Hemocyte RNA-Seq analysis of Indian malarial vectors Anopheles stephensi and Anopheles culicifacies: From similarities to differences.

Anopheles stephensi and Anopheles culicifacies are dominant malarial vectors in urban and rural India, respectively. Both species carry significant biological differences in their behavioral adaptation and immunity, but the genetic basis of these variations are still poorly understood. Here, we uncovered the genetic differences of immune blood cells, that influence several immune-physiological responses. We generated, analyzed and compared the hemocyte RNA-Seq database of both mosquitoes. A total of 5,837,223,769 assembled bases collapsed into 7,595 and 3,791 transcripts, originating from hemocytes of laboratory-reared 3-4 days old naïve (sugar-fed) mosquitoes, Anopheles stephensi and Anopheles culicifacies respectively. Comparative GO annotation analysis revealed that both mosquito hemocytes encode similar proteins. Furthermore, while An. stephensi hemocytes showed a higher percentage of immune transcripts encoding APHAG (Autophagy), IMD (Immune deficiency pathway), PRDX (Peroxiredoxin), SCR (Scavenger receptor), IAP (Inhibitor of apoptosis), GALE (galactoside binding lectins), BGBPs (1,3 beta D glucan binding proteins), CASPs (caspases) and SRRP (Small RNA regulatory pathway), An. culicifacies hemocytes yielded a relatively higher percentage of transcripts encoding CLIP (Clip domain serine protease), FREP (Fibrinogen related proteins), PPO (Prophenol oxidase), SRPN (Serpines), ML (Myeloid differentiation 2-related lipid recognition protein), Toll path and TEP (Thioester protein), family proteins. However, a detailed comparative Interproscan analysis showed An. stephensi mosquito hemocytes encode proteins with increased repeat numbers as compared to An. culicifacies. Notably, we observed an abundance of transcripts showing significant variability of encoded proteins with repeats such as LRR (Leucine rich repeat), WD40 (W-D dipeptide), Ankyrin, Annexin, Tetratricopeptide and Mitochondrial substrate carrier repeat-containing family proteins, which may have a direct influence on species-specific immune-physiological responses. Summarily, our deep sequencing analysis unraveled that An. stephensi evolved with an expansion of repeat sequences in hemocyte proteins as compared to An. culicifacies, possibly providing an advantage for better adaptation to diverse environments.

The number of neurons in Drosophila and mosquito brains.

Various insect species serve as valuable model systems for investigating the cellular and molecular mechanisms by which a brain controls sophisticated behaviors. In particular, the nervous system of Drosophila melanogaster has been extensively studied, yet experiments aimed at determining the number of neurons in the Drosophila brain are surprisingly lacking. Using isotropic fractionator coupled with immunohistochemistry, we counted the total number of neuronal and non-neuronal cells in the whole brain, central brain, and optic lobe of Drosophila melanogaster. For comparison, we also counted neuronal populations in three divergent mosquito species: Aedes aegypti, Anopheles coluzzii and Culex quinquefasciatus. The average number of neurons in a whole adult brain was determined to be 199,380 ±3,400 cells in D. melanogaster, 217,910 ±6,180 cells in Ae. aegypti, 223,020 ± 4,650 cells in An. coluzzii and 225,911±7,220 cells in C. quinquefasciatus. The mean neuronal cell count in the central brain vs. optic lobes for D. melanogaster (101,140 ±3,650 vs. 107,270 ± 2,720), Ae. aegypti (109,140 ± 3,550 vs. 112,000 ± 4,280), An. coluzzii (105,130 ± 3,670 vs. 107,140 ± 3,090), and C. quinquefasciatus (108,530 ±7,990 vs. 110,670 ± 3,950) was also estimated. Each insect brain was comprised of 89% ± 2% neurons out of its total cell population. Isotropic fractionation analyses did not identify obvious sexual dimorphism in the neuronal and non-neuronal cell population of these insects. Our study provides experimental evidence for the total number of neurons in Drosophila and mosquito brains.

