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.     

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 effects of iridovirus disease in a mosquito

Recognition of the importance of debilitating effects of insect virus diseases is currently growing. Commonly observed effects of sublethal infection at the individual level include extended development times, reduced pupal and adult weights, and lowered fecundity. However, for the most part, sublethal infections are assumed to be present in survivors of an inoculum challenge, rather than demonstrated to be present by microscopy or molecular techniques. Invertebrate iridescent viruses are dsDNA viruses capable of causing disease with symptoms obvious to the naked eye, a “patent” infection, that is lethal. Furthermore, inapparent “covert” infections may occur that are non-lethal and which can only be detected using bioassay or molecular techniques. In this study, replication of Invertebrate iridescent virus 6 in Aedes aegypti larvae was demonstrated in the absence of patent disease. A sensitive insect bioassay (using Galleria mellonella) allowed the detection of covert infections, which were more common than patent infections. A concentration-response relationship was detected for the incidence of patent infections. Covert infections were up to 2 orders of magnitude commoner than patent infections, but the prevalence of covert infections did not appear to be related to virus inoculum concentration. Exposure of larvae to virus inoculum resulted in extended juvenile development times. A reduction in the mean and an increase in the variability of fecundity and adult progeny production was observed in females exposed to an inoculum challenge, although formal analysis was not possible. Males appeared capable of passing virus to uninfected females during the mating process. Covertly infected females were smaller and had shorter lifespans than control or virus-challenged females. A conservative estimate for the reduction in the net reproductive rate (R ₀) of such insects was calculated at slightly more than 20% relative to controls. Received: 5 October 1998 / Accepted: 13 February 1999     

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.     

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.     

Age-Dependent Effects of Oral Infection with Dengue Virus on Aedes aegypti (Diptera: Culicidae) Feeding Behavior,Survival, Oviposition Success and Fecundity

Background

Aedes aegypti is the main vector of dengue, a disease that is increasing its geographical range as well as incidence rates. Despite its public health importance, the effect of dengue virus (DENV) on some mosquito traits remains unknown. Here, we investigated the impact of DENV-2 infection on the feeding behavior, survival, oviposition success and fecundity of Ae. aegypti females.

Methods/Principal Findings

After orally-challenging Ae. aegypti females with a DENV-2 strain using a membrane feeder, we monitored the feeding behavior, survival, oviposition success and fecundity throughout the mosquito lifespan. We observed an age-dependent cost of DENV infection on mosquito feeding behavior and fecundity. Infected individuals took more time to ingest blood from anesthetized mice in the 2^nd and 3^rd weeks post-infection, and also longer overall blood-feeding times in the 3^rd week post-infection, when females were around 20 days old. Often, infected Ae. aegypti females did not lay eggs and when they were laid, smaller number of eggs were laid compared to uninfected controls. A reduction in the number of eggs laid per female was evident starting on the 3^rd week post-infection. DENV-2 negatively affected mosquito lifespan, since overall the longevity of infected females was halved compared to that of the uninfected control group.

Conclusions

The DENV-2 strain tested significantly affected Ae. aegypti traits directly correlated with vectorial capacity or mosquito population density, such as feeding behavior, survival, fecundity and oviposition success. Infected mosquitoes spent more time ingesting blood, had reduced lifespan, laid eggs less frequently, and when they did lay eggs, the clutches were smaller than uninfected mosquitoes.     

Fecundity compensation in the three‐spined stickleback Gasterosteus aculeatus infected by the diphyllobothriidean cestode Schistocephalus solidus

Causal explanations for host reproductive phenotypes influenced by parasitism fit into three broad evolutionary models: (1) non‐adaptive side effect; (2) adaptive parasitic manipulation; and (3) adaptive host defence. This study demonstrates fecundity compensation, an adaptive non‐immunological host defence, in the three‐spined stickleback fish (Gasterosteus aculeatus) infected by the diphyllobothriidean cestode Schistocephalus solidus. Both infected and uninfected female sticklebacks produced egg clutches at the same age and size. The reproductive capacity of infected females decreased rapidly with increased parasite : host body mass ratio. Body condition was lower in infected females than uninfected females and decreased with increasing parasite : host mass ratio. Females with clutches had greater body condition than those without clutches. A point biserial correlation showed that there was a body condition threshold necessary for clutch production to occur. Host females apparently had the capacity to produce egg clutches until the prolonged effects of nutrient theft by the parasite and the drain on resources from reproduction precluded clutch formation. Clutch mass, adjusted for female body mass, did not differ significantly between infected and uninfected females. Infected females apparently maintained the same level of reproductive allotment (egg mass as proportion of body mass) as uninfected females. Infected females produced larger clutches of smaller eggs than uninfected females, revealing a trade‐off between egg mass and egg number, consistent with the fecundity compensation hypothesis. The rapid loss of reproductive capacity with severity of infection probably reflects the influence of the parasite combined with a trade‐off between current and future reproduction in the host. Inter‐annual differences in reproductive performance may have reflected ecological influences on host pathology and/or intra‐annual seasonal changes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

West Nile virus cluster analysis and vertical transmission in Culex pipiens complex mosquitoes in Sacramento and Yolo Counties,California, 2011

West Nile virus (WNV) is now endemic in California, with annual transmission documented by the statewide surveillance system. Although much is known about the horizontal avian‐mosquito transmission cycle, less is known about vertical transmission under field conditions, which may supplement virus amplification during summer and provide a mechanism to infect overwintering female mosquitoes during fall. The current study identified clusters of WNV‐infected mosquitoes in Sacramento and Yolo Counties, CA, during late summer 2011 and tested field‐captured ovipositing female mosquitoes and their progeny for WNV RNA to estimate the frequency of vertical transmission. Space‐time clustering of WNV‐positive Culex pipiens complex pools was detected in the northern Elk Grove area of Sacramento County between July 18 and September 18, 2011 (5.22 km radius; p<0.001 and RR=7.80). Vertical transmission by WNV‐infected females to egg rafts was 50% and to larvae was 40%. The estimated minimal filial infection rate from WNV‐positive, ovipositing females was 2.0 infected females/1,000. The potential contribution of vertical transmission to WNV maintenance and amplification are discussed.     

Reduced fitness of the leafhopper vector Scaphoideus titanus exposed to Flavescence dorée phytoplasma

Scaphoideus titanus Ball (Homoptera: Cicadellidae), a specialist and univoltine leafhopper on grapevine (Vitis vinifera L.) (Vitaceae), is a vector of Flavescence dorée phytoplasma (FDP) in vineyards of European temperate areas. Males and females of the leafhopper were exposed to FDP by feeding on infected broad bean (Vicia faba L.) (Fabaceae). Detection of FDP by the amplification of phytoplasma DNA with polymerase chain reaction assays of individual insects revealed an acquisition rate of 91.4% (96/105) after an acquisition access period of 13 days. The adult life span of FD‐exposed males and females was much less than that of leafhoppers fed on healthy broad bean, as revealed by ANOVA on the quartiles of survival distribution and Weibull scale parameter. The progeny of exposed females (number of nymphs emerging from eggs deposited on woody cane segments) was significantly less than the progeny of unexposed females. Eggs produced by FD‐exposed females were slightly but significantly delayed in hatching. Reduced fecundity was confirmed by dissecting FD‐exposed and non‐exposed 42‐day‐old females and counting the number of fully sized eggs in each leafhopper. There was no evidence of transovarial passage of FDP in the offspring of infected females after 72 nymphs were reared on a healthy grapevine until the fifth instar or adult appearance and then confined on broad bean seedlings.