Hermetia illucens in diets for zebrafish (Danio rerio): A study of bacterial diversity by using PCR-DGGE and metagenomic sequencing

In the present research, bacterial diversity was studied during a 6-month feeding trial utilizing zebrafish (Danio rerio) fed Hermetia illucens reared on different substrates with an emphasis on fish gut bacterial diversity. A polyphasic approach based on viable counting, PCR-DGGE and metagenomic 16S rRNA gene amplicon target sequencing was applied. Two different H. illucens groups were reared on coffee by-products (C) or a mixture of vegetables (S). Viable counts showed a wide variability based on substrate. PCR-DGGE and Illumina sequencing allowed the major and minor bacterial taxa to be detected. Both samples of larvae and their frass reared on the S substrate showed the highest richness and evenness of bacterial communities, whereas zebrafish (ZHC) fed H. illucens reared on substrate C and zebrafish (ZHS) fed H. illucens reared on substrate S had the lowest bacterial richness and evenness. A stimulating effect of bioactive compounds from coffee by-products on the occurrence of Lactobacillaceae and Leuconostoccaceae in H. illucens reared on substrate C has been hypothesized. Zebrafish gut samples originating from the two feeding trials showed complex microbial patterns in which Actinobacteria and Alteromonadales were always detected, irrespective of the diet used. Enterobacteriaceae in fish guts were more abundant in ZHS than in ZHC, thus suggesting an influence of the bioactive compounds (chlorogenic and caffeic acids) in the substrate on Enterobacteriaceae in fish guts. ZHC showed a higher abundance of Clostridia than did ZHS, which was likely explained by stimulating activity on the bacteria in this class by the bioactive compounds contained in H. illucens reared on substrate C. An influence of the microbiota of H. illucens or insect-derived bioactive compounds on the gut microbiota of zebrafish has been suggested. The presence of bacteria consistently associated with zebrafish guts has been found irrespective of the diet, thus attesting to the likely stability of the core fish microbiota.本研究开展了一项为期6个月的饲喂试验，以斑马鱼（Danio rerio）为受试对象，投喂以不同基质饲养的黑水虻（Hermetia illucens），重点探究鱼类肠道细菌多样性。本研究采用基于活菌计数、聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）及宏基因组16S rRNA基因扩增子靶向测序的多相分析方法。试验设置两组黑水虻饲养基质：咖啡副产物组（C）与蔬菜混合物组（S）。活菌计数结果显示，细菌群落数量随饲养基质的不同呈现显著差异。PCR-DGGE与Illumina测序技术可检测到鱼类肠道菌群中的主要及次要细菌类群。以蔬菜混合物基质饲养的黑水虻幼虫及其虫粪样本的细菌群落丰富度与均匀度均为最高；而投喂咖啡副产物基质饲养的黑水虻的斑马鱼（ZHC）与投喂蔬菜混合物基质饲养的黑水虻的斑马鱼（ZHS），其肠道细菌丰富度与均匀度均为最低。研究推测，咖啡副产物中的生物活性化合物可促进乳杆菌科（Lactobacillaceae）与明串珠菌科（Leuconostoccaceae）在咖啡副产物基质饲养的黑水虻体内定植。两项饲喂试验的斑马鱼肠道样本均呈现复杂的微生物群落模式，其中放线菌门（Actinobacteria）与交替单胞菌目（Alteromonadales）在所有日粮组中均被检测到，不受饲喂基质的影响。斑马鱼肠道内的肠杆菌科（Enterobacteriaceae）在ZHS组中的丰度高于ZHC组，这表明饲养基质中的生物活性化合物——绿原酸（chlorogenic acid）与咖啡酸（caffeic acid）——可对鱼类肠道内的肠杆菌科丰度产生调控作用。ZHC组斑马鱼肠道内的梭菌纲（Clostridia）丰度高于ZHS组，这一现象可能源于咖啡副产物基质饲养的黑水虻所含的生物活性化合物对该类细菌的增殖促进作用。研究提示，黑水虻的微生物群或昆虫源生物活性化合物可对斑马鱼肠道微生物群产生影响。无论日粮类型如何，均检测到与斑马鱼肠道稳定关联的细菌类群，这证实了鱼类核心微生物群（core fish microbiota）可能具有稳定性。