Lack of avoidance of the fungal entomopathogen, Metarhizium brunneum, by male Agriotes obscurus beetles.

Fungal entomopathogens can greatly reduce the fitness of their hosts, and it is therefore expected that susceptible insects will be selected to avoid exposure to pathogens. Metarhizium brunneum is a fungal pathogen that can infect Agriotes obscurus, which in its larval form is a destructive agricultural pest and is repelled by the presence of M. brunneum conidia. Due to the subterranean nature of larval A. obscurus, recent research has focused on targeting adult A. obscurus with M. brunneum. No-choice and choice behavioural assays were conducted to determine if male adult A. obscurus avoid M. brunneum mycosed cadavers, or conidia applied to either food or soil. To further investigate the response of A. obscurus beetles to conspecific cadavers, the movement and behaviour of beetles placed at the centre of a semi-circular arrangement of mycosed or control cadavers was examined using motion tracking software. We found little evidence to suggest that A. obscurus male beetles avoid M. brunneum conidia or mycosed conspecific cadavers or alter their behaviour in their presence.

Trans-generational viral transmission and immune priming are dose-dependent.

It is becoming increasingly apparent that trans-generational immune priming (i.e. the transfer of the parental immunological experience to its progeny resulting in offspring protection from pathogens that persist across generations) is a common phenomenon not only in vertebrates, but also invertebrates. Likewise, it is known that covert pathogenic infections may become 'triggered' into an overt infection by various stimuli, including exposure to heterologous infections. Yet, rarely have both phenomena been explored in parallel. Using as a model system the African armyworm Spodoptera exempta, an eruptive agricultural pest and its endemic dsDNA virus (Spodoptera exempta nucleopolyhedrovirus, SpexNPV), the aim of this study was to explore the impact of parental inoculating-dose on trans-generational pathogen transmission and immune priming (in its broadest sense). Larvae were orally challenged with one of five doses of SpexNPV and survivors from these treatments were mated and their offspring monitored for viral mortality. Offspring from parents challenged with low viral doses showed evidence of 'immune priming' (i.e. enhanced survival following SpexNPV challenge); in contrast, offspring from parents challenged with higher viral doses exhibited greater susceptibility to viral challenge. Most offspring larvae died of the virus they were orally challenged with; in contrast, most offspring from parents that had been challenged with the highest doses were killed by the vertically transmitted virus (90%) and not the challenge virus. These results demonstrate that the outcome of a potentially lethal virus challenge is critically dependent on the level of exposure to virus in the parental generation-either increasing resistance at very low parental viral doses (consistent with trans-generational immune priming) or increasing susceptibility at higher parental doses (consistent with virus triggering). We discuss the implications of these findings for understanding both natural epizootics of baculoviruses and for using them as biological control agents.

The effect of synthetic female sex pheromone on the transmission of the fungus Metarhizium brunneum by male Agriotes obscurus click beetles.

Autodissemination techniques can potentially be used to distribute insecticides, including microbial insecticides, to cryptic pests. This approach is reliant on the target insect either passing the pathogen passively to other insects or the pathogen cycling within the population after the initial host dies. Here we examine, in small scale experiments, whether male Agriotes obscurus click beetles passively transmit the spores of the fungus Metarhizium brunneum directly, or indirectly via the environment, and whether this is influenced by exposure to synthetic female pheromone. We found that the beetles did not avoid M. brunneum spores and that this behaviour was not affected by pheromone. Exposure to pheromone increased beetle movement and uptake of spores, but this did not result in an increase in infected beetles under our conditions. Beetles were able to transfer spores at high levels via environmental contamination. However, contamination of the environment declined rapidly after exposure to the spores. The results are discussed in the context of developing an autodissemination strategy for click beetles.

Modification of reproductive schedule in response to pathogen exposure in a wild insect: Support for the terminal investment hypothesis.

