Adverse ffects of covert iridovirus infection on life history and demographic parameters of Aedes aegypti

Abstract Sublethal viral infections can cause changes in the body size and demography of insect vectors, with important consequences for population dynamics and the probability that individual mosquitoes will transmit disease. This study examined the effects of covert (sublethal) infection by Invertebrate iridescent virus 6 (IIV‐6) on the demography of female Aedes aegypti and the relationship between key life history parameters in covertly infected female insects compared with healthy (control) insects or non‐infected mosquitoes that had survived exposure to virus inoculum without becoming infected. Of the female mosquitoes that emerged following exposure to virus inoculum and were offered blood meals, 29% (43/150) proved positive for covert IIV‐6 infection. The net reproductive rate (R₀) of covertly infected females was 50% lower for infected females compared to control mosquitoes, whereas non‐infected exposed females had an R₀ approximately 15% lower than that of controls. Reproduction caused a significant decrease of about 13 days in mosquito longevity compared to females that did not reproduce (P < 0.001). Infected females lived 5–8 days less than non‐infected exposed females or controls, respectively (P = 0.028). Infected females and non‐infected exposed females both had significantly shorter wings than control insects (P < 0.001). There was a significant positive correlation between wing length and longevity in covertly infected female mosquitoes but not in control or non‐infected exposed mosquitoes. Longer lived females produced more eggs in all treatments. There were no significant correlations between body size and fecundity or the production of offspring. There was also no correlation between fecundity and fertility, suggesting that sperm inactivation was a more likely cause of decreased fertility in older mosquitoes than sperm depletion. We conclude that covert infection by iridescent virus is likely to reduce the vectorial capacity of this mosquito.     

Effects of an optical brightener and an abrasive on iridescent virus infection and development of Aedes aegypti

The insecticidal properties of certain entomopathogenic viruses can be greatly improved in mixtures with substances that affect the integrity of the insect peritrophic membrane, particularly optical brighteners. We aimed to determine the effect of an optical brightener, Blankophor BBH, and an abrasive compound, silicon carbide, alone and in mixtures, on the prevalence of patent and covert infection of Aedes aegypti (L.) (Diptera: Culicidae) by Invertebrate iridescent virus 6 (IIV‐6) (Iridoviridae). The prevalence of patent infection by IIV‐6 was < 1.5% in all treatments involving virus. Contrary to predictions, there were significantly fewer patent infections in virus treatments involving Blankophor with or without silicon carbide compared with controls. Covert infection of adults detected by insect bioassay was between 6.7 and 12.2%, although no significant differences were observed between treatments. Exposure to IIV‐6 alone or silicon carbide alone did not significantly increase larval mortality compared to the controls, whereas exposure to Blankophor alone, or in any combination with IIV‐6 or silicon carbide, clearly increased larval mortality. These effects did not carry‐over to the pupal stage. Adult females emerged ～1.5 days later than males. Compared to control insects, female development rate was extended by 11.4 and 12.6% in the treatments involving IIV‐6 alone and silicon carbide alone, respectively. The sex ratio at adult emergence did not differ significantly between control insects and those of other treatments. These results support the hypothesis that the gut is unlikely to represent the principal point of infection of mosquito larvae by iridescent viruses.     

Sublethal iridovirus disease of the mosquito Aedes aegypti is due to viral replication not cytotoxicity

Invertebrate iridescent viruses (Iridoviridae) possess a highly cytotoxic protein. In mosquitoes (Diptera: Culicidae), invertebrate iridescent virus 6 (IIV-6) usually causes covert (inapparent) infection that reduces fitness. To determine whether sublethal effects of IIV-6 are principally due to cytotoxicity of the viral inoculum (which inhibits macromolecular synthesis in the host), or caused by replication of the virus larvae of the mosquito Aedes aegypti (L) were exposed to untreated IIV-6 virus that had previously been deactivated by heat or ultraviolet light. Control larvae were not exposed to virus. Larval development time was shortest in control larvae and extended in larvae exposed to untreated virus. Covertly infected mosquitoes laid significantly fewer eggs, produced between 20 and 35% fewer progeny and had reduced longevity compared to other treatments. Wing length was shortest in mosquitoes exposed to heat-deactivated virus. Multivariate analysis of the same data identified fecundity and progeny production as the most influential variables in defining differences among treatments. Overall, viral infection resulted in a 34% decrease in the net reproductive rate (R0) of covertly infected mosquitoes, vs. only 5-17% decrease of R0 following treatments with deactivated virus, compared to controls. Sublethal effects of IIV-6 in Ae. aegypti appear to be mainly due to virus replication, rather than cytotoxic effects of the viral inoculum.     