Diverse cellular morphologies during lumen maturation in Anopheles gambiae larval salivary glands.

Mosquitoes are the greatest animal threat to human health, causing hundreds of millions of infections and around 1 million deaths each year. All mosquito-borne pathogens must traverse the salivary glands (SGs) to be transmitted to the next host, making this organ an ideal target for interventions. The adult SG develops from precursor cells located in the larval SG duct bud. Characterization of the larval SG has been limited. We sought to better understand larval SG architecture, secretion and gene expression. We developed an optimized method for larval SG staining and surveyed hundreds of larval stage 4 (L4) SGs using fluorescence confocal microscopy. Remarkable variation in SG cell and chromatin organization differed among individuals and across the L4 stage. Lumen formation occurred during L4 stage through secretion likely involving a coincident cellular apical lipid enrichment and extracellular vesicle-like structures. Meta-analysis of microarray data showed that larval SG gene expression is divergent from adult SGs, more similar to larval gastric cecae, but different from other larval gut compartments. This work highlights the variable cell architecture of larval Anopheles gambiae SGs and provides candidate targets for genetic strategies aiming to disrupt SGs and transmission of mosquito-borne pathogens.

Anopheles gambiae densovirus (AgDNV) negatively affects Mayaro virus infection in Anopheles gambiae cells and mosquitoes.

BACKGROUND: Recent studies demonstrate that insect-specific viruses can influence the ability of their mosquito hosts to become infected with and transmit arboviruses of medical and veterinary importance. The aim of this study was to evaluate the interactions between Anopheles gambiae densovirus (AgDNV) (Parvoviridae) (a benign insect-specific virus that infects An. gambiae mosquitoes) and Mayaro virus (MAYV) (Togaviridae) (an emerging human pathogen that can be transmitted by An. gambiae) in both insect cell culture and mosquitoes. METHODS: For in vitro studies, An. gambiae Mos55 cells infected or uninfected with AgDNV were infected with MAYV. For in vivo studies, An. gambiae mosquitoes were injected intrathoracically with AgDNV and 4 days later orally infected with MAYV. Mosquitoes were dissected 10 days after MAYV infection, and MAYV titers in the body, legs and saliva samples quantified using focus-forming assay. RESULTS: MAYV virus replication was reduced 10-100-fold in An. gambiae Mos55 cells infected with AgDNV. In mosquitoes, there was a significant negative correlation between AgDNV and MAYV body titers 10 days post-blood meal. CONCLUSIONS: AgDNV infection was associated with reduced production of MAYV in cell culture, and reduced body titers of MAYV in An. gambiae mosquitoes. As densovirus infections are common in natural mosquito populations, these data suggest that they may affect the epidemiology of viruses of medical importance.

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

Premeiotic and meiotic failures lead to hybrid male sterility in the Anopheles gambiae complex.

Hybrid male sterility (HMS) contributes to speciation by restricting gene flow between related taxa. Detailed cytological characterization of reproductive organs in hybrid males is important for identifying phenotypes that can help guide searches of speciation genes. To investigate possible cellular causes of HMS, we performed crosses between closely related species of the Anopheles gambiae complex: An. merus with An. gambiae or An. coluzzii. We demonstrate that HMS in African malaria mosquitoes involves two defects in the reciprocal crosses: a premeiotic arrest of germline stem cells in degenerate testes and a failure of the reductional meiotic division of primary spermatocytes in normal-like testes. The premeiotic arrest in degenerate testes of hybrids is accompanied by a strong suppression of meiotic and postmeiotic genes. Unlike pure species, sex chromosomes in normal-like testes of F1 hybrids are largely unpaired during meiotic prophase I and all chromosomes show various degrees of insufficient condensation. Instead of entering reductional division in meiosis I, primary spermatocytes prematurely undergo an equational mitotic division producing non-motile diploid sperm. Thus, our study identified cytogenetic errors in interspecies hybrids that arise during the early stages of postzygotic isolation.

Inversions are bigger on the X chromosome.

