported to simplify interpretation.

Results
The parasitism percentage of H. hebetor was not affected significantly on irradiated host larvae of P. interpunctella (F = 1.44; df = 5,12; p = 0.28) and G. mellonella (F = 2.17; df = 5,12; p = 0.13). The maximum (98%) parasitism was recorded at 300 Gy for both species while minimum was at the control batch (68%) and 75 Gy (76%) for P. interpunctella and G. mellonella, respectively ( Figure 1). The results also indicated that there was no significant difference regarding the parasitism percentage between the two host species (F = 2.11; df = 1,35; p = 0.16). Nevertheless, the number of H. hebetor larvae that were found to be developed in irradiated larvae of P. interpunctella (F = 3.27; df = 5,12; p = 0.04) and G. mellonella (F = 43.61; df = 5,12; p < 0.001) differed significantly among treatments ( Figure 2). The highest number (58) of H. hebetor larvae was developed from G. mellonella irradiated larvae at 150 Gy, which was higher than the respective figure of P. interpunctella. The production of H. hebetor larvae developing from irradiated host larvae differed significantly between the two host species (F = 23.87; df = 1,35; p < 0.001). Fifth Instar larvae of G. mellonella and P. interpunctella were selected for irradiation prior to the evaluation of the performance of H. hebetor. Larvae of both species were irradiated with different doses including 0 (control), 50, 75, 150, 300 and 500 Gy. After irradiation, the larvae of both species were transferred separately to plastic jars containing a pair of H. hebetor for parasitization under laboratory conditions at 27 ± 1 °C and 50-60% RH. There were three replications (25 larvae per replicate) for each species and gamma radiation dose. The number of fully grown H. hebetor mature larvae developed in irradiated host larvae was counted. After emergence of adult parasitoid, the parasitism percentage, sex ratio and longevity were recorded carefully. The size of H. hebetor adults including body length, head width and wing span was also measured using the ocular and stage micrometer.

Statistical Analysis
Leveneʹs [21] test was used to test the assumptions of normality and homogeneity of variance prior to statistical analysis and then the data were subjected to analysis of variance (ANOVA) using the PROC GLMMIX [22]. Percent parasitism and emergence data were arcsin-transformed for statistical analysis. Means were separated by Tukey's HSD test when the F-test of the ANOVA was significant at the 5% level. Untransformed means and standard errors are reported to simplify interpretation.

Discussion
The results of the present work clearly indicated that some key biology and morphology parameters of H. hebetor were notably affected when the parasitoid was reared on gamma irradiated G. mellonella and P. interpunctella larvae, as compared with non-irradiated larvae. Apart from the apparent effects on mass production, it becomes evident that the quality of H. hebetor was substantially better when the species was reared on irradiated hosts, in comparison with non-irradiated ones. We showed that for many of the combinations tested, larvae at 150 Gy or 300 Gy, greatly enhanced parasitism rate and larval production of the parasitoids than non-irradiated larvae (Figures 1 and 2). The earlier studies by Genchev et al. [23] also showed that 65 Gy of gamma radiation enabled the otherwise marginally suitable factitious host G. mellonella to be used as a highly suitable host for the endoparasitoid Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae). Our results stand in accordance with the above observations and provide the first set of data for designing a mass rearing strategy of H. hebetor on irradiated hosts.
In order for parasitoids to develop successfully on irradiated hosts, two important conditions must be met. First, the radiation cannot substantially diminish the quality of the host as a source of food [24]. Second, particularly in the case of koinobiont endoparasitoids, the host's interior physical and chemical conditions must still provide the cues and hormone required to orchestrate the parasitoid's development. In this regard, there is even some tantalizing evidence that host irradiation could enhance parasitism rates and parasitoid fitness [25]. Several species of insects (e.g., fruit flies etc.) defend themselves against parasitoids through various immune mechanisms, such as encapsulation [26]. In a vast majority of parasitoids, egg and first larval stage development is often very rapid and voracious feeding early in their development may be a means acquiring critical resources before the host can mount a defensive response. If radiation could compromise the host immune system, then a greater proportion of parasitoids might complete their development. It is known that radiation can damage the capacity of certain insect hosts to defend themselves and consequently a parasitoid may not confront fully competent resistance. For example, the irradiation of the lepidopteran hosts of the braconid Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) increased parasitism rates [27]. Some evidence likewise indicates that the larvae of Tephritidae are immunologically compromised, thus radiation can result in a higher percentage of parasitoid emergence. Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) emergence and females-biased sex ratio increased following exposure of both the Mediterranean fruit fly Ceratitis capitata (Wiedemann) and the South American fruit fly Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) hosts to X-ray doses between 20 Gy and 100 Gy [28]. Gamma irradiated C. capitata larvae also supported higher D. longicaudata emergence rates and produced a significantly greater proportion of females [28].
In light of the present findings, it becomes evident that irradiation of larvae of the two host species mostly for G. melonella at the doses of 150 and 300 Gy proved were generally better than the other treatments to enhance the parasitism and adult emergence of H. hebetor. The parasitoid preferred the irradiated larvae and significantly higher numbers of irradiated larvae were parasitized as compared with the untreated (non-irradiated) control. This is particularly important and can be utilized further in mass rearing setups, under the basis of a biocontrol-based strategy. In this effort, we found that G. melonella was more suitable as a host for H. hebetor rearing, as compared with P. interpunctella and this trend was manifested regardless of the treatment (dose). Apparently, G. melonella larvae are larger in size than P. interpunctella and this may partially explain their suitability to host more H. hebetor individuals than P. interpunctella larvae. The interaction of the parasitoids with stored product host larvae has been regarded as a "host regulation" procedure that results in the improvement of the quality of the host [29]. However, host-seeking behavior may result in a considerable loss of energy by the parasitoid, which can lead to a concomitant reduction of the parasitism rate, especially in the case of the synovigenic species [30,31]. Hence, irradiation of larvae, up to a certain level to maintain the irradiated individuals functional as hosts, is likely to alleviate this energy loss and provide increase parasitism success and progeny production.
In the present work, we have developed rearing protocols for parasitoids in stored product protection through the use of irradiated host larvae. We found that certain irradiation doses enhance parasitism rate and larval production of parasitoids and that G. melonella is more suitable as a host species than P. interpunctella. It remains unclear, however, if these findings are mostly related with host preference or with key developmental variables in certain host groups. Moreover, the "quality" of the irradiated hosts, at least in the species' complex that has been examined here, should be further examined in conjunction with the "quality" of the parasitoids that are produced. In other words, parasitization rate and success in adult emergence should be regarded under the basis of the progeny production capacity of the individuals that had been reared in irradiated hosts.

Conclusions
From these experiments, it is concluded that the use of irradiated hosts is fundamental for the production of parasitoids. The irradiation of G. mellonella larvae proved useful to serve better as hosts for the parasitoid H. hebetor. The implementation of these findings would also be helpful for enhancing the mass production of parasitoids and the effectiveness of releases of biocontrol agents for the control of stored product pests as well as other lepidopteran pests.