Rickettsia symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating.

Heritable symbionts are common among animals in nature, but the molecular mechanisms underpinning symbiont invasions of host populations have been elusive. In this study, we demonstrate the spread of Rickettsia in an invasive agricultural pest, the whitefly Bemisia tabaci Mediterranean (MED), across northeastern China from 2018 to 2023. Here, we show that the beneficial symbiont Rickettsia spreads by manipulating host hormone signals. Our analyses suggest that Rickettsia have been horizontally acquired by B. tabaci MED from another invasive whitefly B. tabaci Middle East-Asia Minor 1 during periods of coexistence. Rickettsia is transmitted maternally and horizontally from female B. tabaci MED individuals. Rickettsia infection enhances fecundity and results in female bias among whiteflies. Our findings reveal that Rickettsia infection stimulates juvenile hormone (JH) synthesis, in turn enhancing fecundity, copulation events, and the female ratio of the offspring. Consequently, Rickettsia infection results in increased whitefly fecundity and female bias by modulating the JH pathway. More female progeny facilitates the transmission of Rickettsia. This study illustrates that the spread of Rickettsia among invasive whiteflies in northeastern China is propelled by host hormone regulation. Such symbiont invasions lead to rapid physiological and molecular evolution in the host, influencing the biology and ecology of an invasive species.

Wolbachia symbionts control sex in a parasitoid wasp using a horizontally acquired gene.

Host reproduction can be manipulated by bacterial symbionts in various ways. Parthenogenesis induction is the most effective type of reproduction manipulation by symbionts for their transmission. Insect sex is determined by regulation of doublesex (dsx) splicing through transformer2 (tra2) and transformer (tra) interaction. Although parthenogenesis induction by symbionts has been studied since the 1970s, its underlying molecular mechanism is unknown. Here we identify a Wolbachia parthenogenesis-induction feminization factor gene (piff) that targets sex-determining genes and causes female-producing parthenogenesis in the haplodiploid parasitoid Encarsia formosa. We found that Wolbachia elimination repressed expression of female-specific dsx and enhanced expression of male-specific dsx, which led to the production of wasp haploid male offspring. Furthermore, we found that E. formosa tra is truncated and non-functional, and Wolbachia has a functional tra homolog, termed piff, with an insect origin. Wolbachia PIFF can colocalize and interact with wasp TRA2. Moreover, Wolbachia piff has coordinated expression with tra2 and dsx of E. formosa. Our results demonstrate the bacterial symbiont Wolbachia has acquired an insect gene to manipulate the host sex determination cascade and induce parthenogenesis in wasps. This study reveals insect-to-bacteria horizontal gene transfer drives the evolution of animal sex determination systems, elucidating a striking mechanism of insect-microbe symbiosis.

Insect Bacteriocytes: Adaptation, Development, and Evolution.

Bacteriocytes are host cells specialized to harbor symbionts in certain insect taxa. The adaptation, development, and evolution of bacteriocytes underlie insect symbiosis maintenance. Bacteriocytes carry enriched host genes of insect and bacterial origin whose transcription can be regulated by microRNAs, which are involved in host-symbiont metabolic interactions. Recognition proteins of peptidoglycan, the bacterial cell wall component, and autophagy regulate symbiont abundance in bacteriocytes. Horizontally transferred genes expressed in bacteriocytes influence the metabolism of symbiont peptidoglycan, which may affect the bacteriocyte immune response against symbionts. Bacteriocytes release or transport symbionts into ovaries for symbiont vertical transmission. Bacteriocyte development and death, regulated by transcriptional factors, are variable in different insect species. The evolutionary origin of insect bacteriocytes remains unclear. Future research should elucidate bacteriocyte cell biology, the molecular interplay between bacteriocyte metabolic and immune functions, the genetic basis of bacteriocyte origin, and the coordination between bacteriocyte function and host biology in diverse symbioses.

A chromosome-level genome assembly of the parasitoid wasp Eretmocerus hayati.

Hymenoptera is an order accounting for a large proportion of species in Insecta, among which Chalcidoidea contains many parasitoid species of biocontrol significance. Currently, some species genomes in Chalcidoidea have been assembled, but the chromosome-level genomes of Aphelinidae are not yet available. Using Illumina, PacBio HiFi and Hi-C technologies, we assembled a genome assembly of Eretmocerus hayati (Aphelinidae, Hymenoptera), a worldwide biocontrol agent of whiteflies, at the chromosome level. The assembled genome size is 692.1 Mb with a contig N50 of 7.96 Mb. After Hi-C scaffolding, the contigs was assembled onto four chromosomes with a mapping rate of > 98%. The scaffold N50 length is 192.5 Mb, and Benchmarking Universal Single-Copy Orthologues (BUSCO) value is 95.9%. The genome contains 370.8 Mb repeat sequences and total of 24471 protein coding genes. P450 gene families were identified and analyzed. In conclusion, our chromosome-level genome assembly provides valuable support for future research on the evolution of parasitoid wasps and the interaction between hosts and parasitoid wasps.