In many insects, X-linked inversions fix at a higher rate and are much less polymorphic than autosomal inversions. Here, we report that in Drosophila, X-linked inversions also capture 67% more genes. We estimated the number of genes captured through an approximate Bayesian computational analysis of gene orders in nine species of Drosophila. X-linked inversions fixed with a significantly larger gene content. Further, X-linked inversions of intermediate size enjoy highest fixation rate, while the fixation rate of autosomal inversions decreases with size. A less detailed analysis in Anopheles suggests a similar pattern holds in mosquitoes. We develop a population genetic model that assumes the fitness effects of inversions scale with the number of genes captured. We show that the same conditions that lead to a higher fixation rate also produce a larger size for inversions on the X.

Structural and functional characterization of the contractile aorta and associated hemocytes of the mosquito Anopheles gambiae.

The primary pump of the circulatory system of insects is a dorsal vessel that traverses the length of the insect. The anterior portion, located in the head, neck and thorax, is the aorta, and the posterior portion, located in the abdomen, is the heart. Here, we characterize the structure and function of the aorta and conical chamber of the mosquito, Anopheles gambiae The aorta begins in the head with an excurrent opening located above the dorsal pharyngeal plate and ends at the thoraco-abdominal junction where it joins the conical chamber of the heart. The aorta lacks ostia, and based on the diameter of the vessel as well as the density and helical orientation of muscle, consists of three regions: the anterior aorta, the bulbous chamber, and the posterior aorta. The aorta contracts in the anterograde direction, but these contractions are independent of heart contractions and do not play a major role in hemolymph propulsion. Intravital imaging of the venous channels, the first abdominal segment and the neck revealed that hemolymph only travels through the aorta in the anterograde direction, and does so only during periods of anterograde heart flow. Furthermore, hemolymph only enters the thoraco-abdominal ostia of the conical chamber when the heart contracts in the retrograde direction, propelling this hemolymph to the posterior of the body. Finally, very few hemocytes associate with the aorta, and unlike what is seen in the periostial regions of the heart, infection does not induce the aggregation of hemocytes on the aorta.

A standard photomap of ovarian nurse cell chromosomes and inversion polymorphism in Anopheles beklemishevi.

BACKGROUND: Anopheles beklemishevi is a member of the Maculipennis group of malaria mosquitoes that has the most northern distribution among other members of the group. Although a cytogenetic map for the larval salivary gland chromosomes of this species has been developed, a high-quality standard cytogenetic photomap that enables genomics and population genetics studies of this mosquito at the adult stage is still lacking. METHODS: In this study, a cytogenetic map for the polytene chromosomes of An. beklemishevi from ovarian nurse cells was developed using high-resolution digital imaging from field collected mosquitoes. PCR-amplified DNA probes for fluorescence in situ hybridization (FISH) were designed based on the genome of An. atroparvus. The DNA probe obtained by microdissection procedures from the breakpoint region was labelled in a DOP-PCR reaction. Population analysis was performed on 371 specimens collected in 18 locations. RESULTS: We report the development of a high-quality standard photomap for the polytene chromosomes from ovarian nurse cells of An. beklemishevi. To confirm the suitability of the map for physical mapping, several PCR-amplified probes were mapped to the chromosomes of An. beklemishevi using FISH. In addition, we identified and mapped DNA probes to flanking regions of the breakpoints of two inversions on chromosome X of this species. Inversion polymorphism was determined in 13 geographically distant populations of An. beklemishevi. Four polymorphic inversions were detected. The positions of common chromosomal inversions were indicated on the map. CONCLUSIONS: The study constructed a standard photomap for ovarian nurse cell chromosomes of An. beklemishevi and tested its suitability for physical genome mapping and population studies. Cytogenetic analysis determined inversion polymorphism in natural populations of An. beklemishevi related to this species' adaptation.

Voltage-sensitive potassium channels expressed after 20-Hydroxyecdysone treatment of a mosquito cell line.

