Pathogenesis of Rift Valley fever virus in mosquitoes--tracheal conduits & the basal lamina as an extra-cellular barrier.

Knowledge of the fate of an arbovirus in a mosquito is fundamental to understanding the mosquito's competence to transmit the virus. When a competent mosquito ingests viremic vertebrate blood, virus infects midgut epithelial cells and replicates, then disseminates to other tissues, including salivary glands and/or ovaries. The virus is then transmitted to the next vertebrate host horizontally via bite and/or vertically to the mosquito's offspring. Not all mosquitoes that ingest virus become infected or, if infected, transmit virus. Several "barriers" to arbovirus passage, and ultimately transmission, have been identified in incompetent or partially competent mosquitoes, including, among others, gut escape barriers and salivary gland infection barriers. The extra-cellular basal lamina around the midgut epithelium and the basal lamina that surrounds the salivary glands may act as such barriers. Midgut basal lamina pore sizes are significantly smaller than arboviruses and ultrastructural evidence suggests that midgut tracheae and tracheoles may provide a means for viruses to circumvent this barrier. Further, immunocytochemical evidence indicates the existence of a salivary gland infection barrier in Anopheles stephensi. The basal lamina may prevent access to mosquito cell surface virus receptors and help explain why anopheline mosquitoes are relatively incompetent arbovirus transmitters when compared to culicines.

Effect of the developmental stage at infection on the ability of adult Anopheles stephensi to transmit Rift Valley fever virus.

The ability of adult Anopheles stephensi to transmit Rift Valley fever virus was determined for mosquitoes inoculated at selected times during development. None of 109 female An. stephensi inoculated as adults transmitted virus to hamsters. In contrast, 83% (50 of 60) of those inoculated as larvae transmitted virus by bite to hamsters. Transmission rates decreased as the stage of the mosquito at the time of inoculation changed from larva to pupa to adult. Transmission rates for adult mosquitoes inoculated as larvae, as pupae < 4 hr after pupation, as pupae > 24 hr after pupation, or as adults were 83%, 25%, 11%, and 0%, respectively. Viral titers recovered from mosquitoes were similar for all groups tested, regardless of stage at infection (larva, pupa, or adult) or of transmission status (transmitter or nontransmitter). Thus, differences in transmission rates may have been due to site-specific (i.e., salivary gland) replication, rather than a generalized increase in viral replication in mosquitoes inoculated at an earlier age.

An immunocytochemical study of the distribution of Rift Valley fever virus in the mosquito Culex pipiens.

The dissemination of Rift Valley fever (RVF) virus in females of the epidemic vector Culex pipiens was studied immunocytochemically. Among orally infected specimens, viral antigen was detected in all major midgut regions, although individuals varied with respect to which regions were infected. Among specimens with disseminated infections (infections beyond the midgut epithelium), antigen was detected in most tissues, including those of the nervous and endocrine systems. However, no antigen was found in the ovarian follicular epithelia, oocytes/nurse cells, or female accessory gland. A viral dissemination index (DI) based on several tissues was determined for each orally infected specimen and used to estimate the extent of viral dissemination in the hemocoel. Plots of DI values as a function of time after ingestion of an infectious blood meal indicated that dissemination was sporadic, and that once virus escapes from the midgut, its spread to other tissues is rapid. The most common sequence of tissue/organ infection following escape of virus from the midgut epithelium appears to be as follows: intussuscepted foregut, fat body, salivary glands and thoracic ganglia, epidermis, and ommatidia of the compound eyes. Reduced fecundity and survival identified in other studies of RVF virus-infected Cx. pipiens are probably explainable on the basis of extensive tissue and organ infection resulting in an overall energy drain. Infection of regulatory tissues and organs such as the ganglia of the central nervous system, neurosecretory cells, and the corpora allata may also have detrimental effects on the overall functioning of a mosquito, even in the absence of widespread infection.

Rift Valley fever virus in the cardia of Culex pipiens: an immunocytochemical and ultrastructural study.