Trade-offs in the time and energy allocated to different functions, such as reproductive activities, can be driven by alterations in condition which reduce resources, often in response to extrinsic factors such as pathogens or parasites. When individuals are challenged by a pathogen, they may either reduce reproduction as a cost of increasing defence mechanisms or, alternatively, modify reproductive activities so as to increase fecundity thereby minimizing the fitness costs of earlier death, a behaviour consistent with the terminal investment hypothesis (TIH). The TIH predicts that individuals with decreased likelihood of future reproduction will maximize current reproductive effort, which may include shifts in reproductive timing. We examined how wild, adult female click beetles (Agriotes obscurus) responded after exposure to the fungal pathogen Metarhizium brunneum. Field-collected beetles exposed to a high concentration of M. brunneum died earlier and in greater numbers than those exposed to a low concentration. Using a multivariate approach, we examined the impact of pathogen challenge on lifespan and a suite of reproductive traits. Stepdown regression analysis showed that only female lifespan differed among the fungal treatments. Fungal-induced reductions in lifespan drove changes in the reproductive schedule, characterized by a decrease in preoviposition period. Moving the start of egg laying forward allowed the females to offset the costs of a shortened lifespan. These changes suggest that there is a threshold for terminal investment, which is dependent on strength of the survival threat. From an applied perspective, our findings imply that exposing adult click beetles to M. brunneum to reduce their population density might not succeed and is an approach that needs further investigation.

The impact of baculovirus challenge on immunity: The effect of dose and time after infection.

Understanding how hosts respond to pathogen attack is crucial to disease management. The response of a host can be particularly important if hosts have to defend against multiple pathogens which could either benefit from or be suppressed by prior pathogen exposure. Insect defence against viruses is less well understood than responses to other entomopathogens and much of the information available relates to in vitro studies and model systems. Baculoviruses are natural pathogens of insects, particularly Lepidoptera, and have been well-studied in terms of their ecology, pest control potential and molecular biology. In order to examine how an insect reacts to baculovirus challenge, we measured components of the cellular and humoral immune response of the cabbage looper Trichoplusia ni to Trichoplusia ni SNPV, a narrow-host range nucleopolyhedrovirus (NPV), over four doses and three times after pathogen challenge (18, 42 and 90 h). We found that total haemocyte numbers peaked at 42 h post-exposure at all doses, and declined linearly with increasing dose after the 18 h time point. Two immune-related enzymes, phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD), showed very different responses. PO levels were lowest at the 42 h time point and were not influenced by virus dose when each time point was examined separately. GLD levels declined over time but they interacted with virus dose in a non-linear manner, such that there was an increase in levels at intermediate virus doses after 18 h, no effect at 42 h, and then declined as infection progressed at 90 h post-infection. These data suggest that baculoviruses can rapidly infect haemocytes (or cause a reduction in their numbers) in a dose-dependent manner once the infection is systemic, likely reducing the ability of the host to counter subsequent infections. However, the data do not support a direct role for PO in defence against baculoviruses. Whether GLD plays a role in virus defence is still unclear.

Trade-offs and mixed infections in an obligate-killing insect pathogen.

Natural populations of pathogens are frequently composed of numerous interacting strains. Understanding what maintains this diversity remains a key focus of research in disease ecology. In addition, within-host pathogen dynamics can have a strong impact on both infection outcome and the evolution of pathogen virulence, and thus, understanding the impact of pathogen diversity is important for disease management. We compared eight genetically distinguishable variants from Spodoptera exempta nucleopolyhedrovirus (SpexNPV) isolated from the African armyworm, Spodoptera exempta. NPVs are obligate killers, and the vast majority of transmission stages are not released until after the host has died. The NPV variants differed significantly in their virulence and could be clustered into two groups based on their dose-response curves. They also differed in their speed of kill and productivity (transmission potential) for S. exempta. The mixed-genotype wild-type (WT) SpexNPV, from which each variant was isolated, was significantly more virulent than any individual variant and its mean mortality rate was within the fastest group of individual variants. However, the WT virus produced fewer new infectious stages than any single variant, which might reflect competition among the variants. A survival analysis, combining the mortality and speed of kill data, confirmed the superiority of the genetically mixed WT virus over any single variant. Spodoptera exempta larvae infected with WT SpexNPV were predicted to die 2·7 and 1·9 times faster than insects infected with isolates from either of the two clusters of genotypes. Theory suggests that there are likely to be trade-offs between pathogen fitness traits. Across all larvae, there was a negative linear relationship between virus yield and speed of kill, such that more rapid host death carried the cost of producing fewer transmission stages. We also found a near-significant relationship for the same trend at the intervariant level. However, there was no evidence for a significant relationship between the induced level of mortality and transmission potential (virus yield) or speed of kill.

