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Minute parasitoid wasps in the genus Encarsia are commonly used as biological pest control agents of whiteflies and armored scale insects in greenhouses or in the field. They are also a key host of the bacterial endosymbiont Cardinium hertigii which can cause a suite of reproductive manipulation phenotypes, including parthenogenesis, feminization, and cytoplasmic incompatibility; the last being most thoroughly studied in Encarsia suzannae. Despite their biological and economic importance, there are currently no published Encarsia genomes and only one public transcriptome. In this study, we applied a mapping-and-removal approach to eliminate known contaminants from previously-obtained Illumina sequencing data. We generated de novo transcriptome assemblies for both female and male E. suzannae which contain 45,986 and 54,762 final coding sequences, respectively. Benchmarking Single-Copy Orthologs (BUSCO) results indicate both assemblies are highly complete. Preliminary analyses revealed the presence of homologs of sex-determination genes characterized in other insects and putative venom proteins. These transcriptomes will be valuable tools to better understand the biology of Encarsia wasps and their evolutionary relatives. Furthermore, the separate male and female assemblies will be particularly useful references for studies involving insects of only one sex.

Many beneficial symbioses between bacteria and their terrestrial arthropod hosts are vertically transmitted from mother to offspring, ensuring that the progeny acquire necessary partners. Unusually, in several families of coreoid and lygaeoid bugs (Hemiptera), nymphs must instead ingest the beneficial symbiont, Burkholderia (sensu lato), from the environment early in development. We studied the effects of Burkholderia on development of two species of leaf-footed bug (Coreidae) in the genus Leptoglossus, Leptoglossus zonatus and Leptoglossus phyllopus. We found no evidence for vertical transmission of the symbiont but found stark differences in performance between symbiotic and aposymbiotic individuals. Symbiotic nymphs grew more rapidly, were approximately four times more likely to survive to adulthood than aposymbiotic bugs, and were two times larger. These findings suggest that Burkholderia is an obligate symbiont for the Leptoglossus species. We also tested for variation in fitness effects conferred by four symbiont isolates representing different species within the Burkholderia insect-associated stinkbug beneficial and environmental (SBE) clade. While three isolates conferred similar benefits to hosts, nymphs associated with the fourth isolate grew more slowly and weighed significantly less as adults. The effects of the four isolates were similar for both Leptoglossus species. This work indicates that both Burkholderia acquisition and isolate identity play critical roles in the growth and development of Leptoglossus. IMPORTANCE Leptoglossus zonatus and L. phyllopus are important polyphagous pests, and both species have been well-studied but generally without regard to their dependance on a bacterial symbiont. Our results indicate that the central role of Burkholderia in the biology of these insects, as well as in other leaf-footed bugs, should be considered in future studies of coreid life history, ecology, and pest management. Our work suggests that acquisition of Burkholderia is critical for the growth and development of Leptoglossus species. Further, we found that there was variation in performance outcomes according to symbiont identity, even among members of the stinkbug beneficial and environmental clade. This suggests that although environmental acquisition of a symbiont can provide extraordinary flexibility in partner associations, it also carries a risk if the partner is suboptimal.

Cytoplasmic incompatibility (CI) is a common form of reproductive sabotage caused by maternally inherited bacterial symbionts of arthropods. CI is a two-step manipulation: first, the symbiont modifies sperm in male hosts which results in the death of fertilized, uninfected embryos. Second, when females are infected with a compatible strain, the symbiont reverses sperm modification in the fertilized egg, allowing offspring of infected females to survive and spread the symbiont to high frequencies in a population. Although CI plays a role in arthropod evolution, the mechanism of CI is unknown for many symbionts. Cardinium hertigii is a common CI-inducing symbiont of arthropods, including parasitoid wasps like Encarsia partenopea. This wasp harbors two Cardinium strains, cEina2 and cEina3, and exhibits strong CI. The strains infect wasps at different densities, with the cEina3 present at a lower density than cEina2, and it was previously not known which strain caused CI. By differentially curing wasps of cEina3, we found that this low-density symbiont is responsible for CI and modifies males during their pupal stage. cEina2 does not modify host reproduction and may spread by ‘hitchhiking’ with cEina3 CI or by conferring an unknown benefit. The cEina3 strain also shows a unique localization pattern in male reproductive tissues. Instead of infecting sperm like other CI-inducing symbionts, cEina3 cells are found in somatic cells at the testis base and around the seminal vesicle. This may allow the low-density cEina3 to efficiently modify host males and suggests that cEina3 uses a different modification strategy than sperm-infecting CI symbionts.