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.

Vitellogenin Facilitates Associations between the Whitefly and a Bacteriocyte Symbiont.

Integration between animal reproduction and symbiont inheritance is fundamental in symbiosis biology, but the underlying molecular mechanisms are largely unknown. Vitellogenin (Vg) is critical for oogenesis, and it is also a pathogen pattern recognition molecule in some animals. Previous studies have shown that Vg is involved in the regulation of symbiont abundance and transmission. However, the mechanisms by which an insect and its symbiont contribute to the function of Vg and how Vg impacts the persistence of insect-microbe symbiosis remain largely unclear. Symbionts are transovarially transmitted via maternal inheritance of the bacteriocytes in the whitefly Bemisia tabaci. Surprisingly, Vg is localized in bacteriocytes of whiteflies. Vg could be synthesized in whitefly bacteriocytes by the gene Vg expressed in these cells or exported into bacteriocytes from hemolymph via the Vg receptor. We further found that the juvenile hormone and "Candidatus Portiera aleyrodidarum" (here termed Portiera) control the level and localization of Vg in whiteflies. Immunocapture PCR revealed interactions between Vg and Portiera. Suppressing Vg expression reduced Portiera abundance as well as whitefly oogenesis and fecundity. Thus, we reveal that Vg facilitated the persistence of whitefly-bacteriocyte symbiont associations. This study will provide insight into the key role of Vg in the coevolution of insect reproduction and symbiont inheritance. IMPORTANCE Intracellular heritable symbionts have been incorporated into insect reproductive and developmental biology by various mechanisms. All Bemisia tabaci species harbor the obligate symbiont Portiera in specialized insect cells called bacteriocytes. We report that the whitefly juvenile hormone and Portiera determined vitellogenin (Vg) localization in bacteriocytes of whiteflies. In turn, Vg affected whitefly fecundity as well as fitness and transmission of the symbiont. Our findings show that Vg, a multifunctional protein, is indispensable for symbiont integration into the reproduction and development of insects. This reflects the outcome of long-term coevolution of the insect-microbe symbiosis.

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.

A novel microRNA regulates cooperation between symbiont and a laterally acquired gene in the regulation of pantothenate biosynthesis within Bemisia tabaci whiteflies.

Horizontally transferred genes (HTGs) play a key role in animal symbiosis, and some horizontally transferred genes or proteins are highly expressed in specialized host cells (bacteriocytes). However, it is not clear how HTGs are regulated, but microRNAs (miRNAs) are prime candidates given their previously demonstrated roles in symbiosis and impacts on the expression of host genes. A horizontally acquired PanBC that is highly expressed in whitefly bacteriocytes can cooperate with an obligate symbiont Portiera for pantothenate production, facilitating whitefly performance and Portiera titre. Here, we found that a whitefly miRNA, novel-m0780-5p, was up-regulated and its target panBC was down-regulated in Portiera-eliminated whiteflies. This miRNA was located in the cytoplasmic region of whitefly bacteriocytes. Injection of novel-m0780-5p agomir reduced the expression of PanBC in whitefly bacteriocytes, while injection of novel-m0780-5p antagomir enhanced PanBC expression. Agomir injection also reduced the pantothenate level, Portiera titre and whitefly performance. Supplementation with pantothenate restored Portiera titre and the fitness of agomir-injected whiteflies. Thus, we demonstrate that a whitefly miRNA regulates panBC-mediated host-symbiont collaboration required for pantothenate synthesis, benefiting the whitefly-Portiera symbiosis. Both panBC and novel-m0780-5p are present in the genomes of six Bemisia tabaci species. The expression of a novel miRNA in multiple B. tabaci species suggests that the miRNA evolved after panBC acquisition, and allowed this gene to be more tightly regulated. Our discovery provides the first account of a HTG being regulated by a miRNA from the host genome, and suggests key roles for interactions between miRNAs and HTGs in the functioning of symbiosis.

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.

New insights into the transovarial transmission of the symbiont Rickettsia in whiteflies.

Endosymbiont transmission via eggs to future host generations has been recognized as the main strategy for its persistence in insect hosts; however, the mechanisms for transmission have yet to be elucidated. Here, we describe the dynamic locations of Rickettsia in the ovarioles and eggs during oogenesis and embryogenesis in a globally significant pest whitefly Bemisia tabaci. Field populations of the whitefly have a high prevalence of Rickettsia, and in all Rickettsia-infected individuals, the bacterium distributes in the body cavity of the host, especially in the midgut, fat body, hemocytes, hemolymph, and near bacteriocytes. The distribution of Rickettsia was subjected to dynamic changes in the ovary during oogenesis, and our ultrastructural observations indicated that the bacteria infect host ovarioles during early developmental stages via two routes: (i) invasion of the tropharium by endocytosis and then transmission into vitellarium via nutritive cord and (ii) entry into vitellarium by hijacking bacteriocyte translocation. Most of the Rickettsia are degraded in the oocyte cytoplasm in late-stage oogenesis. However, a few reside beneath the vitelline envelope of mature eggs, spread into the embryo, and proliferate during embryogenesis to sustain high-fidelity transmission to the next generation. Our findings provide novel insights into the maternal transmission underpinning the persistence and spread of insect symbionts.

