Richard Stouthamer

Heteroplasmy, the presence of multiple mitochondrial genotypes (mitotypes) within an individual, has long been thought to be a rare aberrance that is quickly removed by selection or drift. However, heteroplasmy is being reported in natural populations of eukaryotes with increasing frequency, in part due to improved diagnostic methods. Here, we report a seemingly stable heteroplasmic state in California populations of the polyphagous shothole borer (PSHB), Euwallacea fornicatus; an invasive ambrosia beetle that is causing significant tree dieback. We develop and validate a qPCR assay utilizing locked nucleic acid probes to detect different mitotypes, and qualitatively assess heteroplasmy in individual PSHB. We prove the utility of this assay by: (1) mitotyping field-collected PSHB, documenting the prevalence of heteroplasmy across its range in California; and, (2) measuring relative titers of each mitotype across multiple generations of heteroplasmic laboratory colonies to assess the stability of transmission through the maternal germline. We show that our findings are unlikely to be explained by the existence of NUMTs by next generation sequencing of contiguous sections of mitochondrial DNA, where each of the observed heteroplasmic sites are found within fully functional coding regions of mtDNA. Subsequently, we find heteroplasmic individuals are common in Californian field populations, and that heteroplasmy persists for at least 10 generations in experimental colonies. We also looked for evidence of the common occurrence of paternal leakage, but found none. In light of our results, we discuss competing hypotheses as to how heteroplasmy may have arisen, and continues to perpetuate, in Californian PSHB populations.

The Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae: Xyleborini) is a group of four cryptic ambrosia beetle species. Native to Asia, several members of the complex have invaded other continents, where they cause significant economic losses to agricultural crops (e.g., avocado) and natural ecosystems. We were primarily interested in developing management strategies by focusing on the flight behavior of the beetles. Thus, seasonal differences in flight activity were assessed using panel traps baited with a commercial quercivorol lure, placed in infested avocado orchards in Danei, Tainan, Taiwan. Same traps were used to investigate the flight activity of a natural enemy, an undescribed species of the Braconid genus Eucosmophorus sp. Shothole borer species were identified using a DNA-based, high resolution melting assay. Trap data were compared to the predictions of a simple degree-day model, incorporating developmental data and several environmental parameters known to influence flight. Such as the time period representing most of flight activity in a day and temperature-dependent flight propensity. In stark contrast to the degree-day model which predicted the highest emergence, and by extension flight, of shothole borers during spring and summer (May to November), flight activity was actually lowest during these months, and instead, peaked during the winter (October to March). Abundance of the parasitoid wasp closely mirrored flight activity of the shothole borers. The mismatch of trapping and modeling data can have many causes, heavy precipitation and possibly cooperative brood care may suppress the dispersal behavior of the shothole borers during the summer.