Arthropods harbor heritable intracellular symbionts that may manipulate host reproduction to favor symbiont transmission. In cytoplasmic incompatibility (CI), the symbiont sabotages the reproduction of infected males such that high levels of offspring mortality result when they mate with uninfected females. In crosses with infected males and infected females, however (the “rescue” cross), normal numbers of offspring are produced. A common CI-inducing symbiont,Cardinium hertigii, causes variable levels of CI mortality in the parasitoid wasp,Encarsia suzannae.Previous work correlated CI-induced mortality with male development time in this system, although the timing ofCardiniumCI-induction and the relationship between development time and CI mortality was not well understood. Here, using a combination of crosses, manipulation of development time, and fluorescence microscopy, we identify the localization and the timing of the CI-induction step in theCardinium-E. suzannaesystem. Antibiotic treatment of adultCardinium-infected males did not reduce the mortality associated with the CI phenotype, suggesting that CI-alteration occurs prior to adulthood. Our results suggest that the alteration step occurs during the pupal period, and is limited by the duration of pupal development: 1)Encarsiaproduces most sperm prior to adulthood, 2) FISH localization ofCardiniumin testes showed an association with sperm nuclei throughout spermatogenesis but not with mature sperm, and 3) two methods of prolonging the pupal period (cool temperatures and the juvenile hormone analog methoprene) both caused greater CI mortality, suggesting the degree of alteration is limited by the duration of the pupal stage. Based on these results, we compare two models for potential mechanisms ofCardiniumsperm modification in the context of what is known about analogous mechanisms ofWolbachia, a more extensively studied CI-inducing symbiont.

When allopatric species with incomplete prezygotic isolation come into secondary contact, the outcome of their interaction is not easily predicted. The parasitoid wasp Encarsia suzannae (iES), infected by Cardinium inducing cytoplasmic incompatibility (CI), and its sibling species E. gennaroi (EG), not infected by bacterial endosymbionts, may have diverged because of the complementary action of CI and asymmetric hybrid incompatibilities. Whereas postzygotic isolation is now complete because of sterility of F1 hybrid progeny, prezygotic isolation is still incipient. We set up laboratory population cage experiments to evaluate the outcome of the interaction between ES and EG in two pairwise combinations: iES vs EG and cured ES (cES, where Cardinium was removed with antibiotics) vs EG. We also built a theoretical model aimed at exploring the role of life-history differences and asymmetric mating on competitive outcomes. In three of four cages in each treatment, ES dominated the interaction. We found evidence for reproductive interference, driven by asymmetric mating preferences, that gave a competitive edge to ES, the species that better discriminated against heterospecifics. However, we did not find the fecundity cost previously shown to be associated with Cardinium infection in iES. The model largely supported the experimental results. The finding of only a slight competitive edge of ES over EG in population cages suggests that in a more heterogeneous environment the species could coexist. This is supported by evidence that the two species coexist in sympatry, where preliminary data suggest reproductive character displacement may have reinforced postzygotic isolation.

Background Cardinium is an intracellular bacterial symbiont in the phylum Bacteroidetes that is found in many different species of arthropods and some nematodes. This symbiont is known to be able to induce three reproductive manipulation phenotypes, including cytoplasmic incompatibility. Placing individual strains of Cardinium within a larger evolutionary context has been challenging because only two, relatively slowly evolving genes, 16S rRNA gene and Gyrase B, have been used to generate phylogenetic trees, and consequently, the relationship of different strains has been elucidated in only its roughest form. Results We developed a Multi Locus Sequence Typing (MLST) system that provides researchers with three new genes in addition to Gyrase B for inferring phylogenies and delineating Cardinium strains. From our Cardinium phylogeny, we confirmed the presence of a new group D, a Cardinium clade that resides in the arachnid order harvestmen (Opiliones). Many Cardinium clades appear to display a high degree of host affinity, while some show evidence of host shifts to phylogenetically distant hosts, likely associated with ecological opportunity. Like the unrelated reproductive manipulator Wolbachia, the Cardinium phylogeny also shows no clear phylogenetic signal associated with particular reproductive manipulations. Conclusions The Cardinium phylogeny shows evidence of diversification within particular host lineages, and also of host shifts among trophic levels within parasitoid-host communities. Like Wolbachia, the relatedness of Cardinium strains does not necessarily predict their reproductive phenotypes. Lastly, the genetic tools proposed in this study may help future authors to characterize new strains and add to our understanding of Cardinium evolution.

