Hiding in plain sight: Cryptic enemies are found on cochineal (Hemiptera: Dactylopiidae), a scale insect of economic and cultural significance.

Cochineal is the common name for cactus-feeding scale insects in the Dactylopiidae. These ruby-red insects include the domesticated dye insect Dactylopius coccus. Dactylopius coccus and congeners have been introduced around the world, some accidentally, to become pests of prickly pear cactus species (Opuntia), and some intentionally, for dye production or biological control of pest Opuntia. In the northern Sonoran Desert (Tucson, AZ, USA), we studied the enemy complex of D. opuntiae and D. confusus on Opuntia and characterized two cryptic enemies, a coccinellid beetle predator and a parasitoid wasp. (1) Hyperaspis sp. The coccinellid predator Hyperaspis trifurcata shares a niche with a similar, typically all-black beetle. Morphological data, crossing tests, and phylogenetic results showed the black beetle to be a distinct, undescribed species in the genus Hyperaspis, with a rare spotted phenotype that is similar in appearance to H. trifurcata. Crossing tests among black and spotted forms showed the spotted morph is inherited as a single-locus dominant allele. (2) Formicencyrtus thoreauini. Rearing of this ant-like parasitoid wasp (Encyrtidae) in pure culture of D. opuntiae showed it to be a semi-gregarious primary parasitoid of cochineal. To our knowledge, this is the first confirmed instance of a cochineal parasitoid. Observations of development show early instar larvae keep their posterior end within the egg chorion, attached to an aeroscopic plate with a connection to the cochineal body wall. Late instar larvae are eventually surrounded by a membrane, likely of larval origin. Wasps then pupate in a dry air-filled chamber within the desiccated scale remains before chewing out as an adult. Both Hyperaspis sp. and F. thoreauini may have restricted distributions. Hyperaspis sp. does not appear to be a member of the cochineal community in Mexico or Texas, and scant records suggest F. thoreauini may also be restricted to the Southwestern USA.

Quality over quantity: unraveling the contributions to cytoplasmic incompatibility caused by two coinfecting Cardinium symbionts.

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.

Development of Common Leaf-Footed Bug Pests Depends on the Presence and Identity of Their Environmentally Acquired Symbionts.

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. IMPORTANCELeptoglossus 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.

Sex-based de novo transcriptome assemblies of the parasitoid wasp Encarsia suzannae, a host of the manipulative heritable symbiont Cardinium hertigii.

Parasitoid wasps in the genus Encarsia are commonly used as biological pest control agents of whiteflies and armored scale insects in greenhouses or the field. They are also hosts of the bacterial endosymbiont Cardinium hertigii, which can cause reproductive manipulation phenotypes, including parthenogenesis, feminization, and cytoplasmic incompatibility (the last is mainly studied in Encarsia suzannae). Despite their biological and economic importance, there are no published Encarsia genomes and only one public transcriptome. Here, 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 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. Our male and female transcriptomes will be valuable tools to better understand the biology of Encarsia and their evolutionary relatives, particularly in studies involving insects of only one sex.

Cardinium Localization During Its Parasitoid Wasp Host's Development Provides Insights Into Cytoplasmic Incompatibility.

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 of Cardinium CI-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 the Cardinium-E. suzannae system. Antibiotic treatment of adult Cardinium-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) Encarsia produces most sperm prior to adulthood, 2) FISH localization of Cardinium in 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 of Cardinium sperm modification in the context of what is known about analogous mechanisms of Wolbachia, a more extensively studied CI-inducing symbiont.

What Goes Up Might Come Down: the Spectacular Spread of an Endosymbiont Is Followed by Its Decline a Decade Later.

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.

Development of a multi-locus sequence typing system helps reveal the evolution of Cardinium hertigii, a reproductive manipulator symbiont of insects.

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.

Exposure to opposing temperature extremes causes comparable effects on Cardinium density but contrasting effects on Cardinium-induced cytoplasmic incompatibility.

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.

Transcriptome Sequencing Reveals Novel Candidate Genes for Cardinium hertigii-Caused Cytoplasmic Incompatibility and Host-Cell Interaction.

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.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia.

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.

"Darwin's corollary" and cytoplasmic incompatibility induced by Cardinium may contribute to speciation in Encarsia wasps (Hymenoptera: Aphelinidae).

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.

Conditional fitness benefits of the Rickettsia bacterial symbiont in an insect pest.

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.

Dynamics of the endosymbiont Rickettsia in an insect pest.

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.

Characteristics, phenotype, and transmission of Wolbachia in the sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), and its parasitoid Eretmocerus sp. nr. emiratus (Hymenoptera: Aphelinidae).

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.

Bacterial endosymbionts in field-collected samples of Trialeurodes sp. nr. abutiloneus (Hemiptera: Aleyrodidae).

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.

Factors affecting the strength of Cardinium-induced cytoplasmic incompatibility in the parasitic wasp Encarsia pergandiella (Hymenoptera: Aphelinidae).

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.

Comparative genomics suggests an independent origin of cytoplasmic incompatibility in Cardinium hertigii.

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.

Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias.

Maternally inherited bacterial symbionts of arthropods are common, yet symbiont invasions of host populations have rarely been observed. Here, we show that Rickettsia sp. nr. bellii swept into a population of an invasive agricultural pest, the sweet potato whitefly, Bemisia tabaci, in just 6 years. Compared with uninfected whiteflies, Rickettsia-infected whiteflies produced more offspring, had higher survival to adulthood, developed faster, and produced a higher proportion of daughters. The symbiont thus functions as both mutualist and reproductive manipulator. The observed increased performance and sex-ratio bias of infected whiteflies are sufficient to explain the spread of Rickettsia across the southwestern United States. Symbiont invasions such as this represent a sudden evolutionary shift for the host, with potentially large impacts on its ecology and invasiveness.

Almost there: transmission routes of bacterial symbionts between trophic levels.

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.

Population biology of cytoplasmic incompatibility: maintenance and spread of Cardinium symbionts in a parasitic wasp.

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 alpha-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 ( approximately 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.