Madoka Nakai

Genetic and biological comparisons of ten geographic isolates of a nucleopolyhedrovirus that infects Spodoptera litura (Lepidoptera: Noctuidae)

Abstract Nine nucleopolyhedrovirus (NPV) isolates from Japan, Vietnam, Malaysia, and India collected from diseased Spodoptera litura larvae and an Egyptian isolate of Spodoptera littoralis NPV were subjected to genetic and biological comparisons to select a candidate isolate for controlling S. litura. In restriction endonuclease (REN) analysis, submolar bands were detected in 6 of the 10 isolates, showing that these viral populations consisted of a mixture of several genotypes. Based on equimolar bands, the isolates could be broadly divided into two groups: S. litura NPV-type and S. littoralis NPV-type. Four isolates from Southern and Southeast Asia and one from the Ogasawara Islands in Japan had S. litura NPV-type REN profiles. Four isolates from the main island of Japan had REN profiles closely related to the Egyptian isolate, S. littoralis NPV-B, suggesting that S. littoralis NPV-B type is widely distributed in Japan. Droplet feeding bioassays with second and fourth instar larvae showed no evidence of differences in biological activity among the 10 isolates based on the infectivity and survival time of infected larvae.

Oviposition Efficacy of Drosophila suzukii (Diptera: Drosophilidae) on Different Cultivars of Blueberry

ABSTRACT Drosophila suzukii (Matsumura) is an important pest of thin-skinned fruits including blueberry, raspberry, strawberry, and cherry. Blueberry was introduced into Japan in the 1950s, and severe economic losses attributable to D. suzukii were first reported in 2002. The objective of this study was to elucidate whether oviposition behavior varies among blueberry cultivars having different firmness of fruit. Fruit firmness in 12 cultivars of highbush blueberry (Vaccinium corymbosum L.) and rabbiteye blueberry (Vaccinium virgatum Aiton) was determined using a rheometer. More eggs tended to be laid in berries of cultivars possessing softer fruits than in those having firmer fruits. Choice tests, where one female was allowed to oviposit on blueberry fruits with different firmness, showed that softer fruits were more vulnerable to D. suzukii females than firmer fruits.

Viral-enhancing activity of various stilbene-derived brighteners for a Spodoptera litura (Lepidoptera: Noctuidae) nucleopolyhedrovirus

Abstract Previous reports have shown that stilbene-derived optical brighteners enhance viral activity in some baculovirus–lepidopteran host systems. To investigate the relationship between chemical structure and enhancement activity, we synthesized 11 stilbene-derived brighteners and compared their enhancing activity with a nucleopolyhedrovirus (NPV) with that of Tinopal UNPA-GX in common cutworm larvae, Spodoptera litura . Tinopal UNPA-GX significantly increased larval susceptibility to NPV infection, and the LD 50 of NPV was reduced about 2200-fold by the addition of 1% Tinopal to the virus suspension. Five of the 11 brighteners enhanced NPV infection to a similar extent as Tinopal, and one brightener enhanced virus infection more effectively than Tinopal. Replacing the diethyl amino group of Tinopal UNPA-GX with other chemical groups changed the enhancing activity of the brighteners. In addition, disruption of the peritrophic membrane (PM) was observed only when the cutworm larvae were given brighteners that had viral-enhancing activity. This suggests that brighteners disrupt the PM that lines the insect midgut and facilitate NPV infection of midgut epithelial cells.

Novel RNA sequences associated with late male killing in Homona magnanima

Maternally inherited female-biased sex ratios have been documented in many invertebrate species. One cause of such biased sex ratios is male killing, i.e. only males die. In most species, male killing occurs during embryonic stages (early male killing) and is associated with cytoplasmic bacteria, including Wolbachia, Spiroplasma, Rickettsia, Flavobacteria and gamma proteobacteria. However, the oriental tea tortrix, Homona magnanima, is one of the few species in which male death occurs in the larval or pupal stage, and is thus an example of late male killing. We partially purified the agent causing late male killing in H. magnanima and showed that it consists of two RNA sequences. This represents an entirely novel agent causing late male killing.

New Insights into the Evolution of Entomopoxvirinae from the Complete Genome Sequences of Four Entomopoxviruses Infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata

ABSTRACT Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology, structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.

Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host’s immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

Granulovirus prevents pupation and retards development of Adoxophyes honmai larvae

Abstract Larvae of Adoxophyes honmai (Lepidoptera: Tortricidae) infected with granulovirus (AdhoGV) do not pupate; instead, they undergo prolonged larval development and die during the final stadium. Non‐infected larvae, however, pupate after five larval stadia. Insect metamorphosis is regulated by fluctuations of ecdysteroid and Juvenile Hormone (JH). JH esterase activity and titres of ecdysteroid must be measured to understand fully the interaction of an insect virus and its host. JH esterase activity is consistently low in AdhoGV‐infected larvae, which suggests that JH in AdhoGV‐infected larvae is not degraded during the final stadium. The ecdysteroid titre in non‐infected larvae showed a large peak in the final stadium before pupation, whereas that in AdhoGV‐infected larvae increased from day 2 to day 5 in the final stadium, and then remained at a high level until death. Furthermore, an ecdysteroid UDP‐glucosyltransferase (EGT) assay showed that this activity occurs in haemolymph from AdhoGV‐infected larvae, but not in haemolymph of non‐infected larvae. PCR and sequencing analysis revealed that the AdhoGV genome contains an egt gene, which encodes a protein of 445 amino acids, located approximately 1 kbp upstream from the granulin gene. These results suggest that AdhoGV‐infected larvae are prevented from pupating because JHE activity is suppressed and EGT expression inactivates ecdysteroid in the haemolymph.

Late male-killing phenomenon found in a Japanese population of the oriental tea tortrix, Homona magnanima (Lepidoptera: Tortricidae)

A female-biased sex ratio was found in the oriental tea tortrix, Homona magnanima (Lepidoptera: Tortricidae), in Tsukuba, Ibaraki, Japan. There was no difference in mean egg hatch between the all-female and normal strains. Greater than 50% mortality was observed in the all-female strain larvae, suggesting that female-only broods are produced as a result of late male-killing. The female-biased sex ratio was maternally inherited and maintained, even when females were backcrossed with males of the normal strain, thus implicating cytoplasmic parasitism as its cause. The phenomenon was persistent in the presence of antibiotics, and was not due to infection by agents that cause other male-killing phenomena, such as Rickettsia, Wolbachia, Spiroplasma, or protozoan parasites. When a homogenate of dead male larvae of the all-female strain was inoculated in normal-strain larvae, this male-killing trait was transmitted to the next generation; thus, its causative agent is probably transmitted horizontally as well.

Effect of entomopoxvirus infection of the smaller tea tortrix, Adoxophyes sp. on the development of the endoparasitoid, Ascogaster reticulatus

Infection of Adoxophyes sp. (Lepidoptera: Tortricidae) larvae by an entomopoxvirus (AsEPV) adversely affected the development of the endoparasitoid, Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae). Parasitoid larvae developing in AsEPV‐infected hosts grew more slowly and spent more time in their hosts than did parasitoid larvae developing in noninfected hosts. Percentages of emergence of larval parasitoids that developed in AsEPV‐infected hosts were significantly lower than those of parasitoids that developed in noninfected hosts. Parasitoid larvae in AsEPV‐infected host perished when their hosts died of AsEPV infection. Significant numbers of parasitized and infected larvae exhibited apolysis to the final instar, whereas noninfected‐parasitized larvae died in the penultimate instars due to emergence of parasitoids.