Terrestrial arthropods, including insects, commonly harbor maternally inherited intracellular symbionts that confer benefits to the host or manipulate host reproduction to favor infected female progeny. These symbionts may be especially vulnerable to thermal stress, potentially leading to destabilization of the symbiosis and imposing costs to the host. For example, increased temperatures can reduce the density of a common reproductive manipulator, Wolbachia, and the strength of its crossing incompatibility (cytoplasmic incompatibility, or CI) phenotype. Another manipulative symbiont, Cardinium hertigii, infects ~ 6–10% of Arthropods, and also can induce CI, but there is little homology between the molecular mechanisms of CI induced by Cardinium and Wolbachia. Here we investigated whether temperature disrupts the CI phenotype of Cardinium in a parasitic wasp host, Encarsia suzannae. We examined the effects of both warm (32°C day/ 29°C night) and cool (20°C day/ 17°C night) temperatures on Cardinium CI and found that both types of temperature stress modified aspects of this symbiosis. Warm temperatures reduced symbiont density, pupal developmental time, vertical transmission rate, and the strength of both CI modification and rescue. Cool temperatures also reduced symbiont density, however this resulted in stronger CI, likely due to cool temperatures prolonging the host pupal stage. The opposing effects of cool and warm-mediated reductions in symbiont density on the resulting CI phenotype indicates that CI strength may be independent of density in this system. Temperature stress also modified the CI phenotype only if it occurred during the pupal stage, highlighting the likely importance of this stage for CI induction in this symbiosis., Author summary Insects often harbor heritable symbiotic bacteria that infect their cells and/or bodily fluids. These heritable bacteria are passed from mother to offspring and can have substantial effects on host insect biology, and include bacteria like Cardinium that cause mating incompatibilities between symbiont-infected and uninfected individuals. Often, the extent of these symbiont-conferred modifications correlates with the bacterial density in the host. The appearance of these phenotypes is also affected by temperature stress, which often reduces bacterial density. However, here we find that temperature-altered strength of Cardinium-induced mating incompatibility in a whitefly parasitoid wasp can be independent of Cardinium density. While heat treatment reduced the symbiont density and the phenotype, as expected, cold treatment also reduced symbiont density but increased the degree of mating incompatibility. Here, the prolonged duration of the host pupal development in the cold treatments appeared to be more important for phenotype strength. These results suggest that the connection between bacterial density and phenotype strength may not be as general as previously thought. Furthermore, the modification of this manipulative phenotype has implications for the effectiveness of the host, Encarsia suzannae, as a biological control agent.

Facultative, intracellular bacterial symbionts of arthropods may dramatically affect host biology and reproduction. The length of these symbiont-host associations may be thousands to millions of years, and while symbiont loss is predicted, there have been very few observations of a decline of symbiont infection rates. In a population of the sweet potato whitefly species (Bemisia tabaci MEAM1) in Arizona, USA, we documented the frequency decline of a strain of Rickettsia in the Rickettsia bellii clade from near-fixation in 2011 to 36% of whiteflies infected in 2017. In previous studies, Rickettsia had been shown to increase from 1 to 97% from 2000 to 2006 and remained at high frequency for at least five years. At that time, Rickettsia infection was associated with both fitness benefits and female bias. In the current study, we established matrilines of whiteflies from the field (2016, Rickettsia infection frequency = 58%) and studied (a) Rickettsia vertical transmission, (b) fitness and sex ratios associated with Rickettsia infection, (c) symbiont titer, and (d) bacterial communities within whiteflies. The vertical transmission rate was high, approximately 98%. Rickettsia infection in the matrilines was not associated with fitness benefits or sex ratio bias and appeared to be slightly costly, as more Rickettsia-infected individuals produced non-hatching eggs. Overall, the titer of Rickettsia in the matrilines was lower in 2016 than in the whiteflies collected in 2011, but the titer distribution appeared bimodal, with high- and low-titer lines, and constancy of the average titer within lines over three generations. We found neither association between Rickettsia titer and fitness benefits or sex ratio bias nor evidence that Rickettsia was replaced by another secondary symbiont. The change in the interaction between symbiont and host in 2016 whiteflies may explain the drop in symbiont frequency we observed.

Symbioses between bacteria and insects are often associated with changes in important biological traits that can significantly affect host fitness. To a large extent, studies of these interactions have been based on physiological changes or induced phenotypes in the host, and the genetic mechanisms by which symbionts interact with their hosts have only recently become better understood. Learning about symbionts has been challenging in part due to difficulties such as obtaining enough high quality genomic material for high throughput sequencing technology, especially for symbionts present in low titers, and in small or difficult to rear non-model hosts. Here we introduce a new method that substantially increases the yield of bacterial DNA in minute arthropod hosts, and requires less starting material relative to previous published methods.

The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts’ biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes., Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts’ biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium-infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host-symbiont interactions, and can be considered different biological species. One ('E. inaron') is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other ('E. partenopea'). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting 'E. partenopea' induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia. Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae. CI crosses in this host–symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