Chemical and biological stress factors on the activation of nucleopolyhedrovirus infections in covertly infected Spodoptera exigua

Following the consumption of baculovirus occlusion bodies (OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (SeMNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA‐GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus (Chrysodeixis chalcites SNPV) did not result in the activation of SeMNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus‐free controls. In contrast, 0.1% copper sulphate, 1% iron (II) sulphate and 1 mg/l sodium selenite treatments resulted in 12–41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of SeMNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus‐induced disease. However, these substances seem unlikely to prove useful for the activation of covert SeMNPV infections in S. exigua larvae under greenhouse conditions.     

Costs and benefits of sublethal Drosophila C virus infection

Viruses are major evolutionary drivers of insect immune systems. Much of our knowledge of insect immune responses derives from experimental infections using the fruit fly Drosophila melanogaster. Most experiments, however, employ lethal pathogen doses through septic injury, frequently overwhelming host physiology. While this approach has revealed several immune mechanisms, it is less informative about the fitness costs hosts may experience during infection in the wild. Using both systemic and oral infection routes, we find that even apparently benign, sublethal infections with the horizontally transmitted Drosophila C virus (DCV) can cause significant physiological and behavioural morbidity that is relevant for host fitness. We describe DCV‐induced effects on fly reproductive output, digestive health and locomotor activity, and we find that viral morbidity varies according to the concentration of pathogen inoculum, host genetic background and sex. Notably, sublethal DCV infection resulted in a significant increase in fly reproduction, but this effect depended on host genotype. We discuss the relevance of sublethal morbidity for Drosophila ecology and evolution, and more broadly, we remark on the implications of deleterious and beneficial infections for the evolution of insect immunity.     

Effects of triflumuron on the two-spotted spider mite,Tetranychus urticae (Acari: Tetranichydae)

The effects of triflumuron on the mortality, fecundity, and fertility of the two-spotted spider mite, Tetranychus urticae, were evaluated in the laboratory. No differences in toxicity for larvae, protonymphs and deutonymphs were observed, but immature stages were 3.8-times more susceptible than adults at the LC₅₀. The compound exhibited a direct contact ovicidal activity influenced by eggs age. 48–72 hold eggs were significantly more sensitive than eggs of the other age classes. No hatch inhibition was observed in eggs laid by treated adult females using a sublethal dose at two physiological times (<12 and 48–72h old). However, fecundity decreased in younger treated females, but it increased in the older-ones. The toxicity for immatures and eggs, and the sublethal effects described suggest that triflumuron could be an interesting incorporation in integrated pest programs of T. urticae. This revised version was published online in July 2006 with corrections to the Cover Date.     

An iridescent virus and a rickettsia from the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae)

Natural iridescent virus and rickettsia infections of Costelytra zealandica (White) and Odontria sp. indet. larvae were studied at a site in the upper Pareora Gorge scenic reserve, S. Canterbury. By sequentially sampling the site, it was found that neither the iridescent virus nor the rickettsiae appear to give rise to host mortalities that significantly alter the population density. Many larvae were found that appeared healthy, but carried inapparent iridescent virus infections. These diseases are not suitable for biological control of the grass grub.     

Resource limitation and the lethal and sublethal effects of a viral pathogen in the Indian meal moth, Plodia interpunctella

Abstract.

• 1 The effects of resource limitation and the lethal and sublethal effects of a granulosis virus on a lepidopteran host, the Indian meal moth, Plodia interpunctella, were examined.
• 2 The food quality was manipulated by the addition of an inert bulking agent (methyl cellulose) which caused the size, development rate and fecundity of the moths to be reduced.
• 3 The resource quality had no effect on the mortality due to the virus. In contrast, sublethal effects of the virus on pupal weight were more apparent under conditions of resource limitation.
• 4 Considerable variation between the sublethal effects after challenge with different doses of the virus was found. The balance between deleterious sublethal effects of the virus and the selection of more robust individuals by the bioassays is proposed as a mechanism to explain this variation.
• 5 Implications for the dynamics of insect hosts and their pathogens are discussed.

