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

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Abstract

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, Adoxophyes dubia, 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.

Introduction

Entomopoxviruses (EPVs) are large oval-shaped viruses with a linear double-stranded DNA genome and a molecular weight of 200–240 kbp. EPVs belong to one of two Poxviridae subfamilies: Entomopoxvirinae, which are insect poxviruses, or Chordopoxvirinae, which are vertebrate poxviruses (King et al., 1998). Entomopoxvirinae is divided into three genera according to viral morphology, host range, and genome size: Alphaentomopoxvirus, Betaentomopoxvirus, and Ganmaentomopoxvirus. Alphaentomopoxvirus comprise viruses infecting coleopterans, Betaentomopoxvirus infects lepidopterans and orthopterans, while those from Ganmaentomopoxvirus infect dipterans (Buller et al., 2005). Although recent sequencing studies have shown that all members of the two Poxviridae subfamilies have several conserved genes, EPVs are believed to be specific to insects. EPV virions are embedded in occlusion bodies (OBs) or spheroids composed mainly of a highly expressed protein called spheroidin. After ingestion by the insect host, OBs are dissolved by the alkaline-reducing conditions in the midgut and release virions, resulting in viral replication in the midgut epithelial cells, followed by transmission to the internal tissues. Deleting the spheroidin gene has no effect on viral replication in vitro (Palmer et al., 1995); however, spheroidin should have an important function in the viral life cycle in nature by protecting virions from environmental asperity, similar to the polyhedrins of baculoviruses or cypoviruses. EPV pathology depends on the insect host, but the infection course is generally slow (Arif, 1995). For example, larvae of the lepidopteran Choristoneura fumiferana infected with C. fumiferana EPV show few symptoms until late in the infection and die in 1–3 weeks (Palli et al., 2000). The EPV-infected orthopteran Locusta migratoria shows a decreased developmental rate and eventually dies 20–60 days after infection (Jaeger and Langridge, 1984). In the dipterans, Chironomus attenuatus and Goeldichironomus haloprasimus, EPV-infected larvae appear to survive up to 8 weeks after infection (Huger et al., 1970). EPV-infected coleopterans may survive for up to 40 weeks (Goodwin and Roberts, 1975).

The potential use of EPVs as microbial control agents has attracted attention (Mason and Erlandson, 1994, Wegensteiner et al., 1996, Woods et al., 1992). Microbial control agents have been used for integrated pest-management programs with differing strategies (Fuxa, 1987). For example, a pathogen that can kill its insect host shortly after infection could be used in place of a chemical insecticide. In contrast, an appropriate use of a pathogen that needs considerable time to kill its insect host, such as EPVs, may reduce the population growth rate of the insect pest via epizootics. Even small decreases in the population growth rate can affect insect outbreak frequency and, thereby, diminish the need for chemical insecticides (Throne, 1989). This strategy relies on natural epizootics, so it requires persistence and efficient transmission in the target insect pest population and ecosystem. Before applications in nature, it is useful to know the phenotypic characteristics related to viral fitness under laboratory conditions, such as infectivity, kill speed, and viral yield in the target insect pest. Such characteristics pertain to the capability to cause epizootics, which is important in assessing the viral agents for use in the control strategy under consideration. Additionally, comparing viral isolate fitness-related traits aids in the selection of a candidate isolate for insect pest control.

Here, we describe an experiment in which we examined the fitness-related traits of three EPV isolates (hereafter, AdhoEPV) from the smaller tea tortrix, A. honmai, from different localities in Japan to select good potential isolate(s) as a control agent against tortricid insect pests, particularly orchard pests, such as the summer fruit tortrix, Adoxophyes orana, known as a pest of apple and the oriental tea tortrix, Homona magnanima, known as a pest of pea as well as tea. Some tortricid lepidopterans are the principal apple and pear orchard pests in Japan, the United States, and Europe. Although the control of these pests has depended on conventional chemical insecticides, developing insecticide resistance and concerns about product safety and environmental health require the development of new agents. In the United States and Europe, Cydia pomonella granulovirus (CpGV) has been developed as an alternative agent, but it is limited by its narrow host range and lack of persistence in orchards (Cross et al., 1999). These are often cited as shortcomings of microbial control agents. An AdhoEPV isolate from Miyazaki, Japan infected several orchard and tea pest species of the family Tortricidae, but infection was examined using only external EPV disease symptoms (Ishikawa et al., 1983). Furthermore, AdhoEPV disease occurs endemically, and the prevalence has reached high levels in populations of A. honmai and H. magnanima in tea fields (Nakai et al., 1997; Kunimi, unpublished data). Although the degree of persistence depends on the ecosystem, this may suggest that AdhoEPV can persist in tortricid pest populations once applied.

We also subjected the three AdhoEPV isolates to biochemical comparisons to examine their genetic relatedness and sought to determine whether genotypic variation in the related EPVs translates into phenotypic differences that can affect host–EPV dynamics. Genotypic variation and differences in pathogenicity in insect–virus populations have been well demonstrated in baculoviruses at several ecological scales, but very little is known about the evolutionary processes leading to the occurrence and maintenance of genotypic variation and the dynamics of genotypes (Cory et al., 1997). Genotypic variation in EPV populations, however, has received little attention. We hope our results will stimulate studies of genotypic and phenotypic variation in EPV populations.

Section snippets

Insects

We used six species from the family Tortricidae. A. honmai, A. orana, and Pandemis heparana strains were supplied by Agro-Kanesho Co., Ltd. (Saitama, Japan), Nagano Fruit-Tree Experiment Station (Nagano, Japan), and Nippon Kayaku Co., Ltd. (Tokyo, Japan), respectively. The H. magnanima, Adoxophyes dubia, and Archips insulanus strains were originally collected from tea fields in Tokyo, Okinawa Prefecture, and Okinawa Prefecture, Japan, respectively. All colonies were maintained continuously at 25

SDS–PAGE of viral structural proteins

The polypeptide banding patterns of the spheroids and virions obtained using SDS–PAGE were identical irrespective of the AdhoEPV isolate and EPV-propagation host. Examples of AdhoEPVs propagated in H. magnanima are shown in Fig. 2. Two intense bands migrating at apparent molecular weights of 116 and 46 kDa appeared in the samples derived from OB itself (Fig. 2A). The 116-kDa polypeptide may represent spheroidin, as the spheroidin molecular weights from other EPVs have been reported as 100 to 116 

Discussion

The REN analysis was able to differentiate the three AdhoEPV isolates, whereas viral structural protein SDS–PAGE could not, and DNA hybridization showed high genetic similarity among the three isolates. These results indicate that the three isolates could be genotypically distinguished but support the idea that they were related species or variants. The results also indicate that REN analysis is a sensitive method for identifying AdhoEPV isolates. This method has been used in baculoviruses to

Acknowledgments

We thank N. Onigahara, H. Oshikiri, and F. Shimizu for technical assistance. This work was supported by a grant for the Research and Development Program for New Bio-industry Initiatives (Consortium 3: The Development of New Microbial Biopesticides) from the Bio-oriented Technology Research Advancement Institution. JT was supported, in part, by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (No. 21380038).

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