Search

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.

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.

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.

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.

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.

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.

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.

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.