Data_Sheet_1_Spiroplasma as facultative bacterial symbionts of stinkbugs.zip

Many insects are associated with facultative symbiotic bacteria, and their infection prevalence provides an important clue to understand the biological impact of such microbial associates. Here we surveyed diverse stinkbugs representing 13 families, 69 genera, 97 species and 468 individuals for Spiroplasma infection. Diagnostic PCR detection revealed that 4 families (30.8%), 7 genera (10.1%), 11 species (11.3%) and 21 individuals (4.5%) were Spiroplasma positive. All the 21 stinkbug samples with Spiroplasma infection were subjected to PCR amplification and sequencing of Spiroplasma’s 16S rRNA gene. Molecular phylogenetic analysis uncovered that the stinkbug-associated Spiroplasma symbionts were placed in three distinct clades in the Spiroplasmataceae, highlighting multiple evolutionary origins of the stinkbug-Spiroplasma associations. The Spiroplasma phylogeny did not reflect the host stinkbug phylogeny, indicating the absence of host-symbiont co-speciation. On the other hand, the Spiroplasma symbionts associated with the same stinkbug family tended to be related to each other, suggesting the possibility of certain levels of host-symbiont specificity and/or ecological symbiont sharing. Amplicon sequencing analysis targeting bacterial 16S rRNA gene, FISH visualization of the symbiotic bacteria, and rearing experiments of the host stinkbugs uncovered that the Spiroplasma symbionts are generally much less abundant in comparison with the primary gut symbiotic bacteria, localized to various tissues and organs at relatively low densities, and vertically transmitted to the offspring. On the basis of these results, we conclude that the Spiroplasma symbionts are, in general, facultative bacterial associates of low infection prevalence that are not essential but rather commensalistic for the host stinkbugs, like the Spiroplasma symbionts of fruit flies and aphids, although their impact on the host phenotypes should be evaluated in future studies.

诸多昆虫与兼性共生细菌（facultative symbiotic bacteria）存在关联，其感染率是解析这类微生物共生体生物学效应的重要线索。本研究针对涵盖13科、69属、97种共468头臭蝽开展螺原体（Spiroplasma）感染情况调查。经诊断性PCR检测，结果显示有4科（30.8%）、7属（10.1%）、11种（11.3%）以及21头个体（4.5%）呈螺原体感染阳性。我们对全部21份螺原体阳性的臭蝽样本开展了螺原体16S核糖体RNA（16S rRNA）基因的PCR扩增与测序。分子系统发育分析显示，与臭蝽共生的螺原体共生体在螺原体科（Spiroplasmataceae）中可分为3个独立演化支，这表明臭蝽-螺原体共生关系存在多起源演化路径。螺原体的系统发育关系与宿主臭蝽的系统发育并不匹配，这说明二者并未发生宿主-共生体共成种事件。另一方面，隶属于同一臭蝽科的螺原体共生体往往具有较近的亲缘关系，这提示存在一定程度的宿主-共生体特异性，或是存在共生体的生态共享现象。针对细菌16S rRNA基因的扩增子测序、共生体的荧光原位杂交（Fluorescence in situ hybridization, FISH）可视化观察，以及宿主臭蝽的饲养实验结果显示，相较于主要肠道共生细菌，螺原体共生体的丰度普遍更低，且以相对较低的密度定殖于多种组织与器官中，并可通过垂直传播传递给子代。基于上述结果，本研究认为，总体而言螺原体共生体属于感染率较低的兼性共生细菌，对宿主臭蝽并非必需，而是呈现偏利共生关系，这与果蝇和蚜虫的螺原体共生体特征一致，不过其对宿主表型的具体影响仍有待未来研究进一步评估。