In this paper, we consider the movement of Rift Valley fever (RVF) virus from infected mosquito midgut epithelial cells into the hemocoel as an important factor in the ultimate ability of the insect to transmit the virus. Our results are therefore significant in the context of vector competence. The mosquito Culex pipiens was identified as the primary vector of RVF in an epidemic that occurred in Egypt in the 1970s. On this basis, we have carried out several studies of RVF virus in this mosquito species. In the research reported here, we used immunocytochemical and transmission electron microscopic techniques to study the occurrence of RVF virus in the mosquito cardia and aspects of the histology and ultrastructure of this organ. The cardia is a complex organ consisting of both foregut and midgut tissue and is the location of the foregut-midgut junction. The cardia is of interest because it appears to provide routes of RVF virus egress from the midgut lumen and it is consistently infected in mosquitoes with disseminated infections, making it a potentially important site of viral amplification and an ideal site for studying RVF viral morphogenesis. In orally infected mosquitoes, large numbers of RVF virions were observed budding into the basal labyrinth associated with the outer cardial epithelial cells and into the noncellular matrix associated with the inner cardial epithelial cells and the cells of the intussuscepted foregut. In mosquitoes infected by injection of virus into the hemocoel and then held for different incubation periods, viral antigen was first detected in the cells of the intussuscepted foregut in the cardia and later in the cardial epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of Rift Valley fever virus in the mosquito Culex pipiens as revealed by viral titration of dissected organs and tissues.

Distribution of Rift Valley fever virus (RVFV) was studied in the mosquito Culex pipiens. Mosquitoes were dissected on days 1-7 after an infectious bloodmeal, and RVFV plaque assays were performed on the legs, posterior midgut, ovaries, salivary glands, thoracic ganglia, and remaining organs and tissues (remnants). On days 7-12 and 14 following an infectious bloodmeal, mosquitoes were tested for their ability to transmit virus and then dissected. Dissemination (systemic infection) rates averaged 22% on days 1-14 and transmission rates 33% on days 7-14. There were no significant differences in the viral titers of midgut samples among the nondisseminated infected (virus limited to alimentary canal), disseminated infected nontransmitting, and transmitting groups of mosquitoes. The sequence of infection of the organs and tissues studied appeared to be as follows: midgut, hemolymph, remnants: salivary glands, ovaries, and thoracic ganglia. Some individuals were found to have disseminated infections as early as 12 hr following an infectious bloodmeal. Trauma, simulated by vigorous shaking immediately following the viremic bloodmeal, did not affect either infection or dissemination rates.

Histological parameters useful in the identification of multiple bloodmeals in mosquitoes.

Culex nigripalpus females were given double and, in 2 cases, triple (interrupted) bloodmeals separated by various intervals. Mosquitoes given single meals served as controls. Using Azan-stained serial paraffin sections, we could identify multiple meals separated by 1-72 hr in 44 of 53 cases (84.6%). Among the histological parameters of importance in the identification of double and triple bloodmeals are the peritrophic membrane secreted around each bloodmeal, the plug which forms between the anterior and posterior midgut, the layer of heme which forms as a bloodmeal is digested, and remnants of the pupal meconium and/or pupal-pharate adult peritrophic membranes. The parameters we have identified will help determine the incidence of double bloodfeeding in wild populations which, in turn, should enhance our understanding of the transmission of pathogens by mosquitoes.

Use of the avidin-biotin-peroxidase complex immunocytochemical procedure for detection of Rift Valley fever virus in paraffin sections of mosquitoes.

The avidin-biotin-peroxidase complex (ABC) immunocytochemical procedure was used to locate Rift Valley fever (RVF) virus antigen in infected Aedes albopictus C6/36 cultured cells and in serial paraffin sections of intrathoracically-injected Egyptian Culex pipiens. Fixation of the cultured cells in formaldehyde or periodate-lysine-paraformaldehyde, and fixation of whole mosquitoes with formaldehyde resulted in good preservation of morphology and excellent differential staining between uninfected and infected specimens. Two primary antibodies against RVF virus were tested on the cultured cells: a polyclonal rabbit antiserum and a mixture of mouse monoclonal antibodies. Only the mouse monoclonal antibody mixture was tested on the Cx. pipiens. Specific staining of tissues in paraffin sections occurred at dilutions as high as 1:5,000. In both cultured Ae. albopictus cells and sections of Cx. pipiens, only the cytoplasm was positive for antigen. A preliminary list of tissues in Cx. pipiens that exhibited specific or nonspecific staining is given.

Reduced survival of adult Culex pipiens infected with Rift Valley fever virus.