Altered nutrient intake by baculovirus-challenged insects: Self-medication or compensatory feeding?

Infection by parasites can alter the feeding behaviour of hosts. Some animals seek out substances that can therapeutically clear infections (self-medication), some may seek out resources to recoup resources lost while fighting off infection (compensatory feeding) and others may be manipulated to ingest substances that benefit parasite fitness (parasite manipulation of host). Recent studies have indicated that pathogen-challenged insects can self-medicate by increasing their protein intake relative to carbohydrate, which is thought to act by boosting the insect's immune response. However, increased protein intake could also be due to compensatory feeding or pathogen manipulation of the host, and a rigorous examination of all four of the testable predictions, which is necessary for verifying self-medication behaviour, has not been conducted. The therapeutic behaviour must (1) only be employed by infected individuals and (2) alleviate the potential fitness loss of the infected individual. (3) If an uninfected individual engages in the behaviour, they suffer a decrease in fitness, and lastly, (4) the parasite cannot benefit from the behaviour. In response to baculovirus-challenge (AcMNPV) at 24°C, the cabbage looper, Trichoplusia ni, increased proportional protein intake, by increasing protein intake rather than decreasing carbohydrate intake. Increased protein intake did not benefit virus fitness, but it also did not increase the probability of host survival. Increased proportional protein intake did not occur in response to TnSNPV-challenge at 24°C or in response to AcMNPV-challenge at a higher temperature (32°C), indicating that the virus-induced change in nutrient intake depends on virus identity and temperature. Since virus-challenged T. ni did not show the typical costs associated with infection, the altered nutrient intake is likely to be a compensatory response. Understanding the motivation behind pathogen-induced changes in feeding behaviour could have significant implications for determining its importance for species interactions at multiple trophic levels.

Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming.

Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to depend on the host-pathogen system, or the level of pathogen exposure and the type of dietary manipulation. Identifying whether immune priming or transgenerational effects are relevant in field populations, remains a challenge.

Compatibility of the fungus Beauveria bassiana and Trichoplusia ni SNPV against the cabbage looper Trichoplusia ni: crop plant matters.

BACKGROUND: Microbial insecticides are an important weapon in insect pest management, but their use is still relatively limited. One approach for increasing their efficacy and use could be to combine different pathogens to increase pest mortality. However, little is known about whether increasing pathogen diversity will improve pest management. Here, we investigated the compatibility of two pathogens for the management of the cabbage looper, Trichoplusia ni, T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus Beauveria bassiana, on two crops, tomato and broccoli. The pathogens were applied to individual plants using ultra low volume sprays, alone or in combination, either synchronously or asynchronously. Healthy third-instar T. ni larvae were introduced to the plants before application and collected by destructive sampling 24 h after the last pathogen application. RESULTS: Combined applications did not result in an increase in larval mortality compared to TniSNPV alone, although mortality was generally high. B. bassiana was considerably less effective on broccoli compared to tomato. In both the combined treatments, virus-induced mortality was approximately 50% lower when applied together with the fungus, while fungus-induced mortality was not affected by the virus, even when the virus was introduced 24 h before the fungus. CONCLUSION: While our results suggest that applying this combination of entomopathogens would not be beneficial for pest management, this study illustrates the need to consider the target crop as an important driver of the efficacy of both single and mixed pathogen applications in the field. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Warmer temperatures reduce the transmission of a virus in a gregarious forest insect.

