Richard Stouthamer

A new cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera

Vertically transmitted endosymbiotic bacteria, such as Wolbachia, Cardinium and Rickettsia, modify host reproduction in several ways to facilitate their own spread. One such modification results in parthenogenesis induction, where males, which are unable to transmit the bacteria, are not produced. In Hymenoptera, the mechanism of diploidization due to Wolbachia infection, known as gamete duplication, is a post-meiotic modification. During gamete duplication, the meiotic mechanism is normal, but in the first mitosis the anaphase is aborted. The two haploid sets of chromosomes do not separate and thus result in a single nucleus containing two identical sets of haploid chromosomes. Here, we outline an alternative cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera. During female gamete formation in Rickettsia-infected Neochrysocharis formosa (Westwood) parasitoids, meiotic cells undergo only a single equational division followed by the expulsion of a single polar body. This absence of meiotic recombination and reduction corresponds well with a non-segregation pattern in the offspring of heterozygous females. We conclude that diploidy in N. formosa is maintained through a functionally apomictic cloning mechanism that differs entirely from the mechanism induced by Wolbachia.

Molecular and morphological diagnoses of five species of Trichogramma: biological control agents of Chrysodeixis chalcites (Lepidoptera: Noctuidae) and Tuta absoluta (Lepidoptera: Gelechiidae) in the Canary Islands

Prospecting for potential natural enemies of the invasive lepidopteran tomato pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and the banana pest Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae) on the Canary Islands archipelago, where no Trichogramma species were previously recorded, has led to the discovery of five distinct species. T. achaeae Nagaraja & Nagarkatti, T. bourarachae Pintureau & Babault, T. euproctidis (Girault) and T. evanescens Westwood are relatively widespread species. The fifth is close to T. brassicae Bezdenko, but differs sufficiently in the sequence of the ITS2 region of ribosomal RNA to warrant further investigation as a species probably new to science. Each species is treated in detail in order to facilitate identification in future using molecular and/or morphological characters, or a combination of both. All species are newly recorded for the Canary Islands, and the distribution of each within the islands and elsewhere is provided. Known host records are given within the Canary Islands and elsewhere. The most common species found, T. achaeae, is already being used in biological control programmes against T. absoluta in mainland Spain and field trials are ongoing to evaluate its effectiveness as a biological control agent of C. chalcites in banana crops.

Can the destruction of California's oak woodlands be prevented? Potential for biological control of the goldspotted oak borer, Agrilus auroguttatus.

The goldspotted oak borer (GSOB), Agrilus auroguttatus Schaeffer (Coleoptera: Buprestidae), is an introduced and aggressive phloem/wood borer infesting native oaks in southern California. Elevated levels of oak mortality have occurred continually for the last nine years on three oak species in San Diego Co., California, USA. Biological control is being assessed as an option for long-term and widespread management of the invasive population of GSOB. Foreign exploration in the native ranges of GSOB and a related sibling species (Agrilus coxalis Waterhouse) was conducted to determine life history information, to assess the natural enemy complex, and to collect specimens for molecular analyses that could help to identify the area of origin of California’s introduced population. Two species of parasitoids, Calosota elongata Gibson (Eupelmidae) and Atanycolus simplex Cresson (Braconidae), were discovered with GSOB populations in Arizona and California. No insect natural enemies were found with populations of A. coxalis in southern Mexico. However, Quercus conzatti Trel. and Quercus peduncularis Nee in Oaxaca and Chiapas, respectively, were recorded as the first known hosts of A. coxalis. A comparative analysis of our understanding of the natural enemy complexes for other pestiferous Agrilus with that of GSOB suggests that more effort should be directed at uncovering potential egg parasitoids and microbial pathogens of GSOB. Analyses of mitochondrial and nuclear ribosomal DNA (rDNA) revealed that the California population of GSOB was more similar to the Arizona population. Specimens of A. coxalis from southern Mexico were confirmed as a separate species. Additional surveys and sampling are needed across the complete native range of the GSOB species complex to develop a comprehensive inventory of parasitoid species that could be considered for use in a classical biological control program in California and to delineate the area of origin of California’s population.

A molecular key to the common species of Trichogramma of the Mediterranean region.