The effect of Rift Valley fever (RVF) viral infection on the survival of female Culex pipiens was examined. In 3 experiments in which mosquitoes ingested RVF virus, there was a 44% decrease in survival to days 14-16 for transmitting vs. nontransmitting mosquitoes, and a 48% decrease in survival for individuals with disseminated vs. nondisseminated infections. These results were corroborated by other experiments in which survival of mosquitoes intrathoracically inoculated with RVF virus was compared with that of those inoculated with diluent. In both the per os and inoculation tests, uninfected mosquitoes survived significantly longer than infected mosquitoes. Even though mosquitoes with disseminated infections had a lower survival rate than did uninfected mosquitoes, dissemination and transmission rates were similar at days 7 and 14-18 after the infectious bloodmeal. This suggests that nondisseminated individuals were developing disseminated infections and becoming capable of transmitting virus between days 7 and 14-18 at approximately the same rate older transmitters were dying. The decreased survival associated with RVF viral infection should be considered in predictive models of this disease.

The occurrence and fate of the meconium and meconial peritrophic membranes in pupal and adult mosquitoes (Diptera: Culicidae).

In mosquitoes, in addition to larval and adult peritrophic membranes (PMs), a PM (meconial peritrophic membrane or MPM1) forms in the pupa around the meconium, the sloughed, degenerating larval midgut epithelium. Often, a second membrane (MPM2) forms in temporal proximity to adult emergence. Differences in the occurrence, persistence, and timing of disappearance of the meconium/MPMs and gas were studied by dissecting the midgut contents from pupae of known ages postpupation and from adults of known ages postemergence. MPM1 was found in all Anopheles and Culex studied and nearly all Culiseta. The occurrence of MPM1 varied in the Aedes species. In one series of Aedes aegypti (L.) dissections, no fully formed MPM2 was found in any specimens. The occurrence of MPM2 appeared to be associated with adult emergence and varied among and within the seven species studied. It typically was seen in recently emerged adults but was observed occasionally in old pupae. Much of our data supports the idea that MPM2 formation is stimulated by midgut epithelium distention.

Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis.

The location of midgut bacteria relative to meconial peritrophic membranes (MPMs) and changes in bacterial numbers during midgut metamorphosis were studied in Anopheles punctipennis (Say), Culex pipiens (L.), and Aedes aegypti (L.) pupae and newly emerged adults. After adult emergence in Aedes, Anopheles, and most Culex, there were few to no bacteria in the midgut. In most newly emerged adult mosquitoes, few bacteria were found in either the lumen or within the MPMs/meconia. In a few Culex specimens, high numbers of bacteria were found in the MPMs/meconia and low numbers in the lumen. In all three species bacterial counts were high in fourth instars, decreased after final larval defecation, increased in young pupae, and increased further in old pupae. A very effective gut sterilization mechanism is operating during mosquito metamorphosis and adult emergence. This mechanism appears to involve the sequestration of remaining larval gut bacteria within the confines of the meconium and one or two MPMs and the possible bactericidal effect of the exuvial (molting) fluid, which is ingested during the process of adult emergence.

The origin and movement of gas during adult emergence in Aedes aegypti: an hypothesis.

Several events which precede adult emergence in Aedes aegypti were studied, including changes in buoyancy, changes visible through the pupal cuticle and changes at the foregut-midgut junction. Our data suggest tht the gas which is present in the posterior midgut at the time of emergence originates in the tracheal system. During the process of emergence this gas moves into the exuvial space through the adult spiracles and then follows the exuvial fluid into the alimentary canal.

The energetic costs of diving in Aedes aegypti and Aedes albopictus pupae.