Understanding how climate warming will influence species interactions is a key question in ecology and predicting changes in the prevalence of disease outbreaks is particularly challenging. Ectotherms are likely to be more influenced by climatic changes as temperature governs their growth, feeding, development, and behavior. We test the hypothesis that pathogen transmission and host mortality will increase at warmer temperatures using a cyclic forest insect, the western tent caterpillar (WTC), Malacosoma californicum pluviale, and its baculovirus. The virus causes population declines at peak host density. WTC are gregarious and clustering is predicted to increase the risk of within family infection; however, how temperature influences this has not been examined. We investigated the impact of temperature on different components of the transmission process in order to pinpoint the possible mechanisms involved. In the laboratory, leaf consumption increased linearly with rising temperature between 15 and 30°C. Insects died more rapidly from virus infection as temperature increased, but this did not translate into differences in the production of viral transmission stages. To examine the influence of temperature on virus transmission, we created a temperature difference between two greenhouses containing potted red alder trees, Alnus rubra. The cooler greenhouse (mean 19.5°C) was roughly similar to ambient temperatures in the field, while the warmer greenhouse was 10°C higher (mean 29°C). As predicted, both larval movement and feeding were higher at the warmer temperature, while the likelihood of the preinfected, inoculum larvae dying on the tents was twice as high in the cooler greenhouse. This resulted in increased virus mortality and a higher transmission parameter under cooler conditions. Therefore, we suggest that, contrary to our prediction, the reduced movement of infected larvae at colder temperatures increased the risk of infection in these gregarious insects and had a greater impact on virus transmission than the increased activity of the susceptible larvae in warmer conditions. Long-term population data from the field, however, show no relationship between temperature and infection levels, suggesting that local changes in virus transmission might not scale up to population infection levels.

Resource limitation has a limited impact on the outcome of virus-fungus co-infection in an insect host.

Infection by pathogens is strongly affected by the diet or condition of the prospective host. Studies that examine the impact of diet have mainly focused on single pathogens; however, co-infections within a single host are thought to be common. Different pathogen groups might respond differently to resource availability and diverse infections could increase the costs of host defense, meaning the outcome of mixed infections under varying dietary regimes is likely to be hard to predict. We used the generalist cabbage looper, Trichoplusia ni and two of its pathogens, the DNA virus T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus, Beauveria bassiana to examine how nutrient reduction affected the outcome of mixed pathogen infection. We challenged insects with a low or high effective dose of virus, alone or combined with a single dose of fungus. We manipulated food availability after pathogen challenge by diluting artificial diet with cellulose, a non-nutritious bulking agent, and examined its impact on host and pathogen fitness. Reducing diet quantity did not alter overall or pathogen-specific mortality. In all cases, TniSNPV-induced mortality was negatively affected by fungus challenge. Similarly, B. bassiana-induced mortality was negatively affected by TniSNPV challenge, but only at the higher virus dose. Dietary dilution mainly affected B. bassiana speed of kill when mixed with a high dose of TniSNPV, with an increase in the duration of fungal infection when cellulose was low (high quantity). One pathogen dominated the production of transmission stages in the cadavers and co-infection did not affect the yield of either pathogen. There was no evidence that co-infections were more costly to the survivors of pathogen challenge. In conclusion, dietary dilution did not determine the outcome of mixed pathogen infection, but it had more subtle effects, that differed between the two pathogens and could potentially alter pathogen recycling and host-pathogen dynamics.

The effect of food limitation on immunity factors and disease resistance in the western tent caterpillar.

Epizootics of nucleopolyhedrovirus characterize declines of cyclic populations of western tent caterpillars, Malacosoma pluviale californicum. In field populations, infection can be apparently lacking in one generation and high in the next. This may suggest an increase in the susceptibility to infection of larvae at peak density or the triggering of a vertically transmitted virus. Here, we test the hypothesis that reduced food availability, as may occur during population outbreaks of tent caterpillars, influences the immunocompetence of larvae and increases their susceptibility to viral infection. We compared immunity factors, hemolymph phenoloxidase and hemocyte numbers, and the susceptibility to nucleopolyhedroviral infection of fifth instar larvae that were fully or partially fed as fourth instars. To determine if maternal or transgenerational influences occurred, we also determined the susceptibility of the offspring of the treated parents to viral infection. Food limitation significantly reduced larval survival, development rate, larval and pupal mass, moth fecundity and levels of hemolymph phenoloxidase, but not the numbers of hemocytes. Neither the food-reduced larvae nor their offspring were more susceptible to viral infection and were possibly even less susceptible at intermediate viral doses. Food reduction did not activate latent or covert viral infection of larvae as might be expected as a response to stress. We conclude that reducing the food intake of fourth instar larvae to an extent that had measurable and realistic impacts on their life history characteristics was not translated into increased susceptibility to viral infection.

