Yukiko Matsumoto

Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

ABSTRACT Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “Candidatus Cardinium hertigii.”

Molecular Identification of Field-Collected Culicoides Larvae in the Southern Part of Japan

ABSTRACT Although Culicoides biting midges act as a vector of important human and domestic animal diseases, their ecology is poorly understood. The lack of proper identification systems of Culicoides larvae is one of the main obstacles to progress in research. Based on mitochondrial sequences of 19 Japanese Culicoides species, we designed a universal primer set to amplify the partial sequence of the mitochondrial cytochrome c oxidase I (cox 1). The polymerase chain reaction product amplified from extracted DNA of Culicoides larvae using the primer set was directly sequenced, and species identification based on the variation at cox1 was conducted. Using the molecular identification system, we sorted 243 specimens of field-collected larvae from the southern part of Japan into 10 species including Culicoides arakawae (Arakawa), Culicoides oxystoma Kieffer, and Culicoides brevitarsis Kieffer, which are regarded as vectors of important livestock animal diseases. Eight species of Culicoides larvae, including C. arakawae and C. oxystoma, were recovered from active paddy fields and an abandoned paddy field. The result suggests that paddy fields contribute to breeding a variety of Culicoides species and maintenance and spread of Culicoides-borne pathogens. In contrast, larvae of C. brevitarsis were collected from cattle dung in pastures. The molecular identification system described herein using nucleotide sequences successfully achieved larval identification and will be useful for a better understanding of larval habitats of Culicoides biting midges.

Proteome Analysis of Watery Saliva Secreted by Green Rice Leafhopper, Nephotettix cincticeps

The green rice leafhopper, Nephotettix cincticeps, is a vascular bundle feeder that discharges watery and gelling saliva during the feeding process. To understand the potential functions of saliva for successful and safe feeding on host plants, we analyzed the complexity of proteinaceous components in the watery saliva of N. cincticeps. Salivary proteins were collected from a sucrose diet that adult leafhoppers had fed on through a membrane of stretched parafilm. Protein concentrates were separated using SDS-PAGE under reducing and non-reducing conditions. Six proteins were identified by a gas-phase protein sequencer and two proteins were identified using LC-MS/MS analysis with reference to expressed sequence tag (EST) databases of this species. Full -length cDNAs encoding these major proteins were obtained by rapid amplification of cDNA ends-PCR (RACE-PCR) and degenerate PCR. Furthermore, gel-free proteome analysis that was performed to cover the broad range of salivary proteins with reference to the latest RNA-sequencing data from the salivary gland of N. cincticeps, yielded 63 additional protein species. Out of 71 novel proteins identified from the watery saliva, about 60 % of those were enzymes or other functional proteins, including GH5 cellulase, transferrin, carbonic anhydrases, aminopeptidase, regucalcin, and apolipoprotein. The remaining proteins appeared to be unique and species- specific. This is the first study to identify and characterize the proteins in watery saliva of Auchenorrhyncha species, especially sheath-producing, vascular bundle-feeders.

Characterization of Internal Transcribed Spacer (ITS1)-ITS2 Region of Ribosomal RNA Gene From 25 Species of Culicoides Biting Midges (Diptera: Ceratopogonidae) in Japan

ABSTRACT We determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS)1–5.8S-ITS2a-2S-ITS2 region in 103 individuals of 25 Culicoides species (Diptera: Ceratopogonidae) from 11 locations in Japan. Ribosomal RNA genes, 5.8S and 2S rDNA, were highly conserved among the species with few variations. The ITS2a region showed length variation among species. Both ITS1 and ITS2 showed highly varied sequences among species. The noticeable indel regions among ITS1 sequences are present in some Culicoides species, separating species into two types having long or short ITS1 region. However, Culicoides cylindratus Kitaoka possesses both types of ITS1 in each individual; these results seem to indicate that the ITS1-long type was the prototype and the short type was produced through deletion in many Culicoides species. One species, belonging to subgenus Avaritia, possessed an Avaritia-specific sequence in ITS1 and phylogenetically formed a monophyletic group. Geographical genotypes in a species were not clear. Species-specific sequence features were observed, enabling molecular identification of Culicoides species.

Transinfection of Wolbachia in planthoppers: nymphal injection of cultured Wolbachia and infection dynamics.

ABSTRACT Wolbachia species are intracellular symbionts that cause reproductive alterations in arthropods. Transinfection experiments have been performed in many arthropod species to elucidate the interaction between Wolbachia and a new host. To ease transinfection of this bacterium to new arthropod hosts, we introduced two techniques: nymphal injection instead of embryonic injection and the use of a cultured source of Wolbachia instead of direct transfer from donors to recipients. Wolbachia in the small brown planthopper Laodelphax striatellus was cultivated in a cell line and injected into the nymphal body cavity of the brown planthopper Nilaparvata lugens together with the cells. By using these techniques, two transinfected planthopper lines were obtained. In one line, Wolbachia disappeared after several generations; in the other line, Wolbachia was retained for >7 yr. Infection rates in this latter transinfected line were ≈80% in early generations after transinjection but decreased to <10% through 40–60 generations. Subsequent selection for Wolbachia-infected females in this line did not increase the infection rate as a temporary effect. Thus, this transinfected line of N. lugens showed cytoplasmic incompatibility, although the incompatibility level was lower than in L. striatellus, the original host. The method of transinfection presented herein is useful for transmitting intracellular symbionts between small arthropod hosts.

