Keiichiro Matsukura

Dynamics of Southern rice black-streaked dwarf virus in rice and implication for virus acquisition.

A novel viral disease of rice caused by Southern rice black-streaked dwarf virus (SRBSDV) has spread throughout East and Southeast Asia since the mid-2000s. Outbreaks of this viral disease occur yearly in southern parts of Japan concurrently with overseas migration of the planthopper vector Sogatella furcifera from southern China during the rainy season (from late June to early July). We examined the dynamics (changes in titer and localization) of SRBSDV on rice using reverse-transcription real-time polymerase chain reaction and determined the relationship between virus titer in plants and virus acquisition by S. furcifera. Under a constant temperature of 27°C, a substantial increase of SRBSDV titer in the leaf sheath together with typical symptoms (stunted growth and twisting of leaf tips) was observed at 20 days after the end of a 7-day exposure of viruliferous S. furcifera. Approximately 40% of S. furcifera acquired SRBSDV through feeding for 5 days on rice plants that were infected following exposure to viruliferous vectors for 10 to 15 days. These results suggest that rice infected by S. furcifera can be a source of SRBSDV before the next generation of S. furcifera emerges.

Physiological response to low temperature in the freshwater apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae)

SUMMARY Cold hardiness of the freshwater apple snail, Pomacea canaliculata, varies seasonally. We investigated lethal factors and physiological changes arising from exposure of P. canaliculata to low temperatures. Snails did not survive freezing. The supercooling point of cold-acclimated (cold tolerant) snails (–6.6±0.8°C) did not differ significantly from that of non-acclimated ones (–7.1±1.5°C) under laboratory conditions. Furthermore, snails died even under more moderately low temperatures approaching 0°C. These results indicate that indirect chilling injury is a factor in the death of P. canaliculata at low temperatures. Regardless of whether the snails were acclimated to low temperatures, all of the dead, and even some of the snails still alive at 0°C, had injured mantles, indicating that the mantle may be the organ most susceptible to the effects of low temperatures. The concentration of glucose in the posterior chamber of the kidney and concentration of glycerol in the digestive gland were significantly higher in cold-acclimated snails than in non-acclimated ones, suggesting carbohydrate metabolic pathways are altered in snails during cold acclimation.

SEASONAL CHANGES IN COLD HARDINESS OF THE INVASIVE FRESHWATER APPLE SNAIL, POMACEA CANALICULATA (LAMARCK) (GASTROPODA: AMPULLARIIDAE)

We investigated the cold hardiness of a South American freshwater apple snail, Pomacea canaliculata, which began to invade Japanese paddy fields in the early 1980s. Pomacea canaliculata exhibited apparent seasonal fluctuation in its cold hardiness. Snails collected from submerged paddy fields in summer were less tolerant to cold, and none survived exposure to 0°C for five days. With decreasing temperature, together with drainage of its habitat in autumn, P. canaliculata developed cold hardiness, attaining almost 100% survivorship after exposure to 0°C for five days in December. The snails resting in drained fields were approximately nine times more cold tolerant than those collected from submerged fields, based on the time to 50% mortality at 0°C. Snails overwintering in aquatic conditions also acquired cold hardiness.

Host manipulation by the orange leafhopper Cicadulina bipunctata: gall induction on distant leaves by dose-dependent stimulation

The evolution of the gall-inducing ability in insects and the adaptive significance of the galling habit have been addressed by many studies. Cicadulina bipunctata, the maize orange leafhopper, is an ideal study organism for evaluating these topics because it can be mass-reared and it feeds on model plants such as rice (Oryza sativa) and maize (Zea mays). To reveal differences between gall inductions by C. bipunctata and other gall inducers, we conducted four experiments concerning (a) the relationship between the feeding site and gall-induction sites of C. bipunctata on maize, (b) the effects of leafhopper sex and density, (c) the effects of length of infestation on gall induction, and (d) the effects of continuous infestation. C. bipunctata did not induce galls on the leaves where it fed but induced galls on other leaves situated at more distal positions. The degree of gall induction was significantly correlated with infestation density and length. These results indicate that C. bipunctata induces galls in a dose-dependent manner on leaves distant from feeding sites, probably by injecting chemical(s) to the plant during feeding. We suggest that insect galls are induced by a chemical stimulus injected by gall inducers during feeding into the hosts.

Genetic exchange between two freshwater apple snails, Pomacea canaliculata and Pomacea maculata invading East and Southeast Asia

Two species of apple snails, Pomacea canaliculata and Pomacea maculata (formerly Pomacea insularum), have invaded many countries of East and Southeast Asia from their native range in South America. This study investigated the genetic structure of the two species invading these areas. Phylogenetic analysis based on sequences of the nuclear gene elongation factor 1-alpha (EF1α) detected two well-supported clades (Clade C and Clade M). Both P. canaliculata and P. maculata were represented in each clade. Some snails had both Clade C and Clade M EF1α sequences. These results suggest genetic exchange between snails of the two clades. A mating experiment between P. canaliculata with Clade C EF1α sequences and P. maculata with Clade M EF1α sequences resulted in viable F1 progeny under laboratory conditions. The genetic exchange was also inferred in some populations collected from Argentina, suggesting an existence of hybrid in the native range. Simple identification of EF1α types using a restriction enzyme, ApaLI, detected significant geographical structure of the EF1α variants in the invaded area. The divergent geographical structure could have resulted from either the founder effect or the bridgehead effect, although further genetic analysis is needed to clarify this. Average individual egg weight, which is an indicator of egg size, was higher in P. canaliculata than P. maculata in both field and laboratory reared samples, suggesting that some (probably most) P. canaliculata and P. maculata invading East and Southeast Asia still maintain species-specific populations.