Inherited intracellular insect endosymbionts may manipulate host reproduction or provide fitness benefits to their hosts in ways that result in their rapid spread throughout a host population. Fitness benefits in particular can result in the increased pest potential of agriculturally important insects. While benefits due to endosymbiont infection have been well studied in laboratory assays, very little is known about how these benefits translate to insect performance in the field. Laboratory experiments have shown that the maternally inherited bacterial endosymbiont, Rickettsia, increases the fitness of its whitefly host, Bemisia tabaci, through improved fecundity, faster development times and female-biased sex ratios. We conducted field population cage studies to determine whether the benefits conferred by Rickettsia to whiteflies in the laboratory were evident on one of its major hosts, cotton, under field conditions in Arizona, USA. In cages with either Rickettsia-infected or uninfected whiteflies, we observed up to ten-fold higher whitefly egg and nymph densities when whiteflies were Rickettsia-infected compared with uninfected whiteflies throughout the season. We also observed a steep initial increase in Rickettsia frequency in population cages started with either 25% or 50% Rickettsia-infected whiteflies, with the 50% cages approaching fixation within three generations. Using growth rates obtained in the density cages, we calculated and compared an expected trajectory of the frequencies of Rickettsia infection with the observed frequencies. Results showed similar observed and expected frequencies of Rickettsia in the first two generations, followed by a significantly lower than expected frequency in three of four treatment/sample combinations at the end of the season. Taken together, these results confirm the patterns of fecundity and population growth observed in laboratory assays, under field conditions, as well as provides preliminary empirical support for a Rickettsia equilibrium frequency of less than 100%.

Facultative bacterial endosymbionts are common, influential associates of arthropods, yet their movement among host species has not been well documented. Plant-mediated transmission of Rickettsia has been shown for the whitefly Bemisia tabaci. Bemisia tabaci in USA cotton fields harbors the secondary symbionts Rickettsia and Hamiltonella, and co-occurs with Trialeurodes sp. nr. abutiloneus whiteflies. To determine whether symbionts may be shared, the microbial diversity of these whiteflies on cotton across the USA was analyzed. Trialeurodes sp. nr. abutiloneus bore Portiera, Pseudomonas, Serratia, Arsenophonus and Wolbachia. No Rickettsia or Hamiltonella were detected. These results provide no evidence for horizontal transmission of symbionts between these whitefly genera.

Arthropods commonly carry maternally inherited intracellular bacterial symbionts that may profoundly influence host biology and evolution. The intracellular symbiont Rickettsia sp. nr. bellii swept rapidly into populations of the sweetpotato whitefly Bemisia tabaci in the south-western USA. Previous laboratory experiments showed female-bias and fitness benefits were associated with Rickettsia infection, potentially explaining the high frequencies of infection observed in field populations, but the effects varied with whitefly genetic line. Here, we explored whether host extranuclear or nuclear genes influenced the variation in the Rickettsia–host phenotype in two genetic lines of the whitefly host, each with Rickettsia-infected and uninfected sublines. Introgression between the Rickettsia-infected subline of one genetic line and the Rickettsia-uninfected subline of the other was used to create two new sublines, each with the maternally inherited extranuclear genetic lineages of one line (Rickettsia, two other symbionts and the mitochondria) and the nuclear genotype of the other. Performance assays comparing the original and new lines showed that in addition to Rickettsia, the interaction of Rickettsia infection with host nuclear genotype influenced development time and the sex ratio of the progeny, whereas the extranuclear genotype did not. Host nuclear genotype, but not extranuclear genotype, also influenced the titre of Rickettsia. Our results support the hypothesis that differences in host nuclear genotype alone may explain considerable within-population variation in host–symbiont phenotype and may contribute to the observed variation in Rickettsia–whitefly interactions worldwide.

The potential importance of cytoplasmic incompatibility (CI)-inducing bacterial symbionts in speciation of their arthropod hosts has been debated. Theoretical advances have led to a consensus that a role is plausible when CI is combined with other isolating barriers. However, the insect model systems Nasonia and Drosophila are the only two experimental examples documented. Here, we analyzed the components of reproductive isolation between the parasitoid wasp Encarsia suzannae, which is infected by the CI-inducing symbiont Cardinium, and its uninfected sibling species Encarsia gennaroi. Laboratory crosses demonstrated that: (1) sexual isolation is incomplete; (2) hybrid offspring production is greatly reduced in the interspecific CI cross; (3) viable hybrids may be produced by curing E. suzannae males of Cardinium with antibiotics; (4) hybrid offspring production in the reciprocal cross is greatly reduced by hybrid inviability due to genetic incompatibilities; (5) hybrid sterility is nearly complete in both directions at the F1 stage. Thus, asymmetrical hybrid incompatibilities and CI act as complementary isolating mechanisms. We propose a new model for contributions of CI symbionts to speciation, with CI reducing gene flow between species in one direction, and in the other, a symbiont sweep resulting in accelerated mtDNA evolution, negative cytonuclear interactions, and hybrid incompatibilities.