Detection of single and mixed covert baculovirus infections in eastern spruce budworm, Choristoneura fumiferana populations.

We surveyed for covert baculovirus infections in the eastern spruce budworm, Choristoneura fumiferana (Clemens) and compared the prevalence of virus detected in a laboratory and a field population. DNA was extracted from budworm adults and then PCR with degenerate primers was used to identify individuals carrying baculovirus DNA. Multiplex PCR was then applied to the positive samples to distinguish between the multiple baculovirus types that could potentially be found in C. fumiferana populations. Covert infections were found in both the laboratory and the field population of C. fumiferana, although the frequency of infection and the composition of viruses found were very different. Overall 28% of insects from the laboratory population were positive for baculovirus DNA. Individual adults supported both single and mixed covert infections with CfMNPV plus CfDEFNPV, CfDEFNPV plus a GV and mixtures of all three viruses together. However, the majority of insects supported single virus infections, and surprisingly this virus was CfDEFNPV, a virus that is reported not to have per os activity in C. fumiferana larvae. Insects from field populations showed a very different pattern; 70.5% of individuals were baculovirus positive and all of these were positive for CfDEFNPV only.

High levels of genetic diversity in Spodoptera exempta NPV from Tanzania.

The African armyworm, Spodoptera exempta, is a major pest in sub-Saharan Africa. A nucleopolyhedrovirus (NPV) is often recorded in later population outbreaks and can cause very high levels of mortality. Research has been addressing whether this NPV can be developed into a strategic biological control agent. As part of this study, the variation in natural populations of NPV is being studied. An isolate of S. exempta NPV was cloned in vivo and found to contain at least 17 genetically-distinct genotypes. These genotypes varied in size from approximately 115 to 153 kb.

Dose dependency of time to death in single and mixed infections with a wildtype and egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus.

Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster than wild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here, we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpa armigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletion of the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used to determine whether the total number of OBs influenced the response to challenge with a mixture of virus strains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly faster (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival time between the viruses. The survival time after challenge with mixed virus inoculum was significantly different from and intermediate between that of the single viruses at high doses, and not different from that of the single viruses at low doses. No differences in lethal dose were found between single and mixed infections or between virus genotypes. The number of occlusion bodies produced per larva increased with time to death and decreased with virus dose, but no significant differences among virus types were found.

Effect of Collection Month, Visible Light, and Air Movement on the Attraction of Male Agriotes obscurus L. (Coleoptera: Elateridae) Click Beetles to Female Sex Pheromone.

Elaterid female sex pheromone, while currently used for monitoring the adult life stage (click beetle), has only recently been explored as a potential management tool. Consequently, there is little understanding of how abiotic and biotic conditions influence the response of click beetles to the pheromone. We examined whether the response of male Agriotes obscurus L. (Coleoptera: Elateridae) beetles to a cellulose-based formulation of female sex pheromone ('pheromone granules') is influenced by air movement, presence of visible light, and month of beetle collection. In addition, we investigated the distance from which beetles were attracted to the pheromone granules. Click beetle response was determined by measuring movement parameters in free-walking arena experiments. The response to pheromone was not affected by the presence or absence of visible light. We found that beetles collected earlier in the season had increased activity and interaction with pheromone under moving air conditions, compared to beetles collected later. When controlling for storage time, we confirmed that individuals collected in May were less active than beetles collected in March and April. In the field, beetles were recaptured from up to 14 m away from a pheromone granule source, with over 50% being recovered within 4.4 h from a distance of 7 m or less. Understanding how abiotic and biotic factors affect pest response to pheromone can lead to more effective and novel uses of pheromone-based management strategies.

An experimental test of the independent action hypothesis in virus-insect pathosystems.

The 'independent action hypothesis' (IAH) states that each pathogen individual has a non-zero probability of causing host death and that pathogen individuals act independently. IAH has not been rigorously tested. In this paper, we (i) develop a probabilistic framework for testing IAH and (ii) demonstrate that, in two out of the six virus-insect pathosystems tested, IAH is supported by the data. We first show that IAH inextricably links host survivorship to the number of infecting pathogen individuals, and develop a model to predict the frequency of single- and dual-genotype infections when a host is challenged with a mixture of two genotypes. Model predictions were tested using genetically marked, near-identical baculovirus genotypes, and insect larvae from three host species differing in susceptibility. Observations in early-instar larvae of two susceptible host species support IAH, but observations in late-instar larvae of susceptible host species and larvae of a less susceptible host species were not in agreement with IAH. Hence the model is experimentally supported only in pathosystems in which the host is highly susceptible. We provide, to our knowledge, the first qualitative experimental evidence that, in such pathosystems, the action of a single virion is sufficient to cause disease.

