Evaluation Of The Effects Of Egg-Associated Bacteria On Oviposition Behaviour And Development Of Black Soldier Fly,Hermetia Illucens

Black soldier fly (BSF), Hermetia illucens (L.), has been considered as a sustainable method for reducing animal and plant wastes. Larvae reduce dry matter, bacteria, offensive odor and house fly populations. The prepupae can be self-harvested and used as feedstuff for livestock and poultry.While some bacteria species have been cultured and identified from BSF, a true appreciation of fly associated bacterial diversity is not known. On the other hand, the significance of egg-associated bacteria on BSF larval development and what roles they play in the oviposition aggregation behaviour of gravid females are seldom reported up to now. Such information will provide insight into BSF biology and is needed to improve the current waste management system. To address these issues, this study was conducted accordingly. The potential of BSF larval grease to be used as a novel biomass feedstock for biodiesel production was also evaluated in the last chapter.The main results are as follows:(1) Bacterial diversity from successive life stages of black soldier fly.Using16S rDNA454pyrosequencing, we examined bacterial diversity associated with successive life stages of the BSF reared on a Gainesville diet. We obtained4852PCR products from the different life stages of the BSF. The number of sequences obtained from the first generation egg, larva, prepupa, pupa, adult, and second generation egg stages were37,517,1181,2106,883, and128, respectively. The percent of sequences which classified at the phylum, class, order, family, and genus levels were99.65%,97.34%,85.84%,69.31%, and31.55%, respectively, with≥80%bootstrap support. The combined diversity of bacteria classified (99.67%) across all life stages spanned six bacterial phyla with≥80%bootstrap support. Bacteroidetes and Proteobacteria were the most dominant phyla associated with the BSF accounting for two-thirds of the fauna identified. Of the bacteria found associated with the egg stage of development,15%were shared by first and second generation samples suggesting the possibility of vertical transmission and retention of bacteria through successive developmental stages.(2) Egg-associated bacteria-mediated oviposition aggregation by black soldier flies.Oviposition sites preference (OSP) tests were conducted under laboratory conditions to evaluate the roles of different potential factors in the oviposition aggregation behaviour of gravid female BSF. It is indicated that ovipositional substrates acted as a long distance attractant during oviposition site detection and fresh conspecific eggs had an effect of stimulation on oviposition behaviour. Further OSP tests of sterile eggs showed that egg-associated bacteria were the major source of this aggregation stimulus. Interestingly, among the bacteria isolated from BSF eggs, dramatical oviposition stimulation was only detected from BSF-4which is not a sole stain but a bacteria assemblage. Four stains of bacteria were identified as Gordonia sp., Cellulomonas sp., Micrococcus sp., and Microbacterium sp., respectively, by pyrosequencing and16S rDNA sequencing individually. The assemblage showed much stronger stimulation effect than individual ones in comparison OSP tests, although these four bacteria all can enhance oviposition separately. This suggests that synergy may exist in the bacteria assemblage. In addition, some bacteria off the BSF eggs or isolated from competitor insect species exhibited a repellent effect on oviposition of gravid females.(3) Effects of egg associated bacteria on the development of black soldier fly.Growth study with sterile BSF larvae obtained by egg surface sterilization was conducted to determine the changes of their life history traits compared with normal larvae provided with identical sterile artificial diet. The significance of bacteria to the development of BSF was further confirmed by bacteria replenishment to sterile larvae. The results demonstrated that percentage survivorship to prepupal and adult stages of sterile larvae were significantly lower than that of normal larvae. Only57.5%of sterile larvae successfully reached adult stage. Developmental delay was detected with sterile larvae; they took50%longer to develop to prepupal stage (30d). Additionally, sterile larvae also took longer to reach adult stage. Adults resulting from sterile larvae (9d) lived2d less than those from normal larvae. Prepupa and adults resulting from sterile larvae weighed significantly lighter. After bacteria replenishment by adding egg wash or mixed egg-associated bacteria suspension, some of the life history traits of sterile larvae were significantly improved, drawing close to that of normal larvae. It is shown that egg-associated bacteria are very important to the development of BSF larvae.(4) Exploring the potential of black soldier fly larval grease as a novel biomass feedstock for biodiesel production.This study was conducted to evaluate the potential of a secondary biodiesel production from the solid residual fraction of restaurant waste after typical grease extraction (SRF). About23.6g larval grease-based biodiesel was produced from approximately1000larvae grown on1kg of SRF. The weight of SRF was reduced by about61.8%after being fed by the black soldier fly larvae for7d. The amount of biodiesel yield from restaurant waste was nearly doubled (original restaurant waste grease, 2.7%; larval grease,2.4%). Most of the properties of this biodiesel met the specifications of the standard EN14214, including density (860kg/m3), viscosity (4.9mm2/s), flash point (128℃), cetane number (58) and ester contents (96.9%).