Background: Wolbachia bacteria are estimated to occur in more than half of all insect species. In Hymenoptera, Wolbachia often manipulates its host’s reproduction to its own advantage. Wolbachia is likely the reason that males are rare in the uniparental Ooencyrtus mirus Triapitsyn & Power (Hymenoptera: Encyrtidae). The likelihood of producing male offspring can be increased by giving mothers a continuous supply of Bagrada hilaris (Burmeister) (Heteroptera: Pentatomidae) host eggs to parasitize for 2–3 weeks, by feeding the parents antibiotics, or by rearing parent wasps at high temperatures; all variables that have been shown to correlate with depleting Wolbachia titers in other organisms. The purpose of the current study was to determine whether thelytoky in O. mirus is due to Wolbachia, and if so, at what time in development the sex change occurs. We also wished to determine if Wolbachia removal results in the production of intersexes, as in some other hymenopterans. Finally, mating behavior was observed to see if and where it breaks down as a result of the species becoming thelytokous. Methods: Females were collected from parental lines of O. mirus reared at 26, 30, 31, 32, 33, 34, and 36 °C. The offspring of these females were reared at 26 °C, and their sex-ratio was determined. In a subsequent experiment, the parental generation was switched between 26 °C and 36 °C during development to narrow down the critical period at which changes occurred that subsequently affected the sex-ratio of their offspring. Results: The sex ratio was male biased in the offspring of O. mirus parents reared at 34 °C and 36 °C (high temperatures), even if the offspring themselves were reared at 26 °C. The constant temperature at which the percentage of males started to increase after two generations was 31 °C (10% males), rising to 39% males at 33 °C, and 100% males at 34 °C and 36 °C. Lasting more than 2 days, the critical period for the change toward a male biased sex ratio was during the second half of the parent’s development. Molecular diagnostic assays confirmed that O. mirus females contain Wolbachia and males do not. Examination of preserved males and male-female pairs under a dissecting microscope showed no signs of intersex characters. Observation of the mating behavior of live O. mirus showed that males initiate courtship by drumming their antennae on a female’s antennae, but after a few seconds, the females typically turn and walk away. However, a few instances of possible copulation were noted. Conclusions: As hypothesized, the results indicated that thelytoky in O. mirus is likely mediated by Wolbachia bacteria. To maximize the population growth rate without generating males, the best temperature for mass rearing this species is 30 °C.

Species belonging to the Euwallacea fornicatus Eichhoff (Coleoptera: Scolytinae) species complex have invaded the continental U.S. since at least 2003. Three species of this complex are known to have established, two in California (E. fornicatus; and Euwallacea kuroshio), and a third in Florida (Euwallacea perbrevis). Their native ranges are spread across southern and southeast Asia. In Taiwan, all three species occur in sympatry. They attack healthy trees of widely varied species and cause severe damage and death to the trees. The attractant quercivorol is commonly used to promote their detection by passive trapping. Recent studies in Florida have shown that trapping of E. perbrevis can be further improved by adding a synergist, α-copaene, alongside the quercivorol lure. Thus, we were interested in testing the effectiveness of α-copaene for trapping the other invasive members of the complex in California and in an area of Taiwan where all three species co-occur. We found that α-copaene marginally enhanced the trapping of E. perbrevis in Taiwan, but had no effect on the trapping of E. fornicatus or E. kuroshio in either California or Taiwan. We conclude that any enhancing effect of α-copaene is specific to E. perbrevis. This highlights the economic importance of accurate species identification in developing and implementing an efficient, and yet cost-effective, monitoring program for the management of E. fornicatus and E. kuroshio in California and elsewhere.

Wolbachia are maternally transmitted intracellular bacteria that induce a range of pathogenic and fitness-altering effects on insect and nematode hosts. In parasitoid wasps of the genus Trichogramma, Wolbachia infection induces asexual production of females, thus increasing transmission of Wolbachia. It has been hypothesized that Wolbachia infection accompanies a modification of the host epigenome. However, to date, data on genome-wide epigenomic changes associated with Wolbachia are limited, and are often confounded by background genetic differences. Here, we took sexually reproducing Trichogramma free of Wolbachia and introgressed their genome into a Wolbachia-infected cytoplasm, converting them to Wolbachia-mediated asexuality. Wolbachia was then cured from replicates of these introgressed lines, allowing us to examine the genome-wide effects of wasps newly converted to asexual reproduction while controlling for genetic background. We thus identified gene expression and DNA methylation changes associated with Wolbachia-infection. We found no overlaps between differentially expressed genes and differentially methylated genes, indicating that Wolbachia-infection associated DNA methylation change does not directly modulate levels of gene expression. Furthermore, genes affected by these mechanisms exhibit distinct evolutionary histories. Genes differentially methylated due to the infection tended to be evolutionarily conserved. In contrast, differentially expressed genes were significantly more likely to be unique to the Trichogramma lineage, suggesting host-specific transcriptomic responses to infection. Nevertheless, we identified several novel aspects of Wolbachia-associated DNA methylation changes. Differentially methylated genes included those involved in oocyte development and chromosome segregation. Interestingly, Wolbachia-infection was associated with higher levels of DNA methylation. Additionally, Wolbachia infection reduced overall variability in gene expression, even after accounting for the effect of DNA methylation. We also identified specific cases where alternative exon usage was associated with DNA methylation changes with Wolbachia infection. These results begin to reveal distinct genes and molecular pathways subject to Wolbachia induced epigenetic modification and/or host responses to Wolbachia-infection. Wolbachia is an extremely common endosymbiotic infection of arthropods and nematodes. One of the reasons why Wolbachia can so successfully infect diverse species is the bacterium’s ability to profoundly alter the reproductive behavior of its host. It has been proposed that Wolbachia may modify host’s epigenetic programs to alter its reproductive behavior. However, it has been difficult to study how epigenetic programs change with Wolbachia infection, due to the confounding effects of genetic backgrounds. Here, we studied host transcriptome and epigenome changes associated with Wolbachia infection in a homogenous genetic background, by carrying out an innovative introgression scheme. By doing so, we show, for the first time, high-resolution molecular consequences of intracellular infection and offer insights into epigenetic and transcriptomic regulation of invertebrates.

