William S. Romoser

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

Abstract 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.

Evidence for Arbovirus Dissemination Conduits from the Mosquito (Diptera: Culicidae) Midgut

Abstract The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.

Rift Valley fever virus-infected mosquito ova and associated pathology: possible implications for endemic maintenance

Background Endemic/Enzootic maintenance mechanisms like vertical transmission (pathogen passage from infected adults to their offspring) are central in the epidemiology of zoonotic pathogens. In Kenya, Rift Valley fever virus (RVFV) may be maintained by vertical transmission in ground-pool mosquitoes such as Aedes mcintoshi. RVFV can cause serious morbidity and mortality in humans and livestock. Past epidemics/epizootics have occurred in sub-Saharan Africa but, since the late 1970s, RVFV has also appeared in North Africa and the Middle East. Preliminary results revealed RVFV-infected eggs in Ae. mcintoshi after virus injection into the hemocoel after the first of two blood meals, justifying further study. Methods Mosquitoes were collected from an artificially flooded water-catching depression along a stream in Kenya, shipped live to the USA, and studied using an immunocytochemical method for RVFV-antigen localization in mosquito sections. Results and conclusion After virus injection into the hemocoel, RVFV-infected reproductive tissues were found, particularly follicular epithelia and oocyte/nurse cells. Ovarian infection from the hemocoel is a crucial step in establishing a vertically transmitting mosquito line. Ovarian follicles originate from germarial cells, primordia located distally in each ovariole, and infection of these cells is expected to be requisite for long-term vertical transmission. However, no germarial cell infection was found, so establishing a new line of vertically transmitting mosquitoes may require two generations. The findings support the hypothesis that Ae. mcintoshi is involved in the endemic maintenance of RVFV by vertical transmission. Detection of distinct pathology in infected eggs raises the possibility of virus-laden eggs being deposited among healthy eggs, thereby providing an exogenous source of infection via ingestion by mosquito larvae and other organisms. This has potentially significant epidemiological implications. Possible modes of entry of virus from the hemocoel into the ovaries and routes by which larvae might become infected by ingesting virus are discussed.

The Occurrence and Fate of the Meconium and Meconial Peritrophic Membranes in Pupal and Adult Mosquitoes (Diptera: Culicidae)

Abstract 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 matrix structure, formation, and meconial degeneration in mosquito pupae/pharate adults : Histological and ultrastructural aspects

Abstract The noncellular peritrophic matrix (PM) that forms around the food bolus in the midgut of many arthropod species may influence the fate of ingested microbes. In mosquitoes, PMs have been identified in the pupal as well as larval and adult stages. In pupae, the PMs surround the meconium, the sloughed larval midgut epithelium. Meconial PM1 (MPM1) forms early in the pupal stadium, and a second meconial PM (MPM2) sometimes forms around the time of adult emergence. A recent study suggests that MPMs contribute to the sterilization of the adult midgut by sequestering microorganisms ingested during the larval stage, which, along with remaining meconial material, are egested after adult emergence. We have compared MPM1 formation and patterns of meconial degeneration in representative species in five mosquito genera and identified a temporal association between MPM1 formation, meconial degeneration, and apolysis. Ultrastructural study of MPM1 and MPM2 in Aedes aegypti (L.) revealed that MPM1 seems to be structurally different from either the larval or adult PMs, whereas MPM2 more closely resembles PM formed around a bloodmeal in adult females. Our results are consistent with the microbial sequestration role.

Introduction to Arthropods: Structure, Function and Development

The phylum Arthropoda is the largest assemblage in the Animal Kingdom, the number of arthropod species outstripping all others many times over. Of particular interest in medical entomology are the more than 17 thousand species of bloodsucking (hematophagous) insects and the 25 thousand or so species of ticks (Beaty and Marquardt 1996). The enormous success of this group is reflected in the seemingly endless variety of niches occupied, their 600 million-year evolutionary time span, and their high biomass in various ecosystems.