Within and between population variation in disease resistance in cyclic populations of western tent caterpillars: a test of the disease defence hypothesis.

1. Epizootics of nucleopolyhedrovirus (NPV) are an obvious component of the population fluctuations of several species of temperate forest Lepidoptera, including the western tent caterpillar, Malacosoma californicum pluviale (Dyer). An observed relationship between epizootics and the subsequent reduction in fecundity of populations led to the formulation of the disease defence hypothesis. This hypothesis predicts that viral epizootics in peak populations select for more resistant moths and that their reduced fecundity in declining populations reflects a cost of disease resistance. 2. To test the disease defence hypothesis, we carried out bioassays to measure the variation in larval resistance to NPV infection for families of western tent caterpillars from four spatially distinct populations over 3 years of peak and declining host densities. 3. Each female moth lays a single egg mass and larvae are gregarious and remain together through development. We found that the resistance to disease of larvae within families was not related to the number of eggs in the mass from which they hatched (the fecundity of their mother). 4. Disease resistance of larvae varied among populations and over time in a manner consistent with selection for resistance. One population that had not experienced a strong viral epizootic during the last population decline was more susceptible to infection in the first year of the study. Larvae from a second population that experienced an early epizootic became significantly more resistant. The resistance of two other populations increased slightly before the viral epizootic occurred in the field however, and thus could not be explained by selection. 5. As population densities declined from peak density, the background mortality of larvae increased and the fecundity of moths decreased. This indicates a general deterioration in the quality of field populations of tent caterpillars associated with the declining populations. 6. Although some evidence suggests that viral epizootics can select for increased resistance of field populations of tent caterpillars, the general deterioration in quality, elevated background mortality, and the reduced fecundity after the epizootic are stronger influences on the population decline. These are possibly related to sublethal viral infection.

The complete genome sequence of an alphabaculovirus from Spodoptera exempta, an agricultural pest of major economic significance in Africa.

Spodoptera exempta nucleopolyhedrovirus (SpexNPV) is a viral pathogen of the African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), a significant agricultural pest of cereal crops in Africa. SpexNPV has been evaluated as a potential insecticide for control of this pest and has served as the subject of research on baculovirus pathology and transmission. Occlusion bodies (OBs) of SpexNPV isolate 244.1 were examined, and the nucleotide sequence of the genome was determined and characterized. SpexNPV-244.1 OBs consisted of irregular polyhedra with a size and appearance typical for alphabaculoviruses. Virions within the polyhedra contained 1-8 nucleocapsids per unit envelope. The SpexNPV-244.1 genome was comprised of a 129,528 bp circular sequence, in which 139 ORFs were annotated. Five homologous regions (hrs) consisting of a variable number of 28-bp imperfect palindromes were identified in the genome. The genome sequence contained the 38 core genes of family Baculoviridae, as well as three ORFs unique to the SpexNPV sequence and one ORF that was apparently acquired by horizontal gene transfer with a betabaculovirus ancestor. Phylogenetic inference with core gene amino acid sequence alignments placed SpexNPV-244.1 in a lineage containing alphabaculoviruses of Spodoptera frugiperda and Spodopotera exigua which in turn is part of a larger group of alphabaculoviruses from the subfamily Noctuinae in the lepidopteran family Noctuidae. Kimura-2-parameter pairwise nucleotide distances indicated that SpexNPV-244.1 represented a different and previously unlisted species in the genus Alphabaculovirus. Gene parity plots indicated that the gene order of SpexNPV-244.l was extensively collinear with that of Spodoptera exigua NPV (SeMNPV). These plots also revealed a group of 17 core genes whose order was conserved in other alpha- and betabaculoviruses.

Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology.

Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes - that differ only in a short tag sequence for differential PCR recognition - were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.