The goal of this research was to express receptors and ion channels in hormone-treated insect cell lines. Treatment of Anopheles gambiae Sua1B cells with 20-hydroxyecdysone showed an inhibition of cell growth over a time course of three days, with no change in cellular morphology. The effect of 20-hydroxyecdysone was enhanced in the presence of the potassium channel blocker 4-aminopyridine, but not tetraethylammonium. Concentration-response curves of 4-aminopyridine in the presence of 42 µM (1 mg/ml) 20-hydroxyecdysone showed similar IC50 values (6-10 µM) across 3 day exposures. Whole cell patch clamp confirmed the expression of delayed-rectifier (Kv2) potassium channels in hormone-supplemented Sua1B cells, whereas untreated Sua1B cells showed no evidence of Kv2 expression. The hormone-induced expression of Kv2 channels occurred in as little as 4 h after treatment, but were not observed after 24 h of exposure to 20-hydroxyecdysone, suggesting they played a role in cell death. The expressed channels had current-voltage relationships diagnostic for the Kv2 subtype, and were inhibited with an IC50 = 13 mM of tetraethylammonium. Overall, these parameters were similar to Anopheles gambiae Kv2 potassium channels expressed in HEK-293 cells. The induced presence of ion channels (and possibly receptors) in these cells has potential utility for high throughput screening and basic neuroscience research.

Functional integration of the circulatory, immune, and respiratory systems in mosquito larvae: pathogen killing in the hemocyte-rich tracheal tufts.

BACKGROUND: As both larvae and adults, mosquitoes encounter a barrage of immune insults, ranging from microbe-rich communities in larval habitats to ingested blood-borne pathogens in adult blood meals. Given that mosquito adults have evolved an efficient means of eliminating infections in their hemocoel (body cavity) via the coordinated action of their immune and circulatory systems, the goal of the present study was to determine whether such functional integration is also present in larvae. RESULTS: By fluorescently labeling hemocytes (immune cells), pericardial cells, and the heart, we discovered that fourth instar larvae, unlike adults, contain segmental hemocytes but lack the periostial hemocytes that surround the ostia (heart valves) in abdominal segments 2-7. Instead, larvae contain an abundance of sessile hemocytes at the tracheal tufts, which are respiratory structures that are unique to larvae, are located in the posterior-most abdominal segment, and surround what in larvae are the sole incurrent openings for hemolymph entry into the heart. Injection of fluorescent immune elicitors and bacteria into the larval hemocoel then showed that tracheal tuft hemocytes mount rapid and robust immune responses against foreign insults. Indeed, green fluorescent protein-labeled Escherichia coli flowing with the hemolymph rapidly aggregate exclusively at the tracheal tufts, where they are killed within 24 h post-infection via both phagocytosis and melanization. CONCLUSION: Together, these findings show that the functional integration of the circulatory, respiratory, and immune systems of mosquitoes varies drastically across life stages.

"Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi".

BACKGROUND: Anopheles mosquitoes are vectors for malaria, a disease with continued grave outcomes for human health. Transmission of malaria from mosquitoes to humans occurs by parasite passage through the salivary glands (SGs). Previous studies of mosquito SG architecture have been limited in scope and detail. METHODS: We developed a simple, optimized protocol for fluorescence staining using dyes and/or antibodies to interrogate cellular architecture in Anopheles stephensi adult SGs. We used common biological dyes, antibodies to well-conserved structural and organellar markers, and antibodies against Anopheles salivary proteins to visualize many individual SGs at high resolution by confocal microscopy. RESULTS: These analyses confirmed morphological features previously described using electron microscopy and uncovered a high degree of individual variation in SG structure. Our studies provide evidence for two alternative models for the origin of the salivary duct, the structure facilitating parasite transport out of SGs. We compare SG cellular architecture in An. stephensi and Drosophila melanogaster, a fellow Dipteran whose adult SGs are nearly completely unstudied, and find many conserved features despite divergence in overall form and function. Anopheles salivary proteins previously observed at the basement membrane were localized either in SG cells, secretory cavities, or the SG lumen. Our studies also revealed a population of cells with characteristics consistent with regenerative cells, similar to muscle satellite cells or midgut regenerative cells. CONCLUSIONS: This work serves as a foundation for linking Anopheles stephensi SG cellular architecture to function and as a basis for generating and evaluating tools aimed at preventing malaria transmission at the level of mosquito SGs.