Changes in chemical components in the freshwater apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae), in relation to the development of its cold hardiness.

The apple snail, Pomacea canaliculata, is an invasive freshwater snail. It increases its cold hardiness before winter. However, the physiological mechanism of cold hardiness in molluscs is poorly understood, especially in freshwater molluscs. In this study, we examined the changes in low molecular weight compounds, glycogen and lipids, in the body of P. canaliculata in association with the development of cold hardiness. When snails without cold hardiness were experimentally cold-acclimated, the amount of glycerol, glutamine, and carnosine increased, while glycogen and phenylalanine decreased. Overwintering cold-tolerant snails collected from a drained paddy field in November also showed increased glycerol in their bodies with decreasing glycogen concentration, compared to summer snails collected from a submerged field. Water content also decreased during the cold acclimation, although the water loss was minimal. These results indicate that the freshwater snail, P. canaliculata enhances cold hardiness by accumulation of some kinds of low molecular weight compounds in its body as some insects do. However, the actual function of each low molecular compound is still unknown.

Sequence homogeneity of the ψserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regions of 'Candidatus Liberibacter asiaticus' isolates around Okinawa Main Island in Japan.

In Japan and Southeast Asia, ‘Candidatus Liberibacter asiaticus’ (Las) is the dominant causal agent of citrus greening (huanglongbing) disease. Using PCR techniques, we determined the 11168-nucleotide sequence of the ψserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regions for 51 Japanese, four Taiwanese, four Indonesian, and three Vietnamese isolates of Las. The sequence is identical in 62 isolates collected from Japan, Taiwan, Indonesia, and Vietnam, except for nucleotide substitutions at 11 positions. Some Las isolates from Sakishima Islands near Taiwan had unique nucleotide mutations, but all Las isolates around Okinawa Main Island were homologous. On the basis of the pattern of single nucleotide polymorphisms (SNPs) of the 11 nucleotide substitutions, the 62 Las isolates from Japan, Taiwan, Indonesia, and Vietnam could be divided into 12 pattern groups, and the 51 Japanese isolates consisted of six patterns. The results suggested that one unique genetic group is dominant around Okinawa Main Island, whereas several different are commonly distributed around islands near Taiwan.

Both nymphs and adults of the maize orange leafhopper induce galls on their host plant

The maize orange leafhopper, Cicadulina bipunctata is a multivoltine insect that induces galls on various plants of the Poaceae. A previous study revealed that galls produced by this leafhopper were induced by dose-dependent stimulation on distant leaves from the feeding site, probably by chemical(s) injected from adults during feeding. In this paper, we examined the gall-inducing ability of C. bipunctata nymphs. The degree of gall induction gradually increased depending on the number of feeding nymphs and there were no significant differences from the positive control (feeding by five male adults) when seedlings were exposed to five or more nymphs. These results indicate that both adults and nymphs of C. bipunctata have the ability to induce galls on their host plants, a unique feature among gall-inducing insects. This feature may be related to the free-living, multivoltine, and polyphagous habits of C. bipunctata.

Detection and diagnosis of rice-infecting viruses

Rice-infecting viruses have caused serious damage to rice production in Asian, American, and African countries, where about 30 rice viruses and diseases have been reported. To control these diseases, developing accurate, quick methods to detect and diagnose the viruses in the host plants and any insect vectors of the viruses is very important. Based on an antigen–antibody reaction, serological methods such as latex agglutination reaction and enzyme-linked immunosorbent assay have advanced to detect viral particles or major proteins derived from viruses. They aid in forecasting disease and surveying disease spread and are widely used for virus detection at plant protection stations and research laboratories. From the early 2000s, based on sequence information for the target virus, several other methods such as reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription-loop-mediated isothermal amplification have been developed that are sensitive, rapid, and able to differentiate closely related viruses. Recent techniques such as real-time RT-PCR can be used to quantify the pathogen in target samples and monitor population dynamics of a virus, and metagenomic analyses using next-generation sequencing and microarrays show potential for use in the diagnosis of rice diseases.

Quantitative Analysis of Southern rice black-streaked dwarf virus in Sogatella furcifera and Virus Threshold for Transmission.

We investigated Southern rice black-streaked dwarf virus (SRBSDV) accumulation in a vector insect, the whitebacked planthopper (Sogatella furcifera), to elucidate the association of virus accumulation in the vector with virus transmission efficiency. Real-time quantitative reverse transcription polymerase chain reaction analysis confirmed that this virus is transmitted in a persistent-propagative manner. SRBSDV was successfully transmitted by S. furcifera males in which RNA accumulation of the capsid protein gene of SRBSDV was >10(3) in the whole body of S. furcifera, indicating that the threshold accumulation of the virus RNA for virus transmission is 10(3) in an S. furcifera male. The SRBSDV detection rate in the immigrant population of S. furcifera was high in 2011 (39.5%); however, most of the insects contained fewer than 10(3) RNAs of the capsid protein gene. This result indicates that the risk of SRBSDV epidemics could be estimated from the proportion of virus-transmissible S. furcifera (i.e., S. furcifera that contained more than 10(3) RNAs of the virus capsid protein gene) rather than the SRBSDV detection rate in S. furcifera.