     

Transmission dynamics of an iridescent virus in an experimental mosquito population: the role of host density

Abstract.  1. The transmission of insect pathogens cannot be adequately described by direct linear functions of host and pathogen density due to heterogeneity generated from behavioural or physiological traits, or from the spatial distribution of pathogen particles. Invertebrate iridescent viruses (IIVs) can cause patent and lethal infection or a covert sub-lethal infection in insects. Aedes aegypti larvae were exposed to suspensions of IIV type 6 at two densities. High larval density increased the prevalence of aggression resulting in potentially fatal wounding.
2. The overall prevalence of infection (patent + covert) was positively influenced by host density and increased with exposure time in both densities. The survival time of patently infected insects was extended by ≈ 5 days compared with non-infected insects.
3. Maximum likelihood models based on the binomial distribution were fitted to empirical results. A model incorporating heterogeneity in host susceptibility by inclusion of a pathogen-free refuge was a significantly better fit to data than an all-susceptible model, indicating that transmission is non-linear. The transmission coefficient ( υ ) did not differ with host density whereas the faction of the population that occupied the pathogen-free refuge (Π_R) was significantly reduced at high host density compared with the low density treatment.
4. The transmission of free-living infective stages of an IIV in Ae. aegypti larvae is non-linear, probably because of density-related changes in the frequency of aggressive encounters between hosts. This alters host susceptibility to infection and effectively reduces the proportion of hosts that occupy the pathogen-free refuge.     

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 × 10³ DJ per milliliter for S. carpocapsae and 1 to 8 × 10³ DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 × 10³ DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3–6 × 10³ DJs per milliliter in order to obtain >10³ parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 × 10³ DJs per milliliter to reach >2 × 10³ parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 × 10³ and 105 × 10³ per mililiter for S. feltiae.     

Sublethal effects of fenpyroximate and pyridaben on two predatory mite species, <Emphasis Type="Italic">Neoseiulus womersleyi</Emphasis> and <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> (Acari,Phytoseiidae)

Laboratory bioassays were conducted to evaluate the sublethal effects of fenpyroximate and pyridaben on life-table parameters of two predatory mites species, Neoseiulus (= Amblyseius) womersleyi and Phytoseiulus persimilis. In these assays, young adult females were treated with three sublethal concentrations of each acaricide. The life-table parameters were calculated at each acaricide concentration, and compared using bootstrap procedures. For each acaricide, the LC₅₀ estimates for both species were similar, yet the two species exhibited completely different susceptibility when the population growth rate was used as the endpoint. Exposure to both acaricides reduced the net reproduction rate (R _o ) in a concentration-dependent manner and their EC₅₀s were equivalent to less than LC₇. Two different scales of population-level endpoints were estimated to compare the total effect between the species and treatments: the first endpoint values were based on the net reproductive rate (fecundity λ) and the second endpoint values incorporated the mean egg hatchability into the net reproductive rate (vitality λ). The fecundity λ decreased in a concentration-dependent manner for both acaricide treatments, but the vitality λ decreased abruptly after treatment of N. womersleyi with pyridaben. The change in the patterns of λ revealed that the acaricide effects at the population level strongly depended on the life-history characteristics of the predatory mite species and the chemical mode of action. When the total effects of the two acaricides on N. womersleyi and P. persimilis were considered, fenpyroximate was found to be the most compatible acaricide for the augmentative release of N. womersleyi after treatment.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.     

Cross-infectivity and activation studies with four baculoviruses

The NPVs of 3Spodoptera species and 1Heliothis species were bioassay tested for cross-infectivity. The progeny virus from the test insects was purified and examined by specific identification criteria. This demonstrated that activation of virus was much more common than cross-infection. Stress experiments using chemicals and varying environmental conditions failed to activate any virus in the stock insect cultures. Experiments were designed to test for possible mixed inoculum virus as an explanation of the activation effect but this theory was disproved. The mechanism of the activation is unexplained but it seems clear that when independent identification techniques are used it can be demonstrated that infecting an insect larva with a NPV from another host can result in death due to infection with the NPV normally associated with that host rather than that used as inoculum. This can occur even though no latent virus can be detected in the insect population by conventional methods.     