Meconial peritrophic matrix and meconial degradation in the biting midge, Culicoides variipennis (coquillett) (Diptera: ceratopogonidae)

mBackground: The peritrophic matrix (PM), a noncellular layer surrounding the food bolus in the gut, has been described in several invertebrate phyla, including Arthropoda. In arthropod vectors, the PM may be a barrier to pathogens in a meal. In mosquito pupae, a PM forms around the sloughed larval midgut epithelial cells (meconium), and evidence suggests this meconial PM (MPM) protects the developing adult midgut epithelium from microbes ingested during the larval stage. Given the probable protective function of MPM and the possibility that it exists in taxa beyond the Culicidae, we looked for MPM in a representative of a related dipteran family, Certatopogonidae (biting midges). Methods: One hundred thirteen mature Culicoides variipennis (Diptera: Ceratopogonidae) larvae, pupae over the entire pupal stadium, and several adults were paraffin-sectioned, stained, and examined using light microscopy. Results: Near the end of the larval stage, the midgut epithelium sloughs into the lumen forming the meconium and a new epithelium forms from regenerative cell proliferation. The meconium gradually histolyses as indicated by shrinkage, a staining reaction change from red to blue, and loss of cellular structure. A distinct MPM often forms and persists into the adult stage. Shortly after sloughing, blue staining material accumulates around the meconium and then disappears in correlation with MPM formation. Conclusion: Our study supports the hypothesis that MPM occurs in taxa beyond the Culicidae, in this study the Ceratopogonidae, specifically in C. variipennis. As in mosquitoes, MPM occurrence is variable, and its formation may be induced in response to a chemotactic stimulus, possibly of microbial origin. As in mosquitoes, MPM in C. variipennis appears to protect the developing adult midgut by sequestering microorganisms remaining from the larval stage. Consistent with MPM induction and microbial sequestration, we saw in a few older pupae and new adults, brownish material, possibly microbial, within the confines of a distinct MPM.

Comparative Study of the Pathological Effects of Western Equine Encephalomyelitis Virus in Four Strains of Culex tarsalis Coquillett (Diptera: Culicidae).

Early reports suggested that mosquito cells infected with arboviruses remain viable and undamaged. However, more recent experimental evidence suggests that arboviral infection of mosquito tissues might indeed result in pathological changes, with potential implications for vector survival and virus transmission. Here, we compare the pathological effects of western equine encephalomyelitis virus (WEEV) infection in four strains of Culex tarsalis previously reported to differ in their competence as WEEV vectors. Pathological effects were observed in cells of the midgut epithelium, salivary glands, and eggs. Cell rounding and sloughing of midgut epithelial cells was associated with those strains reported to be the least susceptible to WEEV infection, whereas midgut necrosis and vacuolation upon infection were associated with strains showing higher susceptibility. Although pathological effects were sporadically observed in infected salivary glands, further studies are required to evaluate their impact on vector competence. Additionally, the potential implications of observed C. tarsalis egg infection with WEEV are discussed.

Function of the dendritic setae in Aedes aegypti mosquito pupae: float hairs don't float

Correspondence: William S Romoser Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Oh 45701, USA Tel +1 740 592 3417 Fax +1 740 597 2778 email wromoser@gmail.com Purpose: Pupal behavior varies with buoyancy, which changes spontaneously at the water’s surface and during diving. This behavioral regulation is energy conserving, which is a critical need in the nonfeeding but highly motile pupa. Although adult structures are apparent, there are uniquely pupal structures, including setae (most or all mechanoreceptors), ranging from hair-like to the complex, moveable, bilateral “dendritic setae” on the first abdominal tergum. Our aim has been to elucidate the function of the dendritic setae (“float hairs” in earlier studies). Based on the position and shape of dendritic setae plus ultrastructural evidence of mechanoreception, we hypothesized a buoyancy-sensing function related to bending of the setae as water currents flow over them during descent and ascent. Methods: Using Aedes aegypti, we checked to see whether the dendritic setae are hydrophilic and tested the behavioral effects of their removal. In a preliminary closed-system experiment, a pupa was placed in water in a test tube and a syringe attached in continuity with the air space above the water. When pressure was increased by depressing the plunger, a pupa responded by swimming toward the surface. When negative pressure was applied by lifting the plunger, a pupa actively dove. Using a more sophisticated apparatus with a pressure transducer, we tested the effects of dendritic setae removal on behavior in response to pressure changes. Results: The cuticular surface of the dendritic setae is hydrophilic. No significant differences were found in pupae with or without the dendritic setae relative to dive duration, applied pressure duration, or maximum pressure applied, but response time to pressure change in pupae (males and females) without the setae was significantly increased. Conclusion: Hydrophilic dendritic seta cuticle is consistent with our hypothesis but not with a floatation function. Ablation experiments supported our hypothesis that the dendritic setae are involved with buoyancy sensing by bending in response to directional water currents.