A survey of the mycobiota associated with larvae of the black soldier fly (<i>Hermetia illucens</i>) reared for feed production

Feed security, feed quality and issues surrounding the safety of raw materials are always of interest to all livestock farmers, feed manufacturers and competent authorities. These concerns are even more important when alternative feed ingredients, new product developments and innovative feeding trends, like insect-meals, are considered. The black soldier fly (Hermetia illucens) is considered a good candidate to be used as feed ingredient for aquaculture and other farm animals, mainly as an alternative protein source. Data on transfer of contaminants from different substrates to the insects, as well as the possible occurrence of toxin-producing fungi in the gut of non-processed insects are very limited. Accordingly, we investigated the impact of the substrate/diet on the intestinal mycobiota of H. illucens larvae using culture-dependent approaches (microbiological analyses, molecular identification through the typing of isolates and the sequencing of the 26S rRNA D1/D2 domain) and amplicon-based next-generation sequencing (454 pyrosequencing). We fed five groups of H. illucens larvae at the third growing stage on two substrates: chicken feed and/or vegetable waste, provided at different timings. The obtained results indicated that Pichia was the most abundant genus associated with the larvae fed on vegetable waste, whereas Trichosporon, Rhodotorula and Geotrichum were the most abundant genera in the larvae fed on chicken feed only. Differences in the fungal communities were highlighted, suggesting that the type of substrate selects diverse yeast and mold genera, in particular vegetable waste is associated with a greater diversity of fungal species compared to chicken feed only. A further confirmation of the significant influence of diet on the mycobiota is the fact that no operational taxonomic unit common to all groups of larvae was detected. Finally, the killer phenotype of isolated yeasts was tested, showing the inhibitory activity of just one species against sensitive strains, out of the 11 tested species.饲料安全、饲料品质及原料安全相关问题，始终是畜牧养殖户、饲料生产企业及监管主管部门共同关注的核心议题。当涉及替代饲料原料、新产品研发以及昆虫饲料（insect-meals）这类创新性饲喂趋势时，此类关切更显迫切。黑兵蝇（Hermetia illucens）被视为水产养殖与其他畜禽养殖的优质饲料原料候选者，主要作为替代蛋白源。目前关于不同基质向昆虫体内污染物迁移的相关数据，以及未加工昆虫肠道中产毒真菌潜在存在情况的研究资料均极为有限。据此，本研究采用依赖培养的研究方法（包括微生物学分析、菌株分型分子鉴定以及26S rRNA D1/D2结构域测序）以及基于扩增子的下一代测序技术（454焦磷酸测序（454 pyrosequencing）），探究了基质/日粮对黑兵蝇幼虫肠道真菌菌群（mycobiota）的影响。本研究以5组处于第三生长阶段的黑兵蝇幼虫为实验对象，按不同饲喂时序给予两种基质：鸡饲料与/或蔬菜废弃物。研究结果显示，饲喂蔬菜废弃物组的幼虫肠道中，毕赤酵母属（Pichia）为优势菌属；而仅饲喂鸡饲料组的幼虫肠道中，毛孢子菌属（Trichosporon）、红酵母属（Rhodotorula）与地霉属（Geotrichum）为优势菌属。真菌群落结构存在显著差异，表明基质类型可塑造不同的酵母与霉菌菌属组成；具体而言，相较于仅饲喂鸡饲料的组别，蔬菜废弃物组的真菌物种多样性更为丰富。所有幼虫组别均未共享任何操作分类单元（operational taxonomic unit，OTU），这进一步证实了日粮对肠道真菌菌群的显著影响。最后，本研究对分离得到的酵母菌株的杀表型（killer phenotype）进行了检测，结果显示，在11株受试酵母物种中，仅1株对敏感菌株表现出抑制活性。