Trichogramma wasps (Hymenoptera: Trichogrammatidae) are egg parasitoids commonly employed in augmentative biological control releases against a variety of mainly lepidopteran pests. By exploiting the mechanism by which the endosymbiotic bacterium Wolbachia induces parthenogenesis in this genus, we created a set of completely homozygous Wolbachia‐infected recombinant isofemale lines (RILs), each consisting of a different combination of the genome of two well‐characterized lines of Trichogramma pretiosum Riley. We subsequently use 16 of these RILs to investigate the effect of genetic variation on various measures associated with offspring production under laboratory conditions. Unsurprisingly, substantial differences were found between the RILs in their propensity to parasitize hosts, the number of hosts they parasitize, and the levels of mortality in their offspring. Such measures can be used to choose an optimal line for biological control purposes. A method was also developed to characterize the 16 RILs using their allelic state at five loci. Essentially, this binary system uses high‐resolution melt analysis to resolve identity at each locus, with alleles originating from either the grandmaternal or grandpaternal line, and is such that each RIL can be distinguished from each other RIL by their allelic state at one or more loci. The method facilitates the easy diagnosis of line origin when two or more lines are competing with each other in competition assays, allowing for the design of more complicated tests of parasitoid quality. Future field experiments will determine which genetic line performs best under more realistic biological control conditions. The fact that these lines are infected with parthenogenesis‐inducing Wolbachia will allow for prolonged rearing without appreciable change in their genetic makeup, which should lead to a predictable biological control performance.

Mutualistic symbiosis and eusociality have developed through gradual evolutionary processes at different times in specific lineages. Like some species of termites and ants, ambrosia beetles have independently evolved a mutualistic nutritional symbiosis with fungi, which has been associated with the evolution of complex social behaviors in some members of this group. We sequenced the transcriptomes of two ambrosia complexes (Euwallacea sp. near fornicatus–Fusarium euwallaceae and Xyleborus glabratus–Raffaelea lauricola) to find evolutionary signatures associated with mutualism and behavior evolution. We identified signatures of positive selection in genes related to nutrient homeostasis; regulation of gene expression; development and function of the nervous system, which may be involved in diet specialization; behavioral changes; and social evolution in this lineage. Finally, we found convergent changes in evolutionary rates of proteins across lineages with phylogenetically independent origins of sociality and mutualism, suggesting a constrained evolution of conserved genes in social species, and an evolutionary rate acceleration related to changes in selective pressures in mutualistic lineages.

