Confocal Analysis of the Distribution and Persistence of Sindbis Virus (TaV-GFP) Infection in Midguts of Aedes aegypti Mosquitoes.

Biological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

Apoptosis in mosquito salivary glands: Sindbis virus-associated and tissue homeostasis.

Apoptosis is observed during a spectrum of conditions including exogenous virus infection and endogenous cellular turnover. Adult female Aedes albopictus mosquitoes challenged with increasing titres of Sindbis virus (SINV) via intrathoracic inoculation demonstrated that the injection dosage did not result in significantly different levels of virus growth or mosquito survival at day 10 post-infection. Tissues probed for apoptosis using an in situ TUNEL assay revealed SINV-associated apoptotic cells scattered throughout the proximal and distal regions of the salivary gland (SG) lateral lobes but which were not detected in the median lobe or the midgut and hindgut. Apoptosis was also identified in SG duct cells in both infected and uninfected mosquitoes, suggesting routine tissue homeostasis. SINV-associated apoptosis sequestered to the SG lateral lobes indicates a differential epithelial cell response to an arbovirus and provides insight into mosquito defence mechanisms against pathogens and SG infection barriers, hurdles to transmission of arboviruses of public health concern.

Sindbis virus infection alters blood feeding responses and DEET repellency in Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) (Diptera: Culicidae) female mosquitoes infected systemically with Sindbis virus (SINV) took longer than uninfected mosquitoes to locate and fully engorge on blood. On days 7 and 14 postexposure, blood feeding took 1.3 and 1.5 times longer in mosquitoes with a disseminated SINV infection, respectively. SINV dissemination did not affect the average weight of unfed Ae. aegypti, but did result in a 10 and 12% increase in blood imbibed compared with mosquitoes without a positive SINV dissemination and non-SINV-exposed mosquitoes, respectively. Ae. aegypti mosquitoes with a disseminated SINV infection fed an average of 4 h sooner than uninfected mosquitoes when offered a bloodmeal contained inside a DEET (N,N-diethyl-3-methylbenzamide) saturated (30%) bovine sausage casing. Together, these results indicate that behavioral changes in mosquito host-seeking, blood feeding and sensitivity to DEET occurred in mosquitoes after SINV infection and dissemination.

Altered response to DEET repellent after infection of Aedes aegypti (Diptera: Culicidae) with Sindbis virus.

To determine whether a Sindbis virus (family Togaviridae, genus Alphavirus, SINV) infection in Aedes aegypti (L.) (Diptera: Culicidae) affected its response to the repellent DEET, we orally exposed Ae. aegypti to an artificial bloodmeal containing SINV or diluent and then allowed to feed on a 10% sucrose suspension containing 3% DEET. When tested seven or more days after the initial bloodmeal, although none of the diluent-exposed mosquitoes fed on the DEET-sucrose suspension, at least 60% of the SINV-exposed mosquitoes fed on the suspension. When legs from the SINV-exposed mosquitoes were tested to determine dissemination status, 89% of those that fed on the DEET-sucrose suspension contained virus. In contrast, only 34% of the nonfeeders had a disseminated infection. When offered a choice between sucrose with or without DEET, a significantly higher percentage of the SINV-exposed mosquitoes than the control mosquitoes fed on the sucrose containing 3% DEET. Together, these results indicate that mosquitoes with a disseminated SINV infection may be less responsive to DEET than uninfected mosquitoes. Therefore, repellent use may be less effective in deterring infected mosquitoes from biting than previously believed.

Organ-associated muscles in Aedes albopictus (Diptera: Culicidae) respond differentially to Sindbis virus.

Differential host cell responses to the alphavirus Sindbis were observed in visceral muscles of the adult female mosquito Aedes albopictus. Following intrathoracic inoculation with SIN, muscles associated with the midgut, hindgut, and ovary resulted in clearance, persistence, and refractoriness to virus, respectively. Prominent sarcomeres characteristic of myofilaments were identified in muscles associated with these three organs by phalloidin labeling of actin, confirming these cells as muscle. The location of virus antigen mimicked the distribution of actin in both mid- and hindgut-associated muscles. Furthermore, these myofilaments remained intact following virus clearance from midgut muscles and during virus persistence in hindgut muscles. Changes in the temporal onset of virus antigen following high titer inoculum compared with standard titer inoculum was observed in anterior midgut muscles, but not in muscles associated with the posterior midgut or hindgut. Muscle bundles closely approximated the gut surface, while a wispy association was displayed at the ovary surface. Prominent ultrastructural differences were observed in the basal lamina attached to the gut compared with the ovary. Additionally, ultrastructural evidence for virus-associated pathology was observed in gut-associated muscles and gut epithelium. Visceral muscles, all composed of the same tissue type, but associated to three different organs in the insect abdomen, responded differentially to Sindbis. We speculate that variations in structure, function or physiology and ultrastructure inherent to insect host cells or organs interactions reflect the complicated milieu of the organism and contribute to differential virus phenotypic expression in muscle cells.