Presence of a short repeat sequence in internal transcribed spacer (ITS) 1 of the rRNA gene of Sogatella furcifera (Hemiptera: Delphacidae) from geographically different populations in Asia

Nucleotide sequences of the internal transcribed spacer 1 (ITS1)–5.8S–ITS2 region of the nuclear ribosomal RNA gene were determined in the white-backed planthopper (WBPH) Sogatella furcifera (Horváth) to detect molecular variation among regional populations in Asia. We analyzed 932 sequences from 172 individuals (4–9 clones per individual) of 33 populations collected in 1987–2008 from six countries, Japan, China, Taiwan, Vietnam, Philippines, and Papua New Guinea. WBPH showed intra-individual variation in ITS1, which is mainly attributable to the frequency (0–10) of the 66-bp repeat sequence in ITS1. Among the examined clones, the sequences of 5.8S were mostly identical and those of ITS2 were similar. A single planthopper had a maximum of 6 different variants in the number of ITS1 repeats, suggesting highly varied repeat numbers in individual planthoppers. The ITS1 with four repeats was the most frequently (64%) detected. Such a repeat was not observed in two other economically important planthopper species, Nilaparvata lugens (Stål) and Laodelphax striatellus (Fallén). The ITS nucleotide sequences in the WBPH populations in Asia were genetically close and some variations in the sequences were not related to regional populations, indicating that the nucleotide sequences of the ITS region are not useful for geographical discrimination of the WBPH. This closeness seems to be caused by long distance migration and genetic exchange among populations.

The strepsipteran parasite Elenchus japonicus (Strepsiptera, Elenchidae) of planthoppers consists of three genotypes

The twisted-wing parasite Elenchus japonicus is a major parasitoid of rice planthoppers (Hemiptera: Delphacidae), including the brown planthopper Nilaparvata lugens, the white-backed planthopper Sogatella furcifera, and the small brown planthopper Laodelphax striatellus. Another Elenchus species, E. yasumatsui, which is considered to be synonymous with E. japonicus, has also been described in southern Asia. However, limited biological and molecular data on this important parasitoid of rice planthoppers are available. In this study, E. japonicas-stylopized planthoppers were obtained from various regions in Asia and Japan. Sequences of the nuclear 18S ribosomal RNA gene and the mitochondrial cytochrome oxidase I gene of the strepsipterans were analyzed from 40 samples of E. japonicus: 14 from N. lugens, 6 from S. furcifera and 20 from L. striatellus. Sequence analysis of these samples revealed three genotypes of E. japonicus. Of the three types of E. japonicus, one was isolated only from L. striatellus from northern Japan. The other two types were found in all three rice planthopper species collected from a wide area in Asia. These results suggest that the three different genotypes of Elenchus can be associated with a particular geographical region and/or planthopper species. The relationship between two previously described Elenchus species and the present three genotypes needs to be elucidated.

GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE)

RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species.

Passive Long-Distance Migration of Apterous Dryinid Wasps Parasitizing Rice Planthoppers

The wasp family Dryinidae comprises predator and parasitoid wasps of leafand planthoppers (Hemiptera: Auchenorrhyncha). This family is morphologically distinct from other wasps. Females in most subfamilies of Dryinidae have forelegs that are modified into a chela, with an enlarged claw (Fig. 1: green) and 5th tarsomere (Fig. 1: red) that aid in grasping the host insect. The enlarged claw moves widely when the chela opens. Such foreleg morphology is not always the case, such as for females of the subfamily Aphelopinae R.C.L. Perkins and Erwiniinae Olmi & Guglielmino (Olmi & Guglielmino, 2010) that have simple forelegs. Dryinid wasps often show distinctive sexual dimorphism, such as the presence or absence of chela. The subfamily Gonatopodinae Kieffer is one of the extreme cases of sexual dimorphism, but in this case it is because females of most of the species are apterous. The pterothorax becomes so slender in the apterous form (Fig. 2: A, C) that they look like ants. In contrast, males have well developed wings and a pterothorax (Fig. 1: B).

The green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), salivary protein NcSP75 is a key effector for successful phloem ingestion

Nephotettix cincticeps, a prevalent rice pest, injects gelling and watery saliva into plant tissues during the sucking process. Certain components within the saliva are believed to interact with plant cellular constituents and play important roles in overcoming host plant defense responses. Based on our previous analysis of the salivary gland transcriptome and secreted saliva proteome of N. cincticeps, in this study, we analyzed the biological functions of salivary protein, NcSP75 (N. cincticeps salivary protein 75 kD). NcSP75, a salivary glands-specific gene, showed low similarities to any previously reported sequences. Knockdown of NcSP75 by RNA interference (RNAi) reduced the longevity of treated nymphs to approximately half of the longevity of controls and caused severe developmental retardation. Furthermore, the knockdown of NcSP75 decreased the survival rate of adults, and reduced the number of deposited eggs and hatched nymphs. Thus, the adverse effects caused by the knockdown of NcSP75 were observed throughout the lifetime of N. cincticeps, when feeding on rice plants. In contrast, no reduction was observed in the survival rate of the knockdown of NcSP75 adults fed on an artificial diet. Electrical penetration graph measurements taken from adult females feeding on rice plants showed a significantly shorter duration of phloem ingestion associated with the knockdown of NcSP75 than the knockdown of the enhanced green fluorescent protein (EGFP). Furthermore, the total sugar content of the honeydew was lower when NcSP75 was knocked down. These results suggest that the NcSP75 protein contribute to successful and sustainable ingestion from the sieve elements of rice plants. The NcSP75 protein of N. cincticeps can, accordingly, be considered as a key effector for establishing compatible interaction with rice plants and could be a potential target for controlling this species.