A bacteriocyte symbiont determines whitefly sex ratio by regulating mitochondrial function.

Nutritional symbionts influence host reproduction, but the underlying molecular mechanisms are largely unclear. We previously found that the bacteriocyte symbiont Hamiltonella impacts the sex ratio of the whitefly Bemisia tabaci. Hamiltonella synthesizes folate by cooperation with the whitefly. Folate deficiency by Hamiltonella elimination or whitefly gene silencing distorted whitefly sex ratio, and folate supplementation restored the sex ratio. Hamiltonella deficiency or gene silencing altered histone H3 lysine 9 trimethylation (H3K9me3) level, which was restored by folate supplementation. Genome-wide chromatin immunoprecipitation-seq analysis of H3K9me3 indicated mitochondrial dysfunction in symbiont-deficient whiteflies. Hamiltonella deficiency compromised mitochondrial quality of whitefly ovaries. Repressing ovary mitochondrial function led to distorted whitefly sex ratio. These findings indicate that the symbiont-derived folate regulates host histone methylation modifications, which thereby impacts ovary mitochondrial function, and finally determines host sex ratio. Our study suggests that a nutritional symbiont can regulate animal reproduction in a way that differs from reproductive manipulators.

Bacteriocyte development is sexually differentiated in Bemesia tabaci.

Some symbiotic microbes are restricted to specialized host cells called bacteriocytes. However, the molecular and cellular mechanisms underlying the development of bacteriocytes are largely obscure. We find that maternally inherited bacteriocytes proliferate in adult females but degenerate in adult males of the whitefly Bemisia tabaci. Single-cell transcriptomics and immunohistochemistry reveal that cell division only occurs in the bacteriocytes of adult females, whereas autophagy and apoptosis are induced in the bacteriocytes of adult males. A transcription factor, Adf-1, enriched in bacteriocytes, is highly expressed in female bacteriocytes relative to male bacteriocytes. Silencing Adf-1 reduces the bacteriocyte number and Portiera titer and activates autophagy and apoptosis in females. The differential dynamics of both cell division and death in bacteriocytes and distinct expression of Adf-1 in bacteriocytes between whitefly sexes underlie the sexual differentiation of bacteriocyte development. Our study reveals that insect sex affects the development of bacteriocytes by cellular and molecular remodeling.

Autophagy Regulates Whitefly-Symbiont Metabolic Interactions.

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders. These symbionts can provide essential nutrients to the host. However, the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear. The whitefly Bemisia tabaci MEAM1 hosts "Candidatus Portiera aleyrodidarum" (here, "Ca. Portiera") and "Candidatus Hamiltonella defensa" (here, "Ca. Hamiltonella") bacteria in the same bacteriocyte. In this study, the induction of autophagy by chemical treatment and gene silencing decreased symbiont titers and essential amino acid (EAA) and B vitamin contents. In contrast, the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers, and amino acid and B vitamin contents. Furthermore, dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes, elevated TOR (target of rapamycin) expression, and increased symbiont titers. TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins. These data suggest that "Ca. Portiera" and "Ca. Hamiltonella" evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients. Taken together, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. Therefore, this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies. The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes. IMPORTANCE Nutritional symbionts, which are restricted to specialized host cells called bacteriocytes, can provide essential nutrients for many hosts. However, the cellular mechanisms of regulation of animal-symbiont metabolic associations have been largely unexplored. Here, using the whitefly-"Ca. Portiera"/"Ca. Hamiltonella" endosymbiosis, we demonstrate autophagy regulates the symbiont titers and thereby alters the essential amino acid and B vitamin contents. For persistence in the whitefly bacteriocytes, "Ca. Portiera" and "Ca. Hamiltonella" alleviate autophagy by activating the TOR (target of rapamycin) pathway through providing essential nutrients. Therefore, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. This study also provides insight into the cellular basis of bacteriocyte evolution and symbiosis persistence in the whitefly. The mechanisms underlying the role of autophagy in whitefly symbiosis could be widespread in many insect nutritional symbioses. These findings provide a new avenue for whitefly control via regulating autophagy in the future.

Lysine provisioning by horizontally acquired genes promotes mutual dependence between whitefly and two intracellular symbionts.

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.

Pantothenate mediates the coordination of whitefly and symbiont fitness.

Intracellular symbionts in insects often have reduced genomes. Host acquisition of genes from bacteria is an important adaptation that supports symbionts. However, the function of horizontally transferred genes in insect symbiosis remains largely unclear. The primary symbiont Portiera housed in bacteriocytes lacks pantothenate synthesis genes: panB and panC, which is presumably complemented by a fused gene panB-panC (hereafter panBC) horizontally transferred from bacteria in Bemisia tabaci MEAM1. We found panBC in many laboratory cultures, and species of B. tabaci shares a common evolutionary origin. We demonstrated that complementation with whitefly panBC rescued E. coli pantothenate gene knockout mutants. Portiera elimination decreased the pantothenate level and PanBC abundance in bacteriocytes, and reduced whitefly survival and fecundity. Silencing PanBC decreased the Portiera titer, reduced the pantothenate level, and decreased whitefly survival and fecundity. Supplementation with pantothenate restored the symbiont titer, PanBC level, and fitness of RNAi whiteflies. These data suggest that pantothenate synthesis requires cooperation and coordination of whitefly PanBC expression and Portiera. This host-symbiont co-regulation was mediated by the pantothenate level. Our findings demonstrated that pantothenate production, by the cooperation of a horizontally acquired, fused bacteria gene and Portiera, facilitates the coordination of whitefly and symbiont fitness. Thus, this study extends our understanding on the basis of complex host-symbiont interactions.