The Alphavirus Sindbis Infects Enteroendocrine Cells in the Midgut of Aedes aegypti.

Transit of the arthropod-borne-virus (arbovirus) Sindbis (SINV) throughout adult female mosquitoes initiates with its attachment to the gut lumen, entry and amplification in midgut cells, followed by dissemination into the hemolymph. Free-mated adult females, aged day 5-7, were proffered a viremic blood suspension via sausage casings containing SINV-TaV-Green Fluorescent Protein (GFP) at a final titer of 106 PFU/mL. Midguts (MGs) from fully engorged mosquitoes were resected on days 5 and 7 post-bloodmeal, and immunolabeled using FMRFamide antibody against enteroendocrine cells (ECs) with a TX-Red secondary antibody. Following immunolabeling, the organs were investigated via laser confocal microscopy to identify the distribution of GFP and TX-Red. Infection using this reporter virus was observed as multiple GFP expression foci along the posterior midgut (PMG) epithelium and ECs were observed as TX-Red labeled cells scattered along the entire length of the MG. Our results demonstrated that SINVGFP did infect ECs, as indicated by the overlapping GFP and TX-Red channels shown as yellow in merged images. We propose that ECs may be involved in the SINV infection pathway in the mosquito MG. Due to the unique role that ECs have in the exocytosis of secretory granules from the MG and the apical-basolateral position of ECs in the PMG monolayer, we speculate that these cells may assist as a mechanism for arboviruses to cross the gut barriers. These findings suggest that MG ECs are involved in arbovirus infection of the invertebrate host.

ImageJ analysis of dentin tubule distribution in human teeth.

Mapping the distribution of dentin tubules is vital to understanding the structure-function relationship of dentin, an important indicator of tooth stability. This study compared the distances between and density of tubules in the external dentin located in the crown region of an adult human incisor and molar to determine if analysis could be conducted using light-level microscopy. Teeth were processed for routine histology, cut in cross-section, images captured using Advanced SPOT Program, and microstructure was analyzed using ImageJ (NIH). Intratubular (peritubular) dentin with or without odontoblast processes were observed and although incisor and molar images appeared visually similar, plot profile graphs differed. Distance-intervals between tubules in the incisor (5.45-7.67 µm) had an overall range of 2.22 µm and in the molar (7.43-8.42 µm) an overall range of 0.99 µm. While molar tubule distribution displayed a tighter overall range, there was a smaller distance between most incisor tubules. The average densities observed in incisors were 15,500 tubules/mm(2), compared with 20,100 tubules/mm(2) in molars. ImageJ analysis of prepared histology microscopic slides provides researchers with a rapid, inexpensive assessment tool when compared with advanced/ultrastructural methodologies. By combining routine histological processing and light microscopic observations followed by ImageJ analysis, tooth structure can be converted into numerical data and easily mastered by laboratory personnel.

Avian fissura prima: differential accumulation of extracellular matrix at a fold.

Extracellular matrix components that flank the fissura prima, a primary surface infolding of the cerebellum in birds and mammals, were examined in the embryonic chick using light and transmission electron microscopy. Cerebella dissected from Day 10 embryos were perfused with a paraformaldehyde-glutaraldehyde-tannic acid primary fixative and sectioned in the sagittal plane through the mid-vermis. Ultrastructural analysis revealed a distinct, continuous basal lamina separating the organ parenchyma (epithelia) from pia mater (mesenchyme) at the fissure surface (arbitrarily labeled; fissure floor, folia wall, and folia apex). The basal lamina was significantly thicker (P < 0.001) at the fissure floor compared to that found at the folia wall, which was significantly thicker (P < 0.001) than that observed at the folia apex. Folds in the basal lamina were observed exclusively at the fissure floor. Surface-associated collagen fibrils were distributed in an aligned, relatively dense manner at the fissure floor, compared with fibrils observed in various orientations and widely separated or absent at the folia wall and folia apex. Metachromasia was more pronounced in the fissure floor than in either the folia wall or folia apex in methylene blue-stained tissue sections. Together, the thicker, folded basal lamina and densely aligned collagen fibrils at the fissure floor provide a chemical rationale for this color change. These findings suggest that the differential accumulation of extracellular matrix at the fissura prima is positioned to play a structural and/or biochemical role in the maintenance of this fold.

Sindbis virus-associated pathology in Aedes albopictus (Diptera: Culicidae).