Intracellular symbionts drive sex ratio in the whitefly by facilitating fertilization and provisioning of B vitamins.

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.

Biotin provisioning by horizontally transferred genes from bacteria confers animal fitness benefits.

Insect symbionts are widespread in nature and lateral gene transfer is prevalent in insect symbiosis. However, the function of horizontally transferred genes (HTGs) in insect symbiosis remains speculative, including the mechanism that enables insects to feed on plant phloem deficient in B vitamins. Previously, we found there is redundancy in biotin synthesis pathways from both whitefly Bemisia tabaci and symbiotic Hamiltonella due to the presence of whitefly HTGs. Here, we demonstrate that elimination of Hamiltonella decreased biotin levels but elevated the expression of horizontally transferred biotin genes in whiteflies. HTGs proteins exhibit specific expression patterns in specialized insect cells called bacteriocytes housing symbionts. Complementation with whitefly HTGs rescued E. coli biotin gene knockout mutants. Furthermore, silencing whitefly HTGs in Hamiltonella-infected whiteflies reduced biotin levels and hindered adult survival and fecundity, which was partially rescued by biotin supplementation. Each of horizontally transferred biotin genes are conserved in various laboratory cultures and species of whiteflies with geographically diverse distributions, which shares an evolutionary origin. We provide the first experimental evidence that biotin synthesized through acquired HTGs is important in whiteflies and may be as well in other animals. Our findings suggest that B vitamin provisioning in animal-microbe symbiosis frequently evolved from bacterial symbionts to animal hosts through horizontal gene transfer events. This study will also shed light on how the animal genomes evolve through functional transfer of genes with bacterial origin in the wider contexts of microbial ecology.

CRISPR-Cas9-Based Genome Editing in the Silverleaf Whitefly (Bemisia tabaci).

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject and which have high mortality post injection. We developed a CRISPR-Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.

Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae).

BACKGROUND: Conogethes pinicolalis (Lepidoptera: Crambidae), is similar to Conogethes punctiferalis (yellow peach moth) and its host plant is gymnosperms, especially for masson pine. So far, less literature was reported on this pest. In the present study, we sequenced and characterized the antennal transcriptomes of male and female C. pinicolalis for the first time. RESULTS: Totally, 26 odorant-binding protein (OBP) genes, 19 chemosensory protein (CSP) genes, 55 odorant receptor (OR) genes and 20 ionotropic receptor (IR) genes were identified from the C. pinicolalis antennae transcriptome and amino sequences were annotated against homologs of C. punctiferalis. The neighbor-joining tree indicated that the amino acid sequence of olfactory related genes is highly homologous with C. punctiferalis. Furthermore, the reference genes were selected, and we recommended the phosphate dehydrogenase gene (GAPDH) or ribosomal protein 49 gene (RP49) to verify the target gene expression during larval development stages and RP49 or ribosomal protein L13 gene (RPL13) for adult tissues. CONCLUSIONS: Our study provides a starting point on the molecular level characterization between C. pinicolalis and C. punctiferalis, which might be supportive for pest management studies in future.

Patterns of host cell inheritance in the bacterial symbiosis of whiteflies.

Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria.

The inherited bacterial symbiont Hamiltonella influences the sex ratio of an insect host.

In many intracellular symbioses, the microbial symbionts provide nutrients advantageous to the host. However, the function of Hamiltonella defensa, a symbiotic bacterium localized in specialized host cells (bacteriocytes) of a whitefly Bemisia tabaci, is uncertain. We eliminate this bacterium from its whitefly host by two alternative methods: heat treatment and antibiotics. The sex ratio of the host progeny and subsequent generations of Hamiltonella-free females was skewed from 1 : 1 (male : female) to an excess of males, often exceeding a ratio of 20 : 1. B. tabaci is haplodiploid, with diploid females derived from fertilized eggs and haploid males from unfertilized eggs. The Hamiltonella status of the insect did not affect copulation frequency or sperm reserve in the spermathecae, indicating that the male-biased sex ratio is unlikely due to the limitation of sperm but likely to be associated with events subsequent to sperm transfer to the female insects, such as failure in fertilization. The host reproductive response to Hamiltonella elimination is consistent with two alternative processes: adaptive shift in sex allocation by females and a constitutive compensatory response of the insect to Hamiltonella-mediated manipulation. Our findings suggest that a bacteriocyte symbiont influences the reproductive output of female progeny in a haplodiploid insect.