Symbionts can regulate animal reproduction in multiple ways, but the underlying physiological and biochemical mechanisms remain largely unknown. The presence of multiple lineages of maternally inherited, intracellular symbionts (the primary and secondary symbionts) in terrestrial arthropods is widespread in nature. However, the biological, metabolic, and evolutionary role of co-resident secondary symbionts for hosts is poorly understood. The bacterial symbionts Hamiltonella and Arsenophonus have very high prevalence in two globally important pests, the whiteflies Bemisia tabaci and Trialeurodes vaporariorum, respectively. Both symbionts coexist with the primary symbiont Portiera in the same host cell (bacteriocyte) and are maternally transmitted. We found that elimination of both Hamiltonella and Arsenophonous by antibiotic treatment reduced the percentage of female offspring in whiteflies. Microsatellite genotyping and cytogenetic analysis revealed that symbiont deficiency inhibited fertilization in whiteflies, leading to more haploid males with one maternal allele, which is consistent with distorted sex ratio in whiteflies. Quantification of essential amino acids and B vitamins in whiteflies indicated that symbiont deficiency reduced B vitamin levels, and dietary B vitamin supplementation rescued fitness of whiteflies. This study, for the first time, conclusively demonstrates that these two intracellular symbionts affect sex ratios in their whitefly hosts by regulating fertilization and supplying B vitamins. Our results reveal that both symbionts have the convergent function of regulating reproduction in phylogenetically-distant whitefly species. The 100% frequency, the inability of whiteflies to develop normally without their symbiont, and rescue with B vitamins suggests that both symbionts may be better considered co-primary symbionts.

共生体可以通过多种方式调节动物繁殖，但其潜在的生理和生化机制仍然很大程度上未知。陆地节肢动物中多种母系遗传的细胞内共生体（初级和次级共生体）的存在在自然界中广泛存在。然而，人们对宿主共存的次生共生体的生物学、代谢和进化作用知之甚少。细菌共生体汉密尔顿菌和砷菌在两种全球重要害虫（烟粉虱和烟粉虱）中的流行率非常高。两种共生体与初级共生体Portiera共存于同一宿主细胞（细菌细胞）中，并通过母系传播。我们发现，通过抗生素治疗消除汉密尔顿菌和蠹虫可降低粉虱雌性后代的比例。微卫星基因分型和细胞遗传学分析表明，共生体缺陷抑制了粉虱的受精，导致更多具有一种母体等位基因的单倍体雄性，这与粉虱性别比例扭曲的情况一致。对粉虱体内必需氨基酸和 B 族维生素的定量表明，共生体缺乏会降低 B 族维生素水平，而膳食 B 族维生素补充剂可恢复粉虱的健康。这项研究首次最终证明，这两种细胞内共生体通过调节受精和提供 B 族维生素来影响粉虱宿主的性别比例。我们的结果表明，两种共生体都具有调节系统发育较远的粉虱物种繁殖的趋同功能。100% 的频率、粉虱在没有共生体的情况下无法正常发育以及 B 族维生素的拯救表明，两种共生体可能更好地被视为共同主要共生体。