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.

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.

Breakdown of a defensive symbiosis, but not endogenous defences, at elevated temperatures.

Environmental factors, including temperature, can have large effects on species interactions, including mutualisms and antagonisms. Most insect species are infected with heritable bacterial symbionts with many protecting their hosts from natural enemies. However, many symbionts or their products are thermally sensitive; hence, their effectiveness may vary across a range of temperatures. In the pea aphid, Acyrthosiphon pisum, the bacterial symbiont Hamiltonella defensa and its associated APSE bacteriophages confer resistance to this aphid's dominant parasitoid, Aphidius ervi. Here, we investigate the effects of temperature on both endogenous and symbiont-based protection against this parasitoid. We also explored the defensive properties of the X-type symbiont, a bacterium hypothesized to shape aphid defence when co-occurring with H. defensa. We show that H. defensa protection fails at higher temperatures, although some aphid genotype and H. defensa strain combinations are more robust than others at moderately warmer temperatures. We also found that a single X-type strain neither defended against parasitism by A. ervi nor rescued lost H. defensa protection at higher temperatures. In contrast, endogenous aphid resistance was effective across temperatures, revealing that these distinct defensive modes are not equally robust to changing environments. Through a survey of field-collected pea aphids, we found a negative correlation between H. defensa frequencies and average daily temperatures across North American locales, fitting expectations for reduced symbiont benefits under warm climates. Based on these findings, we propose that rising global temperatures could promote the widespread breakdown of defensive mutualisms, a prospect with implications for both human and ecosystem health.

Multi-modal defences in aphids offer redundant protection and increased costs likely impeding a protective mutualism.

The pea aphid, Acyrthosiphon pisum, maintains extreme variation in resistance to its most common parasitoid wasp enemy, Aphidius ervi, which is sourced from two known mechanisms: protective bacterial symbionts, most commonly Hamiltonella defensa, or endogenously encoded defences. We have recently found that individual aphids may employ each defence individually, occasionally both defences together, or neither. In field populations, Hamiltonella-infected aphids are found at low to moderate frequencies and while less is known about the frequency of resistant genotypes, they show up less often than susceptible genotypes in field collections. To better understand these patterns, we sought to compare the strengths and costs of both types of defence, individually and together, in order to elucidate the selective pressures that maintain multi-modal defence mechanisms or that may favour one over the other. We experimentally infected five aphid genotypes (two lowly and three highly resistant), each with two symbiont strains, Hamiltonella-APSE8 (moderate protection) and Hamiltonella-APSE3 (high protection). This resulted in three sublines per genotype: uninfected, +APSE8 and +APSE3. Each of the 15 total sublines was first subjected to a parasitism assay to determine its resistance phenotype and in a second experiment, a subset was chosen to compare fitness (fecundity and survivorship) in the presence and absence of parasitism. In susceptible aphid genotypes, parasitized sublines infected with Hamiltonella generally showed increased protection with direct fitness benefits, but clear infection costs to fitness in the absence of parasitism. In resistant genotypes, Hamiltonella infection rarely conferred additional protection, often further reduced fecundity and survivorship when enemy challenged, and resulted in constitutive fitness costs in the absence of parasitism. We also identified strong aphid genotype × symbiont-strain interactions, such that the best defensive strategy against parasitoids varied for each aphid genotype; one performed best with no protective symbionts, the others with particular strains of Hamiltonella. This surprising variability in outcomes helps explain why Hamiltonella infection frequencies are often intermediate and do not strongly track parasitism frequencies in field populations. We also find that variation in endogenous traits, such as resistance, among host genotypes may offer redundancy and generally limit the invasion potential of mutualistic microbes in insects.

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.

Aphid Heritable Symbiont Exploits Defensive Mutualism.

Insects and other animals commonly form symbioses with heritable bacteria, which can exert large influences on host biology and ecology. The pea aphid, Acyrthosiphon pisum, is a model for studying effects of infection with heritable facultative symbionts (HFS), and each of its seven common HFS species has been reported to provide resistance to biotic or abiotic stresses. However, one common HFS, called X-type, rarely occurs as a single infection in field populations and instead typically superinfects individual aphids with Hamiltonella defensa, another HFS that protects aphids against attack by parasitic wasps. Using experimental aphid lines comprised of all possible infection combinations in a uniform aphid genotype, we investigated whether the most common strain of X-type provides any of the established benefits associated with aphid HFS as a single infection or superinfection with H. defensa We found that X-type does not confer protection to any tested threats, including parasitoid wasps, fungal pathogens, or thermal stress. Instead, component fitness assays identified large costs associated with X-type infection, costs which were ameliorated in superinfected aphids. Together these findings suggest that X-type exploits the aphid/H. defensa mutualism and is maintained primarily as a superinfection by "hitchhiking" via the mutualistic benefits provided by another HFS. Exploitative symbionts potentially restrict the functions and distributions of mutualistic symbioses with effects that extend to other community members.IMPORTANCE Maternally transmitted bacterial symbionts are widespread and can have major impacts on the biology of arthropods, including insects of medical and agricultural importance. Given that host fitness and symbiont fitness are tightly linked, inherited symbionts can spread within host populations by providing beneficial services. Many insects, however, are frequently infected with multiple heritable symbiont species, providing potential alternative routes of symbiont maintenance. Here we show that a common pea aphid symbiont called X-type likely employs an exploitative strategy of hitchhiking off the benefits of a protective symbiont, Hamiltonella Infection with X-type provides none of the benefits conferred by other aphid symbionts and instead results in large fitness costs, costs lessened by superinfection with Hamiltonella These findings are corroborated by natural infections in field populations, where X-type is mostly found superinfecting aphids with Hamiltonella Exploitative symbionts may be common in hosts with communities of heritable symbionts and serve to hasten the breakdown of mutualisms.

Aphid-encoded variability in susceptibility to a parasitoid.

BACKGROUND: Many animals exhibit variation in resistance to specific natural enemies. Such variation may be encoded in their genomes or derived from infection with protective symbionts. The pea aphid, Acyrthosiphon pisum, for example, exhibits tremendous variation in susceptibility to a common natural enemy, the parasitic wasp Aphidius ervi. Pea aphids are often infected with the heritable bacterial symbiont, Hamiltonella defensa, which confers partial to complete resistance against this parasitoid depending on bacterial strain and associated bacteriophages. That previous studies found that pea aphids without H. defensa (or other symbionts) were generally susceptible to parasitism, together with observations of a limited encapsulation response, suggested that pea aphids largely rely on infection with H. defensa for protection against parasitoids. However, the limited number of uninfected clones previously examined, and our recent report of two symbiont-free resistant clones, led us to explicitly examine aphid-encoded variability in resistance to parasitoids. RESULTS: After rigorous screening for known and unknown symbionts, and microsatellite genotyping to confirm clonal identity, we conducted parasitism assays using fifteen clonal pea aphid lines. We recovered significant variability in aphid-encoded resistance, with variation levels comparable to that contributed by H. defensa. Because resistance can be costly, we also measured aphid longevity and cumulative fecundity of the most and least resistant aphid lines under permissive conditions, but found no trade-offs between higher resistance and these fitness parameters. CONCLUSIONS: These results indicate that pea aphid resistance to A. ervi is more complex than previously appreciated, and that aphids employ multiple tactics to aid in their defense. While we did not detect a tradeoff, these may become apparent under stressful conditions or when resistant and susceptible aphids are in direct competition. Understanding sources and amounts of variation in resistance to natural enemies is necessary to understand the ecological and evolutionary dynamics of antagonistic interactions, such as the potential for coevolution, but also for the successful management of pest populations through biological control.

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.