Microbiome of Zoophytophagous Biological Control Agent Nesidiocoris tenuis.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

Egg parasitoids of Arboridia apicalis (Nawa, 1913) (Hemiptera, Cicadellidae), a leafhopper pest of grapevines in Japan, with description of a new species of Anagrus Haliday, 1833 (Hymenoptera, Mymaridae).

Several species of egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of the leafhopper pest of grapevines in Japan, Arboridia (Arboridia) apicalis (Nawa) (Hemiptera, Cicadellidae), were reared and identified for the first time. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) arboridiae Triapitsyn & Adachi-Hagimori, sp. nov. (Mymaridae) is described and illustrated from Honshu Island (Shimane Prefecture) and Kyushu Island (Miyazaki Prefecture). It is shown to be different from Anagrus (Anagrus) japonicus Sahad and A. flaviapex Chiappini & Lin, to which it is most similar; the latter species was originally described from China and is newly recorded here from Okinawa Island, Japan. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A. arboridiae from A. flaviapex, A. japonicus, and some other members of the Anagrus (Anagrus) atomus (L.) species group. Two other species of Anagrus Haliday, A. (Anagrus) avalae Soyka and A. atomus, are also identified in Japan from eggs of the leafhoppers Edwardsiana ishidae (Matsumura) and Eurhadina ? betularia Anufriev, respectively. An updated key to females of the Japanese species of Anagrus is given. Oligosita pallida Kryger (a new record for Japan), Oligosita sp., and an Aphelinoidea (Aphelinoidea) sp. (Trichogrammatidae) were the other, although much less abundant, apparent egg parasitoids of A. apicalis in Shimane Prefecture, mainly in non-organic vineyards.

Egg parasitoids of the tea green leafhopper Empoascaonukii (Hemiptera, Cicadellidae) in Japan, with description of a new species of Anagrus (Hymenoptera, Mymaridae).

Fairyfly (Hymenoptera, Mymaridae) egg parasitoids of the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda (Hemiptera, Cicadellidae), an economically important pest in Asia of the tea plant, Camelliasinensis, were identified from specimens reared in Japan. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) rugmanjonesi Triapitsyn & Adachi-Hagimori, sp. n., is described and illustrated. It is shown to be different from the most similar A.turpanicus Triapitsyn & Hu, an egg parasitoid of a leafhopper pest of cultivated grapes which is known from Xinjiang Uyghur Autonomous Region in China. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A.rugmanjonesi from A.turpanicus and other members of the Anagrusincarnatus Haliday species complex. A key to females of the Japanese species of Anagrus Haliday is given. Two other species of Mymaridae, Aresconenocki (Subba Rao & Kaur) and Stethyniumempoascae Subba Rao, are also identified, albeit the latter one only tentatively. Both latter taxa are newly recorded from Japan, and E.onukii represents their new host association.

Release of the Flightless Strain of Harmonia axyridis (Coleoptera: Coccinellidae) Against Two Aphid Species on Komatsuna (Brassica rapa var. perviridis) in Open Fields.

Myzus persicae (Sulzer) (Hemiptera: Aphididae) and Lipaphis erysimi (Kaltenbach) are important pests of Brassica leafy vegetables, especially in Japan, the United States, and India. In Japan, because most of the nonheading Brassica vegetables are considered minor crops, the number of commercially available pesticides against these aphids is limited. Here, we evaluated the effect of releasing adults of a flightless strain of the multicolored Asian ladybird, Harmonia axyridis Pallas, on these aphid species on a nonheading Brassica cultivar in open fields. Three weeks after, ladybirds were released onto aphid-infested plants at a rate of two adults per m2, only 4-12% equal numbers of aphids were found on plants with ladybirds as without ladybirds in all three trials. The result indicates that H. axyridis adults are the effective biocontrol agents against aphids on the plants.

Multiplex PCR Method for Discriminating Between Sphaerophoria macrogaster (Diptera: Syrphidae) and Sphaerophoria indiana (Diptera: Syrphidae).

Hoverflies are potential candidates for biological control of aphid populations; however, identification of closely related hoverfly species by using morphological characteristics is quite difficult. For instance, adults of Sphaerophoria macrogaster (Thomson) (Diptera: Syrphidae)-the predominant species early in the vegetable production season in Japan-are morphologically indistinguishable from those of Sphaerophoria indiana Bigot (Diptera: Syrphidae) without comparison of the male genitalia. Here, we investigated genetic variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene of the two species by DNA sequencing and developed a multiplex PCR method for differentiating the two species. Alignment of COI sequences revealed 1.2% nucleotide variance between the two species. The COI sequence of S. macrogaster collected from Japan was 99.8% identical to those of S. macrogaster collected from India. The COI sequence of S. indiana collected from Japan was 100% identical to that of Sphaerophoria philanthus (Meigen) (Diptera: Syrphidae) collected from Canada. The sizes of the multiplex PCR products differentiated following gel electrophoresis were 162 bp for S. macrogaster and 607 bp for S. macrogaster and S. indiana. The accuracy rate of multiple PCR was 100%. Use of this method will facilitate further research into the characteristics of hoverflies and will improve the efficacy of biological control using hoverflies on vegetable crops.

Gene flow between sexual and asexual strains of parasitic wasps: a possible case of sympatric speciation caused by a parthenogenesis-inducing bacterium.

Sympatric speciation is strictly defined as the emergence of two species from a population in which mating has been random with respect to the place of birth of the mating partners. Mathematical models have shown that sympatric speciation is possible, but very few examples have been documented in nature. In this article, we demonstrate that arrhenotokous and thelytokous strains of a parasitic wasp, Neochrysocharis formosa, speciated sympatrically through infection by a symbiotic bacterium Rickettsia for the following reasons: First, Rickettsia infection was detected in all of the thelytokous strains collected throughout Japan. Second, the arrhenotokous and thelytokous strains have been collected sympatrically. Third, crossing experiments between the two strains did not result in fertilized offspring. In addition, the two strains were genetically isolated at the nuclear and mitochondrial genes. Fourth, the two strains showed a sister relationship in nuclear 28S rRNA gene. Finally, thelytokous females treated with antibiotics produced Rickettsia-free male offspring of the same reproductive form as arrhenotokous females indicating that the thelytokous strain could have speciated sympatrically from an individual of the arrhenotokous strain.

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.

Development of a multiplex method to discriminate between Neochrysocharis formosa (Hymenoptera: Eulophidae) reproductive modes.

A multiplex polymerase chain reaction (PCR) method was applied to differentiate thelytokous and arrhenotokous strains of Neochrysocharis formosa (Westwood). Alignment of strain first internal transcribed spacer regions revealed high nucleotide variability and the strain-specific primer sequence used. Strains were easily differentiated after gel electrophoresis of multiplex PCR products because arrhenotokous specimens produced a 500-bp fragment as well as the 800-bp fragment common to both strains. This method successfully distinguished N. formosa strains regardless of collection site across Japan; thus, it is probably suitable for similar applications in Turkey, Italy, and elsewhere.

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.