Anopheles gambiae Ag55 cell line as a model for Lysinibacillus sphaericus Bin toxin action.

Binary toxin (Bin) produced by Lysinibacillus sphaericus is toxic to Culex and Anopheles mosquito larvae. It has been used world-wide for control of mosquitoes that vector disease. The Bin toxin interacts with the glucosidase receptor, Cpm1, in Culex and its orthologue, Agm3, in Anopheles mosquitoes. However, the exact mechanism of its mode of action is not clearly understood. It is essential to understand mode of action of Bin toxin to circumvent the resistance that develops over generations of exposure. A suitable model cell line will facilitate investigations of the molecular action of Bin toxin. Here we report Bin toxin activity on Ag55 cell line that has been derived from an actual target, Anopheles gambiae larvae. The Bin toxin, both in pro and active forms, kills the Ag55 cells within 24h. Bin toxin internalizes in Ag55 cells and also induces vacuolation as tracked by Lysotracker dye. The dose response studies showed that 1.5nM of Bin toxin is sufficient to induce vacuolation and Ag55 cell death. Presence of α-glucosidase gene (Agm3) expression in the Ag55 cells was also confirmed. Thus, Ag55 cells constitute an appropriate model system to decipher the mode of Bin action in mosquito larvae.

In vitro and in vivo host range of Anopheles gambiae densovirus (AgDNV).

AgDNV is a powerful gene transduction tool and potential biological control agent for Anopheles mosquitoes. Using a GFP reporter virus system, we investigated AgDNV host range specificity in four arthropod cell lines (derived from An. gambiae, Aedes albopictus and Drosophila melanogaster) and six mosquito species from 3 genera (An. gambiae, An. arabiensis, An. stephensi, Ae. albopictus, Ae. aegypti and Culex tarsalis). In vitro, efficient viral invasion, replication and GFP expression was only observed in MOS55 An. gambiae cells. In vivo, high levels of GFP were observed in An. gambiae mosquitoes. Intermediate levels of GFP were observed in the closely related species An. arabiensis. Low levels of GFP were observed in An. stephensi, Ae. albopictus, Ae. aegypti and Cx. tarsalis. These results suggest that AgDNV is a specific gene transduction tool for members of the An. gambiae species complex, and could be potentially developed into a biocontrol agent with minimal off-target effects.

Characterization of Anopheles gambiae (African Malaria Mosquito) Ferritin and the Effect of Iron on Intracellular Localization in Mosquito Cells.

Ferritin is a 24-subunit molecule, made up of heavy chain (HC) and light chain (LC) subunits, which stores and controls the release of dietary iron in mammals, plants, and insects. In mosquitoes, dietary iron taken in a bloodmeal is stored inside ferritin. Our previous work has demonstrated the transport of dietary iron to the ovaries via ferritin during oogenesis. We evaluated the localization of ferritin subunits inside CCL-125 [Aedes aegypti Linnaeus (Diptera: Culicidae), yellow fever mosquito] and 4a3b [Anopheles gambiae Giles (Diptera: Culicidae), African malaria mosquito] cells under various iron treatment conditions to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of the intracellular ferritin subunits following iron administration. Deconvolution microscopy captured 3D fluorescent images of iron-treated mosquito cells to visualize the ferritin HC and LC homologue subunits (HCH and LCH, respectively) in multiple focal planes. Fluorescent probes were used to illuminate cell organelles (i.e., Golgi apparatus, lysosomes, and nuclei) while secondary probes for specific ferritin subunits demonstrated abundance and co-localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin under conditions of iron exposure, and to advance novel hypotheses for the crucial role of iron in mosquito vectors.