Undisturbed mosquito pupae rest at the water surface and respond to passing shadows or vibrations by diving. Pupae do not feed and rely solely on energy stored from the larval stage. The ability of a newly emerged adult mosquito to survive, and therefore to transmit disease, depends on these energy reserves. Earlier studies of diving behavior in Aedes aegypti, Ae. albopictus, and Ae. triseriatus pupae provided evidence that pupae sense their state of buoyancy and modify their diving behavior accordingly. With strong stimulation pupae tend to dive to a depth where they become neutrally or negatively buoyant and commonly rest on the bottom. This behavior, as well as the tendency to rest when not disturbed, may logically be viewed as energy-conserving. The results of these studies also generated the hypothesis that the diving behavior displayed by these container-breeding mosquitoes helps them avoid being washed from their container by overflowing water during rainfall. Rainfall stimulates diving and logically, prolonged, heavy rainfall stimulates excessive diving, a likely drain on energy reserves. Our objectives were to determine, in Ae. aegypti and Ae. albopictus, the energetic costs associated with resting behavior, with frequent diving, and with buoyancy reduction. Using survival rate, mean survival after adult emergence, and measurement of total calories, we found a clear energetic cost associated with frequent diving. In contrast, relative to diving, essentially no energy cost was associated with buoyancy reduction, that is, pupae behave in response to variations in buoyancy in a way that does not impact significantly on energy reserves.

Buoyancy and diving behavior in mosquito pupae.

Mosquito pupal diving behavior has been studied mostly in Aedes aegypti and in this species pupal buoyancy varies relative to several factors. The research reported herein addresses the 2 following questions. Does diving behavior vary among different mosquito genera and species? How is diving behavior influenced by variation in buoyancy? Depth and duration of dive, and dive pattern, were compared among Ae. aegypti, Culex pipiens, Anopheles stephensi, Aedes albopictus, and Aedes triseriatus. In response to the stimulation associated with transferring pupae between containers, diving behavior varied dramatically among the different genera studied. Culex pipiens and An. stephensi make short-duration, shallow dives and remain positively buoyant. The 3 aedine species studied make longer-duration dives, typically to a depth at which they become neutrally or negatively buoyant. Buoyancy reduction effects were studied in the 3 aedine species. Normally buoyant pupae tend to dive to greater depths and for longer periods of time than reduced-buoyancy pupae. Aedine pupal diving behavior clearly is closely regulated relative to buoyancy variation. To the earlier hypotheses that pupal behavior may help avoid predation and be energy-conserving, we add the suggestion that the diving behavior displayed by the container-breeding aedine pupae we studied represents an adaptation that helps keep them from being washed from their container habitat by overflowing water during rainfall. We also suggest that the diving behavior of all the species studied may help pupae survive heavy, pelting rainfall by enabling them to avoid the mechanical shock of a direct hit by a raindrop, which could cause disruption of the gas in the ventral air space, thereby causing the loss of hydrostatic balance and drowning.

Effect of mechanical shock on hydrostatic balance and survival of mosquito pupae.

The presence and distribution of ventral air space gas assures that mosquito pupae are positively buoyant and that they float, dive, and ascend in an upright, balanced orientation. Our objective was to test the effects of mechanical shocks of varying magnitude on mosquito pupae representing 3 genera. Forces that disrupt the pupa's buoyancy and/or hydrostatic balance are of a much lower magnitude than those that would cause tissue damage. Once hydrostatic balance and/or buoyancy are compromised, pupae are unable to restore them and eventually drown. This could represent a weak link in the mosquito life cycle. It is possible that mechanical shock or sound-generating devices could be designed that could break this link.

A behavioral mechanism for resting site selection by pupae in three mosquito species.

Pupae of Aedes aegypti, Ae. triseriatus and Culex restuans dive less frequently when resting in a concave meniscus than when resting in open water. They also tend to terminate diving after contacting submerged vertical surfaces, increasing their chances of surfacing in a concave meniscus. As a result pupae tend to rest in concave menisci associated with emergent vertical surfaces, a behavioral adaptation by which they probably conserve energy and avoid predation.

Impact of stressful conditions on the survival of Culex pipiens exposed to Rift Valley fever virus.

Several groups of Rift Valley fever (RVF) virus-exposed and unexposed Culex pipiens were allowed differential access to a carbohydrate food source and their survival monitored. When stressed by deprivation of a carbohydrate source, mean survival times of RVF virus-exposed mosquitoes were consistently higher than those of unexposed mosquitoes in each of the carbohydrate-deficient experiments. These differences were statistically significant when mosquitoes were provided 5% sucrose for 24 hours. Mosquitoes that were provided access to a carbohydrate source for 24 h after a bloodmeal and then were denied access survived significantly longer than did those mosquitoes denied access, regardless of their exposure to RVF virus. When not stressed, RVF virus-exposed individuals had slightly higher daily survival rates than did unexposed individuals.