A molecular key for the identification of common Trichogramma (Hymenoptera: Trichogrammatidae) species found in agricultural settings around the Mediterranean is developed based on the sequence of the internal transcribed spacer 2 of the ribosomal cistron. Using the size of the ITS2 PCR product and restriction fragment length polymorphisms of the amplicon, ten Trichogramma species (T. bourarachae Pintureau and Babault, T. brassicae Bezdenko, T. cacoeciae Marchal/T. embryophagum Hartig, T. cordubensis Vargas and Cabello, T. dendrolimi Matsumura, T. euproctidis Girault, T. evanescens Westwood, T. nerudai Pintureau and Gerding, T. oleae Voegelé and Pointel, and T. pintoi Voegelé) can be distinguished.

The origin of a selfish B chromosome triggering paternal sex ratio in the parasitoid wasp Trichogramma kaykai

This study uses molecular and cytogenetic methods to determine the origin of a B chromosome in some males of the wasp Trichogramma kaykai. This so-called paternal sex ratio (PSR) chromosome transmits only through sperm and shortly after fertilization triggers degeneration of the paternal genome, while keeping itself intact. The resulting embryos develop into haploid B-chromosome-carrying males. Another PSR chromosome with a very similar mode of action is found in the distantly related wasp Nasonia vitripennis and its origin was traced by transposon similarity to the genus Trichomalopsis, which is closely related to Nasonia. To determine whether both PSR chromosomes have a similar origin we aimed to reveal the origin of the Trichogramma PSR chromosome. Using fluorescent in situ hybridization, we discovered a major satellite repeat on the PSR chromosome, the 45S ribosomal DNA. Analysis of the internal transcribed spacer 2 (ITS2) of this repeat showed the presence of multiple ITS2 sequences on the PSR chromosome resembling either the ITS2 of T. oleae or of T. kaykai. We therefore conclude that the Trichogramma PSR chromosome originates from T. oleae or a T. oleae-like species. Our results are consistent with different origins for the PSR chromosomes in Trichogramma and Nasonia.

Characterization of intersex production in Trichogramma kaykai infected with parthenogenesis-inducing Wolbachia

Sexually aberrant individuals, displaying both male and female characteristics, are rare in occurrence but are documented throughout the animal kingdom. In parasitoid wasps of the genus Trichogramma, such individuals typically appear as a result of rearing Wolbachia-infected thelytokous wasps at high temperatures. Sexually aberrant Trichogramma have been referred to interchangeably in the literature as gynandromorphs, sexual mosaics and intersexes. However, accurately used, the terms “gynandromorph” and “sexual mosaic” describe an individual composed of a mixture of genetically distinct tissues corresponding to the sexual phenotypes observed, while “intersex” refers to an individual having a uniform genetic constitution but with some tissues exhibiting sexual phenotypes conflicting with the associated genotype. Here, we investigate the heat-induced production of sexually aberrant offspring by thelytokous Trichogramma kaykai. Aberrant individuals were rare, but each was characterized as one of 11 morphotypes ranging from very feminine to very masculine. Overall, the production of aberrant individuals increased with time from the onset of maternal oviposition. However, while the production of males also increased with time, the degree of masculinity of aberrant individuals did not; the different morphotypes appeared to be produced haphazardly. We conclude that the aberrant individuals produced by T. kaykai are actually intersexes and not gynandromorphs. The wasps close association with Wolbachia and the absence of intersexes in uninfected populations allow us to discuss a possible origin of the condition.

Iranian Trichogramma : ITS2 DNA characterization and natural Wolbachia infection

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) are important natural enemies of many lepidopterous pests in agricultural and forest areas. Because the use of indigenous Trichogramma populations/species can significantly increase the biological control efficiency, the characterization of endemic populations and precise species identification is important. In this study, Trichogramma wasps were collected from parasitized eggs of several lepidopteran pests in Northern and Central Iran. Seven Trichogramma species were identified based on the morphology and the nucleotide sequence, size and restriction profile (PCR-RFLP) of the internal transcribed spacer 2 (ITS2) region of the rDNA of T. brassicae Bezdenko, T. cacoeciae Marchal, T. embryophagum Hartig, T. evanescens Westwood, T. euproctidis Girault, T. pintoi Voegelé and T. tshumakovae Sorokina. This is the first report of T. euproctidis in Iran. Additionally, natural populations were surveyed for the prevalence of Wolbachia. Two populations of T. brassicae were found to be infected by a single Wolbachia strain which was identified by using a multi-locus sequencing approach.