1. Bacterial symbionts have the potential to alter insect fitness, which could influence insect competitive ability. To investigate this possibility, within-host competitions were staged between individuals of the parasitoid, Encarsia inaron Walker, that were differentially infected with the bacterial symbionts Cardinium and Wolbachia. 2. When parasitoids of different infection status parasitised the same whitefly host, there was no evidence that symbiont infection influenced the outcome of competition. 3. Using symbionts as markers, a significant advantage for the second (superparasitising) wasp was detected when eggs were deposited within 4 h of one another, probably as a result of ovicidal behaviour by the superparasitoid, but not when eggs were deposited 1 day apart. Additionally, the emerged sex ratio of superparasitoid offspring was male biased when eggs were deposited 4 h after the initial eggs, but had an even sex ratio when deposited 1 day later. 4. The present study demonstrates the potential utility of symbiont infection as a marking system for investigating within-host competition among parasitoids.

Bacteria that cause cytoplasmic incompatibility (CI) are perhaps the most widespread parasites of arthropods. CI symbionts cause reproductive failure when infected males mate with females that are either uninfected or infected with a different, incompatible strain. Until recently, CI was known to be caused only by the α-proteobacterium Wolbachia. Here we present the first study of the population biology of Cardinium, a recently discovered symbiont in the Bacteroidetes that causes CI in the parasitic wasp Encarsia pergandiella (Hymenoptera: Aphelinidae). Cardinium occurs at high frequency (∼92%) in the field. Using wasps that were recently collected in the field, we measured parameters that are crucial for understanding how CI spreads and is maintained in its host. CI Cardinium exhibits near-perfect rates of maternal transmission, causes a strong reduction in viable offspring in incompatible crosses, and induces a high fecundity cost, with infected females producing 18% fewer offspring in the first 4 days of reproduction. We found no evidence for paternal transmission or horizontal transmission of CI Cardinium through parasitism of an infected conspecific. No evidence for cryptic parthenogenesis in infected females was found, nor was sex allocation influenced by infection. We incorporated our laboratory estimates into a model of CI dynamics. The model predicts a high stable equilibrium, similar to what we observed in the field. Interestingly, our model also predicts a high threshold frequency of CI invasion (20% for males and 24% for females), below which the infection is expected to be lost. We consider how this threshold may be overcome, focusing in particular on the sensitivity of CI models to fecundity costs. Overall our results suggest that the factors governing the dynamics of CI Wolbachia and Cardinium are strikingly similar.

1. Studies of inter-specific competition in parasitoids have largely focused on the outcome of within-host competition and the behavioural mechanisms by which female parasitoids prevent competition. Another, less well studied, possibility is oviposition preceded by ‘heterospecific ovicide’, the destruction of the other species’ egg. Heterospecific ovicide essentially eliminates within-host competition. 2. This study investigated the mechanisms and outcome of within-host competition in Encarsia formosa and Encarsia luteola, solitary endoparasitoids of whitefly pests. These species are known to commit ovicide of conspecific eggs. 3. Competition experiments indicated that the offspring of second-ovipositing females had an apparent advantage in competition, regardless of whether the second female was E. formosa or E. luteola. 4. Observations of ovipositor movement through the cuticle of host whitefly nymphs showed that both species often committed heterospecific ovicide and then oviposited or host-fed. Multiparasitism and heterospecific host discrimination were less common and absent respectively. 5. Heterospecific ovicide appears to explain the second-female advantage in competition between these species. Second-female advantage is contrary to the paradigmatic view of multiparasitism, where the first-ovipositing female has an advantage in competition or one of the species consistently prevails in competition.

Inherited bacterial symbionts are common in arthropods and can have strong effects on the biology of their hosts. These effects are often mediated by host ecology. The Rickettsia symbiont can provide strong fitness benefits to its insect host, Bemisia tabaci, under laboratory and field conditions. However, the frequency of the symbiont is heterogeneous among field collection sites across the USA, suggesting that the benefits of the symbiont are contingent on additional factors. In two whitefly genetic lines collected from the same location, we tested the effect of Rickettsia on whitefly survival after heat shock, on whitefly competitiveness at different temperatures, and on whitefly competitiveness at different starting frequencies of Rickettsia. Rickettsia did not provide protection against heat shock nor affect the competitiveness of whiteflies at different temperatures or starting frequencies. However, there was a strong interaction between Rickettsia infection and whitefly genetic line. Performance measures indicated that Rickettsia was associated with significant female bias in both whitefly genetic lines, but in the second whitefly genetic line it conferred no significant fitness benefits nor conferred any competitive advantage to its host over uninfected whiteflies in population cages. These results help to explain other reports of variation in the phenotype of the symbiosis. Furthermore, they demonstrate the complex nature of these close symbiotic associations and the need to consider these interactions in the context of host population structure.