Physicochemical properties of tipula iridescent virus

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10⁸, with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm²/mg of virus and a light-scattering corrected absorbance of 9.8 cm²/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.     

Expression of Poplar Chitinase in Tomato Leads to Inhibition of Development in Colorado Potato Beetle

The previously described poplar chitinase, WIN6, is induced during infestation by gypsy moth (Lymantria dispar L.) larvae, thus suggesting a role in defense against insect pests. To test this hypothesis, we produced tomato seedlings infected with a recombinant potato virus X (PVX), which produces WIN6, and tested its insecticidal properties on Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)], which is a serious pest of tomatoes and other crops. The advantage of PVX is that plant material is ready for insect bioassay within 3–4 weeks of constructing the recombinant virus. Considering that production of transgenic tomato seedlings using Agrobacterium takes at least 6 months, this hastens the rate at which genes can be examined. Upon insect bioassay, only 47% CPB neonates feeding on leaves containing >0.3% w/w WIN6 developed to 2nd instar while 93% of controls reached 2nd instar. To our knowledge this is the first plant chitinase that retards development of an insect pest. Revisions requested 12 December 2005; Revisions received 18 January 2006     

Influence of honey and maternal age on egg load of lab-cultured <Emphasis Type="Italic">Cotesia marginiventris</Emphasis>

Laboratory experiments were conducted to determine the impact of feeding status and maternal age on egg load of Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), a solitary, koinobiont endoparasitoid of noctuid pests. Egg load was defined as the number of mature (i.e., fully-chorionated) eggs found in the ovaries and oviducts. Significantly more mature eggs were stored in honey-fed than starved females. For honey-fed females, egg load increased within several days of isolation from hosts. This study suggests that C. marginiventris is weakly synovigenic because females emerge with a considerable number of mature eggs and are capable of maturing many more eggs. Feeding on a suitable source of carbohydrate should increase the egg load (i.e., potential fecundity) of this insect within 3–4 days in an in vivo rearing system.     

Virulence is context-dependent in a vertically transmitted aquatic host–microparasite system

Recent work has suggested that the outcomes of host–symbiont interactions can shift between positive, neutral and negative depending on both biotic and abiotic conditions. Even organisms traditionally defined as parasites can have positive effects on hosts under some conditions. For a given host–parasite system, the effects of infection on host fitness can depend on host vigour, route of transmission and environmental conditions. We monitored sublethal microsporidian infections in populations of Gammarus pseudolimnaeus (Amphipoda: Gammaridae) from four cool water streams in southwestern Michigan, USA. Our objectives were to: (i) infer the mechanism of transmission (horizontal, vertical or mixed) from observed effects of infection on host fitness, (ii) determine if the magnitude of the effects on host fitness is a function of parasite load (infection intensity) compared with simple presence or absence of infection, and (iii) determine if there is variation in parasite effects on host fitness in isolated populations. PCR and DNA sequence analyses revealed that there were two microsporidia present among the four host populations: Dictyocoela sp. and Microsporidium sp. PCR screening of a subset of infected hosts showed that Dictyocoela sp. accounted for 90% of infections and was present in all four G. pseudolimnaeus populations, while Microsporidium sp. was found in two populations but was only relatively common in one. We found very low prevalence in males (∼5%), but high prevalence in females (range: 37–85%). Female fitness was positively associated with infection in two streams, resulting from either higher fecundity or more reproductive bouts. Infection had a negative effect on the number of reproductive bouts in a third population, and no effect on fecundity in a fourth population. Infection intensity explained additional variation in fecundity in one population; females with intermediate infection intensity had higher fecundity than females with either light or heavy infection intensity. Given the high prevalence of infection in females compared with males and the generally weak negative fitness effects coupled with some positive fitness effects, it is likely that both Dictyocoela sp. and Microsporidium sp. are primarily vertically transmitted, feminizing microsporidia. Our results suggest that microsporidian effects on G. pseudolimnaeus fitness were context-dependent and varied with host sex and local environment.     

Virus infections reduce in vitro multiplication of ‘Malling Landmark’ raspberry

Summary Virus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Anderson's medium with 8.9 μM N⁶-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants. Part of a thesis submitted by C.-W.V.T. in partial fulfilment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.