Taxon-specific multiplex-PCR for quick, easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves

Citricola scale, Coccus pseudomagnoliarum Kuwana (Hemiptera: Coccidae), is a serious pest of citrus in California’s San Joaquin Valley, but not in southern California where a complex of Metaphycus spp. Mercet (Hymenoptera: Encyrtidae) suppress it. This has created interest in using these (and other Metaphycus) species for biological control in the San Joaquin Valley. A critical step in assessing an organism’s potential for biological control is the ability to accurately identify it. For Metaphycus spp., this currently requires slide mounted adult specimens and expert taxonomic knowledge. We present a simple, quick and accurate method to identify any life stage of the ten major parasitoids of soft scales in California citrus, based on amplification of ribosomal DNA, using the polymerase chain reaction (PCR). Three multiplex-PCR protocols amplify products of taxon-specific sizes, allowing direct diagnosis of taxa accommodated by the PCR, and reducing identification time to a fraction of that of existing methods.

Population genetics of goldspotted oak borer, Agrilus auroguttatus Schaeffer (Coleoptera: Buprestidae): investigating the origin of an invasive pest of native oaks in California

The goldspotted oak borer, Agrilus auroguttatus Schaeffer, is an invasive woodborer in California USA that is native to oak woodlands across southern Arizona USA. Developing a classical biological control program for this pest in southern California is a high priority due to the continuing ecological and economic damage caused by this insect since its recent introduction into the area. In an attempt to determine the area of origin for this invasive beetle, analyses of the mitochondrial cytochrome oxidase and ribosomal nuclear D2 domain of the 28S gene regions were undertaken and provided insight into the phylogeographic relationship between and within populations of A. auroguttatus in Arizona and California. The area of origin for the invasive population of goldspotted oak borer in California was not determined conclusively, although our molecular data suggests the Dragoon Mountains in Cochise Co., Arizona as a possible source for the California population of A. auroguttatus. Results also confirmed that individuals collected from populations across southern Arizona and California are all A. auroguttatus, and are not part of a cryptic species complex comprised of the morphologically similar A. coxalis. Future surveys for natural enemies of A. auroguttatus will focus on the Dragoon Mountains as a potential source for co-evolved enemies for use in a classical biological control program against this invasive woodborer in southern California.

Distribution Patterns of Wolbachia Endosymbionts in the Closely Related Flower Bugs of the Genus Orius: Implications for Coevolution and Horizontal Transfer

Vertical transmission is the primary route of the endosymbiont Wolbachia for its own spread among invertebrate hosts, but horizontal transmission between different hosts is believed to have occurred multiple times. However, it is not well known how Wolbachia commonly spread among closely related hosts. We focused on the closely related species of the minute pirate bugs belonging to the genus Orius, which are important biological control agents in agricultural crops because they are the most useful natural enemy of various tiny pests, such as thrips. Here, we examined five Orius species (Orius sauteri, Orius nagaii, Orius minutus, Orius strigicollis, and Orius tantillus) from eight geographic localities in Japan for Wolbachia infection. Two distinct strains, wOus1 and wOus2, were detected based on Wolbachia surface protein (wsp) gene sequencing. Furthermore, multilocus sequence typing revealed that each of the strains comprised two variants that differed in a single nucleotide. The overall distribution patterns of the two Wolbachia strains were found to differ among host species: prevalent double infection with wOus1 and wOus2 in O. strigicollis; fixation of single infection with wOus2 in O. nagaii; occurrence of single infection with wOus1 in O. sauteri; prevalence of single infection with wOus1 in O. minutus with an exception in a single population; and lack of Wolbachia infection in O. tantillus. Such differences in the distribution patterns of Wolbachia may reflect the evolutionary history of Wolbachia infection among Orius species and/or ecological and physiological differences among the Orius species that determine the invasiveness and maintenance of the two Wolbachia strains.