Terrestrial arthropods are commonly infected with maternally inherited symbionts that cause reproductive incompatibilities between hosts with differing infection status. Such symbionts can have major effects on the efficacy of a biological control program if releases are comprised of mixtures of differentially infected individuals. In this study, the ash whitefly parasitoid, Encarsia inaron (Hymenoptera: Aphelinidae) from Arizona was surveyed for the presence of heritable bacterial symbionts; experiments were also performed to test for two phenotypes known to be caused by Encarsia symbionts—cytoplasmic incompatibility and changes in oviposition behavior and host use. E. inaron has successfully reduced ash whitefly to non-pest status in all three locations it has been released (California, Arizona, and North Carolina) and is also notable as one of the only Encarsia species that is not autoparasitic, with both male and female wasps developing as primary parasitoids of whiteflies. We show that E. inaron is infected with both Wolbachia and Cardinium. While there was no effect of the symbionts on oviposition behavior or host use, crosses between doubly infected male wasps and uninfected females resulted in a severe reduction in the number of female offspring; male offspring production was unaffected. This study thus serves as a further warning that ascribing a phenotype to a symbiont with confidence depends on eliminating the possibility of a mixed infection, and establishes E. inaron as a useful model for dissecting Wolbachia–Cardinium interactions.

Vertically transmitted symbionts of arthropods have been implicated in several reproductive manipulations of their hosts. These include cytoplasmic incompatibility (CI), parthenogenesis induction in haplodiploid species (PI), feminization and male killing. One symbiont lineage in the alpha-Proteobacteria, Wolbachia, is the only bacterium known to cause all of these effects, and has been thought to be unique in causing CI, in which the fecundity of uninfected females is reduced after mating with infected males. Here, we provide evidence that an undescribed symbiont in the Bacteroidetes group causes CI in a sexual population of the parasitic wasp Encarsia pergandiella. Wasps were crossed in all four possible combinations of infected and uninfected individuals. In the cross predicted to be incompatible, infected (I) males x uninfected (U) females, progeny production was severely reduced, with these females producing only 12.6% of the number of progeny in other crosses. The incompatibility observed in this haplodiploid species was the female mortality type; dissections showed that most progeny from the incompatible cross died as eggs. The 16S rDNA sequence of this symbiont is 99% identical to a parthenogenesis-inducing symbiont in other Encarsia, and 96% identical to a feminizing symbiont in haplodiploid Brevipalpus mites. Thus, this recently discovered symbiont lineage is capable of inducing three of the four principal manipulations of host reproduction known to be caused by Wolbachia.

Theory predicts that intraguild consumers such as predators or parasitoids may displace more specialized heterospecific competitors and thereby actually increase the population densities of a shared host or prey. We tested this idea with a native primary parasitoid, Eretmocerus eremicus, and an exotic autoparasitoid Encarsia sophia, both at- tacking the sweetpotato whitefly Bemisia tabaci. Autoparasitoids are intraguild consumers that attack and kill both the immature stages of hemipteran hosts, such as whiteflies, and heterospecific and conspecific parasitoids. In population cages on cotton plants in the field in 1997 and 1998, we introduced whiteflies and then parasitoids in a replacement design with constant total numbers of parasitoids, as follows: (1) control (whiteflies only), (2) E. eremicus only, (3) E. sophia only, and (4) both E. eremicus and E. sophia. Destructive samples of plants were taken 2, 4, and 6 wk after wasp releases, and immature whitefly and wasp stages were censused. In 1997, there were no significant differences in whitefly densities among treatments. In 1998, the control treatment densities were significantly greater than parasitoid treatments, but there was no difference among the parasitoid treatments, indicating equivalent sup- pression of whitefly populations by the two parasitoid species. In both years, similar patterns in parasitoid dynamics were observed. Densities of E. eremicus were significantly higher in the absence of E. sophia. In contrast, E. sophia densities were unaffected by the presence of E. eremicus. The results suggest that interference by the autoparasitoid reduced primary parasitoid density, but with no concommitant disruption of host suppression. The results support theoretical predictions that no disruption should occur when both parasitoids are equally efficient and suggest that an autoparasitoid may be as efficient as a primary par- asitoid in suppressing host densities.

Recent population dynamic theory predicts that disruption of biological control may occur when one parasitoid species' superiority in intrinsic competition is associated with a lower ability to find and exploit hosts (i.e., ability in extrinsic competition). One might expect such a trade-off, for instance, if parasitoids with larger (and fewer) eggs are more likely to prevail in intrinsic competition than species with smaller (and more numerous) eggs. We tested the idea that relative egg size could be used to predict the outcome of intrinsic competition in two closely related endoparasitoids, Encarsia pergandiella Howard and Encarsia formosa Gahan. Contrary to expectation, the parasitoid species with smaller eggs, E. pergandiella, prevailed in intrinsic competition, regardless of the order that hosts were exposed to the two species. In a literature survey, we found four studies of competing pairs of endoparasitoid species for which: (a) egg size estimates were available and (b) one species was consistently superior in intrinsic competition. In three of the four studies, the small-egged species prevailed in intrinsic competition, as we also found. Although E. formosa lost in intrinsic competition, this species negatively affected E. pergandiella's progeny production by host feeding on and killing hosts containing E. pergandiella eggs. E. formosa females also host fed on conspecific-parasitized hosts. As a mechanism of both intra- and interspecific interference competition, host feeding on parasitized hosts contradicts assumptions about the nature of interference competition in existing population dynamics models.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such asWolbachia. Matings between males carrying CI-inducingWolbachiaand uninfected females, or between males and females infected with differentWolbachiastrains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachiacause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other thanWolbachiaaffect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium,Cardinium, which naturally infects a parasitic wasp,Encarsia suzannae. CI crosses in this host–symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