Virus dissemination and associated pathology were examined in Aedes albopictus after intrathoracic inoculation of Sindbis virus (SIN), the prototypic Alphavirus. At 10 days postinfection, virus RNA was detected in all three-body segments of the insect. Colocalization of virus antigen with structural pathology was observed in mosquito salivary glands and midgut-associated visceral muscles, representing yet another example of arbovirus-associated pathology in a mosquito host. SIN antigen and gross pathology were detected in lateral lobes, but not the medial lobe of salivary glands, whereas virus antigen, vacuolated cytoplasm, and myofilament misalignment were detected in the visceral muscles at the midgut exterior surface. Early in the midgut infection, virus antigen was localized in small foci on the organ surface that progressed to a grate work-like banding pattern that eventually cleared. Both the salivary glands and the midgut are essential to insect survival and reproduction. Additionally, these organs provide a pathway for virus transmission in nature. Although SIN infection may not shorten the mosquito life span, persistent coexistence could permit survival of both host and microbe as well as contribute to alterations in insect behavior.

Heparan sulfate proteoglycan: an arbovirus attachment factor integral to mosquito salivary gland ducts.

Variants of the prototype Alphavirus, Sindbis (SINV), were used in per os infections of adult female mosquitoes to investigate arbovirus interaction with the salivary gland (SG). Infection of Aedine mosquitoes with AR339, a heparan sulfate proteoglycan (HSPG)-dependent variant, resulted in gross pathology in the SG lateral lobes while infection with TR339, a HSPG-independent variant, resulted in minimal SG pathology. HSPG was detected in the internal ducts of the SG lateral lobes by immunolabeling but not in the median lobe, or beyond the triad structure and external ducts. Reports that human lactoferrin interacts with HSPG, suggested an interference with virus attachment to receptors on vertebrate cells. Pre-incubation of Aedes albopictus cultured C7-10 cells with bovine lactoferrin (bLF) followed by adsorption of SINV resulted in earlier and greater intensity of cytopathic response to TR339 compared with AR339. Following pre-treatment of C7-10 cells with bLF, plaques from tissue culture-adapted high-titer SINVTaV-GFP-TC were observed at 48 h post-infection (p.i.), while plaques from low-titer SINVTaV-GFP-TC were not observed until 120 h p.i. Confocal optics detected this reporter virus at 30 days p.i. in the SG proximal lateral lobe, a region of HSPG-immunolocalization. Altogether these data suggest an association between SINV and HSPG in the host mosquito.

Altered behavioral responses of Sindbis virus-infected Aedes aegypti (Diptera: Culicidae) to DEET and non-DEET based insect repellents.

Changes in the time to first bite (TFB) and the bloodfeeding behavior of adult female Aedes aegypti (L.) mosquitoes following dissemination of Sindbis virus (SINV) were observed after exposure to repellents with the active ingredients (AI) DEET, picaridin, 2-undecanone (2-U), and oil of lemon eucalyptus. Dissemination of SINV significantly decreased (P<0.0001) the TFB of DEET (15%) and picaridin (15%) by 46% and 37%, respectively. Significant (P<0.0001) changes in activation, probing, and engorgement times were observed in SINV infected mosquitoes after exposure to the four repellents compared to uninfected mosquitoes. Taken together, a decrease in TFB and time to complete the four bloodfeeding stages will lessen the prey-status, and enhance both the chances of mosquito survival and arbovirus transmission.

Blood feeding position increases success of recalcitrant mosquitoes.

Aedes aegypti and Aedes albopictus are competent natural and laboratory vectors for numerous arthropod-borne viruses (arboviruses), many of which pose global public health concerns. Efficiently imbibing a blood meal from an artificial membrane feeder, Ae. aegypti is an easy feeder: ∼ 96% success. Alternatively, Ae. albopictus is known to be a difficult feeder imbibing poorly: ∼ 20% success. Adult female mosquitoes were grouped in cohorts of 50, proffered a bovine blood meal, and challenged with experimental variables, and feeding success was documented. Controls included Ae. aegypti and the artificial glass membrane feeder: topside presentation (upside-down feeding position only). Variables included lambskin versus bovine collagen sausage membranes, presence or absence of gentle motion, filial generations, and large or small blood packets positioned differently: horizontal presentation (right side-up or nose-up feeding position) and vertical presentation (nose-up feeding position only). Both species preferred sausage casings, and ultrastructural analysis revealed that sausage casings had a textured gripping surface not observed on lambskin membranes. Neither filial generations nor gentle motion improved feeding; however, a 32%-46% increase in blood feeding was observed when Ae. albopictus fed on large horizontal and large or small vertical blood packets. Upside-down feeding of Ae. albopictus with a blood suspension of Sindbis virus heat resistant (SVHR) and the original isolate (AR339) resulted in virus dissemination of 10% and 50%, respectively. Use of bovine collagen sausage membranes in a vertical feeding position will increase the number of engorged females, thereby substantially increasing the number of arbovirus-exposed organisms in the laboratory. Differences in blooding success in response to feeding position further separates the behavior attributes of two Aedine species. Blood meal presentation facilitates gravity and we suggest this is a deciding factor in the feeding success of Ae. albopictus.