Yasuhisa Kunimi

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.

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.

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.

Effect of entomopoxvirus infection of Pseudaletia separata larvae on the oviposition behavior of Cotesia kariyai

Parasitoid behavior and oviposition were monitored to investigate the ability of Cotesia kariyai (Watanabe) (Hymenoptera: Braconidae) to distinguish between entomopoxvirus‐infected and noninfected larvae of Pseudaletia separata Walker (Lepidoptera: Noctuidae). Comparison of individual means revealed that searching time was not significantly different between treatments. Ovipositional time, however, differed significantly after day 8 post virus inoculation. The parasitoid adopted ovipositional posture on infected and noninfected host irrespective of the stage of infection and did not distinguish infected from noninfected larvae up to 4 days post infection. Mean number of larvae stung by the parasitoid was not influenced by the length of time interval between exposure to the virus. However, duration of ovipositor insertion was influenced by the time interval between exposure to the virus and subsequent exposure to parasitoid females. Female parasitoids did not completely reject infected larvae as unsuitable for egg deposition. However, 5 days after virus inoculation they rejected significantly more infected than noninfected larvae. Furthermore, significantly more eggs were laid in noninfected than infected larvae from day 5 following virus administration. These observations suggest that probing by Cotesia kariyai may provide information concerning host suitability.

Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan

Three entomopoxviruses (EPVs) isolated from diseased Adoxophyes honmai larvae at different localities (Tsukuba, Itsukaichi, and Miyazaki) in Japan were compared for biochemical identity and key parameters of virus fitness, fatal infection, speed of kill, and virus yield. When the structural peptides of occlusion bodies (OBs) and occlusion-derived viral particles were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no difference in banding patterns was observed. However, DNA restriction endonuclease analysis showed that the three isolates were genotypically different, but many commonly sized DNA fragments were observed. Five tortricid species, A. honmai, Adoxophyes orana, Adoxophyesdubia, Homona magnanima, and Archips insulanus were susceptible to all isolates. No significant differences in the key viral fitness parameters were detected among the isolates in A. orana. However, the Miyazaki isolate had a different effect on H. magnanima; it allowed infected insects to survive longer and develop to a larger size, but had a lower yield of OBs per larva at any given time to death. OB yields per unit cadaver weight for the Miyazaki isolate, which indicate the conversion rate of the insect to virus, were lower over time compared to the other two isolates. The implications for selecting a candidate isolate to control tortricid pests are discussed.