Data_Sheet_1_New Insights Into Culturable and Unculturable Bacteria Across the Life History of Medicinal Maggots Lucilia sericata (Meigen) (Diptera: Calliphoridae).pdf

Because of the nutritional ecology of dung- and carrion-feeding, bacteria are the integral part of Lucilia sericata life cycle. Nevertheless, the disinfected larvae of the blowfly are applied to treat human chronic wounds in a biosurgery named maggot debridement therapy (MDT). To realize the effects of location/diet on the gut bacteria, to infer the role of bacteria in the blowfly ecology plus in the MDT process, and to disclose bacteria circulating horizontally in and vertically between generations, bacterial communities associated with L. sericata specimens from various sources were investigated using culture-based and culture-independent methods. In total, 265 bacteria, including 20 families, 28 genera, and 40 species, were identified in many sources of the L. sericata. Culture-dependent method identified a number of 144 bacterial isolates, including 21 species, in flies reared in an insectary; specimens were collected from the field, and third-instar larvae retrieved from chronic wounds of patients. Metagenetic approach exposed the occurrences of 121 operational taxonomic units comprising of 32 bacterial species from immature and adult stages of L. sericata. Gammaproteobacteria was distinguished as the dominant class of bacteria by both methods. Bacteria came into the life cycle of L. sericata over the foods and transovarially infected eggs. Enterococcus faecalis, Myroides phaeus, Proteus species, Providencia vermicola, and Serratia marcescens were exchanged among individuals via transstadial transmission. Factors, including diets, feeding status, identification tool, gut compartment, and life stage, governed the bacteria species. Herein, we reemphasized that L. sericata is thoroughly connected to the bacteria both in numerous gut compartments and in different life stages. Among all, transstadially transmitted bacteria are underlined, indicating the lack of antagonistic effect of the larval excretions/secretions on these resident bacteria. While the culture-dependent method generated useful data on the viable aerobic gut bacteria, metagenomic method enabled us to identify bacteria directly from the tissues without any need for cultivation and to facilitate the identification of anaerobic and unculturable bacteria. These findings are planned to pave the way for further research to determine the role of each bacterial species/strain in the insect ecology, as well as in antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities.

由于食粪与食腐的营养生态特征，细菌是丝光绿蝇（Lucilia sericata）生命周期中不可或缺的组成部分。不过，该丽蝇的消毒幼虫可应用于名为蛆虫清创疗法（maggot debridement therapy, MDT）的生物外科手术，用于治疗人类慢性伤口。为阐明生境与饮食对其肠道菌群的影响、推断细菌在该丽蝇生态及蛆虫清创疗法过程中的作用，并揭示细菌在种群内的水平传播以及代际间的垂直传播规律，研究人员采用基于培养的方法（culture-based method）与非培养依赖的方法（culture-independent method），对不同来源的丝光绿蝇样本关联的细菌群落开展了分析。本次研究共计从多来源的丝光绿蝇样本中鉴定出265株细菌，隶属于20个科、28个属及40个物种。其中，基于培养的方法从昆虫饲养室饲养的蝇类、野外采集的样本以及从患者慢性伤口中分离的三龄幼虫中，共获得144株细菌分离株，涵盖21个物种。宏基因组方法则检测到121个操作分类单元（operational taxonomic units, OTUs），涵盖丝光绿蝇未成熟阶段与成虫阶段的32个细菌物种。两种方法均鉴定出γ-变形菌纲（Gammaproteobacteria）为优势细菌类群。细菌可通过食物途径与经卵感染的卵进入丝光绿蝇的生命周期。粪肠球菌（Enterococcus faecalis）、淡黄色杆菌（Myroides phaeus）、变形杆菌属（Proteus）物种、嗜虫普罗维登斯菌（Providencia vermicola）及粘质沙雷氏菌（Serratia marcescens）可通过经龄期传递在个体间进行传播。饮食、进食状态、鉴定工具、肠道区段以及发育阶段等因素，均会对细菌物种的组成产生调控作用。本研究再次证实，丝光绿蝇的多个肠道区段及不同发育阶段均与细菌存在紧密关联。其中，经龄期传递的细菌受到重点关注，这表明幼虫的排泄物/分泌物对这些定殖细菌不存在拮抗作用。尽管基于培养的方法可获得有关可培养需氧肠道细菌的有效数据，但宏基因组方法可直接从组织样本中鉴定细菌，无需进行培养，同时也助力了厌氧及不可培养细菌的鉴定工作。本研究结果可为后续研究奠定基础，以明确各细菌物种/菌株在昆虫生态，以及抗菌、抗生物膜、抗炎及伤口愈合相关活性中的具体作用。