This study evaluated the flight propensity of 6 isofemale lines of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) using a flight test developed as a quality control measure for mass reared Trichogramma wasps. The same isofemale lines were used in earlier work to determine fecundity in the laboratory and performance in the field. We used 5 isofemale lines from a Mediterranean climate, collected in Irvine, California, USA, and 1 isoline from a humid subtropical climate, collected in Piracicaba, São Paulo, Brazil. The flight propensity was assessed with a short-range flight test of the different isofemale lines at 2 different relative humidity levels, 80% and 30%. Relative humidity (RH) affected only the isofemale line from Piracicaba, and about 30% fewer insects flew in high RH conditions compared with low RH conditions. Relative humidity conditions did not affect the California isofemale lines, which showed a flight propensity above 75% in both RH conditions. These results showed that for some lines, environmental conditions at the release sites might adversely affect the flight propensity. However, just the flight test method was not sensitive enough to discriminate the lines with different field performances. Este estudio evaluó la propensidad al vuelo de seis líneas isohembras de Trichogramma pretiosum Riley, utilizando una prueba de vuelo desarrollada como medida de control de calidad para las avispas Trichogramma criadas en masa. Se utilizaron las mismas líneas isohembras en un trabajo anterior para determinar su fecundidad en el laboratorio y su desempeño en el campo. Utilizamos cinco líneas isohembras de clima mediterráneo, recolectada en Irvine, California, EE. UU. y una isolínea de un clima subtropical húmedo, recolectadas en Piracicaba, São Paulo, Brasil. Se evaluó la propensidad al vuelo con una prueba de vuelo de corto alcance de las diferentes líneas isohembras bajo una humedad relativa (HR) del 80% y 30%. La HR solo afectó a la línea isohembra de Piracicaba y aproximadamente un 30% más de insectos no volaron en condiciones de baja HR. Las condiciones de HR no afectaron las líneas isohembras de California, que mostraron una propensidad de vuelo superior al 75% en ambas condiciones de humedad relativa. Estos resultados mostraron que en algunas líneas, las condiciones ambientales en los sitios de liberación podrían afectar negativamente la propensidad a volar de los insectos. Sin embargo, la prueba de vuelo por sí sola no fue lo suficientemente sensible como para discriminar las líneas con diferentes actuaciones en el campo.

Trichogrammatids are minute parasitoid wasps that develop within other insect eggs. They are less than half a millimeter long, smaller than some protozoans. The Trichogrammatidae are one of the earliest branching families of Chalcidoidea: a diverse superfamily of approximately half a million species of parasitoid wasps, proposed to have evolved from a miniaturized ancestor. Trichogramma are frequently used in agriculture, released as biological control agents against major moth and butterfly pests. Additionally, Trichogramma are well known for their symbiotic bacteria that induce asexual reproduction in infected females. Knowledge of the genome sequence of Trichogramma is a major step towards further understanding its biology and potential applications in pest control. We report the 195-Mb genome sequence of Trichogramma pretiosum and uncover signatures of miniaturization and adaptation in Trichogramma and related parasitoids. Comparative analyses reveal relatively rapid evolution of proteins involved in ribosome biogenesis and function, transcriptional regulation, and ploidy regulation. Chalcids also show loss or especially rapid evolution of 285 gene clusters conserved in other Hymenoptera, including many that are involved in signal transduction and embryonic development. Comparisons between sexual and asexual lineages of Trichogramma pretiosum reveal that there is no strong evidence for genome degradation (e.g., gene loss) in the asexual lineage, although it does contain a lower repeat content than the sexual lineage. Trichogramma shows particularly rapid genome evolution compared to other hymenopterans. We speculate these changes reflect adaptations to miniaturization, and to life as a specialized egg parasitoid. The genomes of Trichogramma and related parasitoids are a valuable resource for future studies of these diverse and economically important insects, including explorations of parasitoid biology, symbiosis, asexuality, biological control, and the evolution of miniaturization. Understanding the molecular determinants of parasitism can also inform mass rearing of Trichogramma and other parasitoids for biological control.