A new heritable bacterial association can bring a fresh set of molecular capabilities, providing an insect host with an almost instantaneous genome extension. Increasingly acknowledged as agents of rapid evolution, inherited microbes remain underappreciated players in pest management programs. A Rickettsia bacterium was tracked sweeping through populations of an invasive whitefly provisionally described as the “B” or “MEAM1” of the Bemisia tabaci species complex, in the southwestern USA. In this population, Rickettsia provides strong fitness benefits and distorts whitefly sex ratios under laboratory conditions. In contrast, whiteflies in Israel show few apparent fitness benefits from Rickettsia under laboratory conditions, only slightly decreasing development time. A survey of B. tabaci B samples revealed the distribution of Rickettsia across the cotton-growing regions of Israel and the USA. Thirteen sites from Israel and 22 sites from the USA were sampled. Across the USA, Rickettsia frequencies were heterogeneous among regions, but were generally very high, whereas in Israel, the infection rates were lower and declining. The distinct outcomes of Rickettsia infection in these two countries conform to previously reported phenotypic differences. Intermediate frequencies in some areas in both countries may indicate a cost to infection in certain environments or that the frequencies are in flux. This suggests underlying geographic differences in the interactions between bacterial symbionts and this serious agricultural pest.

Wolbachia is a common intracellular bacterial endosymbiont of insects, causing a variety of effects including reproductive manipulations such as cytoplasmic incompatibility (CI). In this study, we characterized Wolbachia in the whitefly Bemisia tabaci and in the whitefly parasitoid Eretmocerus sp. nr. emiratus. We also tested for horizontal transmission of Wolbachia between and within trophic levels, and we determined the phenotype of Wolbachia in E. sp. nr. emiratus. Using multilocus sequence typing and phylogenetic analyses, we found that B. tabaci and E. sp. nr. emiratus each harbor a different and unique strain of Wolbachia. Both strains belong to the phylogenetic supergroup B. No evidence for horizontal transmission of Wolbachia between and within trophic levels was found in our study system. Finally, crossing results were consistent with a CI phenotype; when Wolbachia-infected E. sp. nr. emiratus males mate with uninfected females, wasp progeny survival dropped significantly, and the number of females was halved. This is the first description of CI caused by Wolbachia in the economically important genus Eretmocerus. Our study underscores the expectation that horizontal transmission events occur rarely in the dynamics of secondary symbionts such as Wolbachia, and highlights the importance of understanding the effects of symbionts on the biology of natural enemies.

Encarsia transvenais an ‘autoparasitoid’ in the hymenopteran family Aphelinidae. In this species, female eggs are laid in whitefly nymphs. Male eggs are laid externally on immature parasitoids enclosed within the whitefly integument, either their own species, or other primary parasitoids. We explored parasitism by E. transvenaof conspecific female immatures and those of a native primary parasitoid, Eretmocerus eremicus, in laboratory experiments. In the first experiment, female E. transvenawere offered different combinations of two stages of E. transvena(late larvae ‐ prepupae (ET2), and early pupae (ET3)), and one stage of E. eremicus (prepupae ‐ early pupae (EE2)) in paired choice tests. The results indicated very little parasitism of ET3 relative to the host it was paired with, either EE2 or ET2. However, when EE2 was offered with ET2, there was no statistically significant difference in parasitism. In a no-choice experiment in which oviposition patterns and male progeny development were examined in four stages of both species of wasp, clear differences were observed between the host species. Only one stage of E. transvena (ET2) was parasitized and supported development of male E. transvena to any significant degree. In contrast, in E. eremicus, EE2, EE3 (red-eyed pupae), and EE4 (late pupae) were all parasitized, and male E. transvenaemerged from all three stages, although fewer males emerged from EE4. In both species, wasp larvae that were still enclosed within the wet whitefly remains (ET1 and EE1) were parasitized at a very low rate. Lastly, an experiment that determined the length of the later developmental stages of E. transvenaand E. eremicussuggested that the duration of the period in which E. transvena is susceptible to parasitism by conspecific females is less than half the period of susceptibility of E. eremicus. These results taken together suggest the potential for interference of E. eremicus by E. transvena, but other factors not examined here may also influence the outcome of interactions in the field.

