Diversity and composition of the microbiome associated with eggs of the Southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae)

Background: Although microbial communities of insects from larval to adult stage have been increasingly investigated in recent years, little is still known about the diversity and composition of egg-associated microbiomes. In this study, we used high-throughput amplicon sequencing and qPCR to get a better understanding of the microbiome of insect eggs and how they are established using the Southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) as study object. First, in order to determine the bacterial community composition, egg masses from two natural populations in Belgium and Italy were examined. Subsequently, microbial community establishment was assessed by studying stinkbug eggs of different ages obtained from laboratory strains (unlaid eggs collected from the ovaries, eggs less than 24 hours old, and eggs collected four days after oviposition). Both the external and internal egg-associated microbiome were analyzed by investigating egg washes and surface-sterilized washed eggs, respectively. Results: Eggs from the ovaries were completely devoid of bacteria, indicating that egg-associated bacteria were deposited on the eggs during or after oviposition. Bacterial diversity of deposited eggs was very low, with on average 6.1 zOTUs in the external microbiome and 1.2 zOTUs in internal samples of egg masses collected from the field. Bacterial community composition and density did not change significantly over time, suggesting limited bacterial growth. A Pantoea-like symbiont previously found in the midgut of N. viridula was found in every sample and generally occurred at high relative and absolute densities, especially in the internal egg samples. Additionally, some eggs harbored a Sodalis symbiont, which has previously been found in the abdomen of several insects, but so far not in N. viridula populations. Conclusions: We conclude that the egg-associated bacterial microbiome of N. viridula is species-poor and dominated by a few symbionts, particularly the species-specific obligate Pantoea-like symbiont.

研究背景：近年来，学界对昆虫从幼虫至成虫阶段的微生物群落研究日益深入，但针对与虫卵相关的微生物组（microbiome）的多样性与组成，目前仍知之甚少。本研究以南方绿蝽（Nezara viridula (L.)，半翅目（Hemiptera）蝽科（Pentatomidae））为研究对象，采用高通量扩增子测序与定量聚合酶链式反应（quantitative PCR，qPCR）技术，解析昆虫虫卵相关微生物组及其构建过程。首先，为明确细菌群落组成，本研究对采自比利时与意大利的两个自然种群的虫卵块进行了检测；随后，通过分析实验室种群中不同发育时长的蝽虫卵（包括从卵巢中采集的未产出卵、产出不足24小时的卵，以及产卵四天后采集的卵），评估微生物组的构建动态。研究分别通过虫卵清洗液与经表面灭菌后的清洗虫卵，解析虫卵相关微生物组的外部与内部组分。
研究结果：卵巢内的虫卵完全不含细菌，表明与虫卵相关的细菌是在产卵过程中或产卵后附着于虫卵的。野外采集的虫卵块中，产出卵的细菌多样性极低，外部微生物组平均包含6.1个零半径操作分类单元（zero-radius operational taxonomic units，zOTUs），内部样本平均含1.2个zOTUs。细菌群落组成与密度随时间未发生显著变化，提示细菌增殖程度有限。所有样本中均检测到此前在稻绿蝽中肠内发现的泛菌属（Pantoea）类似共生菌，其相对与绝对丰度普遍较高，尤其在虫卵内部样本中更为显著。此外，部分虫卵中存在索氏菌属（Sodalis）共生菌，该类共生菌此前仅在多种昆虫的腹部中被检测到，目前尚未在稻绿蝽种群中发现。
研究结论：综上，本研究认为稻绿蝽的虫卵相关细菌微生物组物种丰富度较低，且以少数共生菌为主导，尤其是该物种特异性专性泛菌属类似共生菌。