Bacteria that cause cytoplasmic incompatibility (CI) are among the most common maternally transmitted parasites of insects. In CI, uninfected females produce few or no offspring when they mate with infected males and, as a result, are often at a reproductive disadvantage relative to infected females. Two different bacteria are known to cause CI, Wolbachia and Cardinium. CI Cardinium was discovered more recently and has been little studied. Here, factors that could influence the reduction in reproductive output in a CI cross, or CI “strength,” were explored in the parasitic wasp Encarsia pergandiella. Cardinium in this wasp exhibits variable CI strength. Experiments tested the effect of male age, male size, male host species, Cardinium density, and male development time on CI strength. We found a striking effect of male development time, with males that took longer to develop exhibiting stronger CI when mated to uninfected females. Male age had little effect; although in one experiment, the oldest males exhibited stronger CI. Male size, host species, and bacterial density had no effect on the strength of CI. Identifying the factors that control CI are crucial for understanding the dynamics of infection, as well as the success of strategies that aim to use CI microbes to control insect pests and disease vectors.

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI–inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI., Author Summary Many arthropods are infected with bacterial symbionts that are maternally transmitted and have a great impact on their hosts' biology, ecology, and evolution. One of the most common phenotypes of facultative symbionts appears to be cytoplasmic incompatibility (CI), a type of reproductive failure in which bacteria in males modify sperm in a way that reduces the reproductive success of uninfected female mates. In spite of considerable interest, the genetic basis for CI is largely unknown. Cardinium hertigii, a symbiont of tiny parasitic wasps, is the only bacterial group other than the well-studied Wolbachia that is known to cause CI. Analysis of the Cardinium genome indicates that CI evolved independently in Wolbachia and Cardinium. However, a suite of shared proteins was likely involved in mediating host cell interactions, and CI shows functional overlap in both lineages. Our analysis suggests the presence of an unusual phage-derived, putative secretion system and reveals that Cardinium encodes biosynthetic pathways that suggest a potential role in host nutrition. Our findings provide a novel comparative context for understanding the mechanistic basis of CI and substantially increase our knowledge on reproductive manipulator symbionts that do not only severely affect population genetic structure of arthropods but may also serve as powerful tools in pest management.

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts.

Many bacterial endosymbionts of insects are capable of manipulating their host's reproduction for their own benefit. The most common strategy of manipulation is cytoplasmic incompatibility (CI), in which embryonic mortality results from matings between uninfected females and infected males. In contrast, embryos develop normally in infected females, whether or not their mate is infected, and infected progeny are produced. In this way, the proportion of infected females increases in the insect population, thereby promoting the spread of the maternally inherited bacteria. However, what happens when multiple endosymbionts inhabit the same host? The parasitoid wasp Encarsia inaron is naturally infected with two unrelated endosymbionts, Cardinium and Wolbachia, both of which have been documented to cause CI in other insects. Doubly infected wasps show the CI phenotype. We differentially cured E. inaron of each endosymbiont, and crossed hosts of different infection status to determine whether either or both bacteria caused the observed CI phenotype in this parasitoid, and whether the two symbionts interacted within their common host. We found that Wolbachia caused CI in E. inaron, but Cardinium did not. We did not find evidence that Cardinium was able to modify or rescue Wolbachia-induced CI, nor did we find that Cardinium caused progeny sex ratio distortion, leaving the role of Cardinium in E. inaron a mystery.

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts – Rickettsia and Hamiltonella – from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.

Previously, analysis of 16S rDNA sequences placed a newly discovered lineage of bacterial symbionts of arthropods in the ‘Bacteroidetes’. This symbiont lineage is associated with a number of diverse host reproductive manipulations, including induction of parthenogenesis in severalEncarsiaparasitoid wasps (Hymenoptera: Aphelinidae). In this study, electron microscopy and phylogenetic analysis of the 16S rRNA andgyrBgenes of symbionts fromEncarsia hispidaandEncarsia pergandiellaare used to describe and further characterize these bacteria. Phylogenetic analyses based on these two genes showed that theEncarsiasymbionts are allied with theCytophaga aurantiacalineage within the ‘Bacteroidetes’, with their closest described relative being the acanthamoeba symbiont ‘CandidatusAmoebophilus asiaticus’. TheEncarsiasymbionts share 97 % 16S rDNA sequence similarity withBrevipalpusmite andIxodestick symbionts and 88 % sequence similarity with ‘CandidatusA. asiaticus’. Electron microscopy revealed that many of the bacteria found in the ovaries of the twoEncarsiaspecies contained a regular, brush-like array of microfilament-like structures that appear to be characteristic of the symbiont. Finally, the role of this bacterium in parthenogenesis induction inE. hispidawas confirmed. Based on phylogenetic analyses and electron microscopy, classification of the symbionts fromEncarsiaas ‘CandidatusCardinium hertigii’ is proposed.

The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia , a genus of parasitoid wasps. Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the “ Encarsia bacterium” (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia . Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia . Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.