A standard photomap of ovarian nurse cell chromosomes in the European malaria vector Anopheles atroparvus.

Anopheles atroparvus (Diptera: Culicidae) is one of the main malaria vectors of the Maculipennis group in Europe. Cytogenetic analysis based on salivary gland chromosomes has been used in taxonomic and population genetic studies of mosquitoes from this group. However, a high-resolution cytogenetic map that could be used in physical genome mapping in An. atroparvus is still lacking. In the present study, a high-quality photomap of the polytene chromosomes from ovarian nurse cells of An. atroparvus was developed. Using fluorescent in situ hybridization, 10 genes from the five largest genomic supercontigs on the polytene chromosome were localized and 28% of the genome was anchored to the cytogenetic map. The study established chromosome arm homology between An. atroparvus and the major African malaria vector Anopheles gambiae, suggesting a whole-arm translocation between autosomes of these two species. The standard photomap constructed for ovarian nurse cell chromosomes of An. atroparvus will be useful for routine physical mapping. This map will assist in the development of a fine-scale chromosome-based genome assembly for this species and will also facilitate comparative and evolutionary genomics studies in the genus Anopheles.

A standard cytogenetic map for Anopheles sinensis and chromosome arm homology between the subgenera Anopheles and Cellia.

Anopheles sinensis (Diptera: Culicidae) is an important vector of Plasmodium vivax in Southeast Asia. To facilitate population genetic and genomic studies of An. sinensis, we developed a standard cytogenetic photomap for this species. The polytene chromosomes were straightened and divided into 39 numbered divisions and 116 lettered subdivisions. The chromosomal localizations of 13 DNA probes were determined by fluorescent in situ hybridization. A comparison of the physical map for An. sinensis with the genome map for Anopheles gambiae revealed a whole-arm autosomal translocation between the two species. Specifically, the 2R arm of An. gambiae corresponds to the 3R arm of An. sinensis and the pattern of correspondence of the other chromosome arms remains regular. We mapped the breakpoints of the polymorphic paracentric chromosomal inversion 3Ra to subdivisions 28A and 31A. The standard cytogenetic map developed in this study will be useful for detailed comparative genome mapping and population genetic studies of An. sinensis.

Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles.

Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.

Cytotoxicity of the Bacillus thuringiensis Cry4B toxin is mediated by the cadherin receptor BT-R3 of Anopheles gambiae.

We demonstrate for the first time the selective cytotoxicity of Bacillus thuringiensis subsp. israelensis Cry4B toxin mediated by BT-R3 using a cell-based system, which employs High Five insect cells stably expressing BT-R3. Discovery and validation of BT-R3 as the Cry4B receptor was accomplished using a web-based computational pipeline platform that facilitates high-throughput insecticidal target identification utilizing the Anopheles gambiae genome. Once the Cry4B toxin binds to the BT-R3 receptor, a cell death pathway is manifested by sequential cytological changes that include membrane blebbing, cell swelling and lysis. Cry4B toxin associates with cell membrane in both oligomeric and monomeric forms. Monomeric toxin binds specifically to BT-R3 whereas oligomer interacts with cell membrane non-specifically. Cytotoxicity and cell death are the direct result of binding of toxin monomer to BT-R3. The oligomeric form of Cry4B toxin is not involved in cell death. Both the location of the toxin-binding region within BT-R3 and its structural motif are critical to the binding affinity and specificity of the toxin. The toxin-binding region of BT-R3 appears to be located in EC11, the most membrane proximal EC module within the extracellular domain. It is characterized by the presence of two highly conserved amino acid sequences within their N- and C-termini that flank EC11. These sequences represent signature motifs that mark the toxin-binding function in BT-R3. The two sequences form two adjacent ß-strands within the ß-barrel of EC11, the positioning of which is a hallmark of all Cry toxin receptors thus far reported.

Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection.

BACKGROUND: Mosquitoes respond to infection by mounting immune responses. The primary regulators of these immune responses are cells called hemocytes, which kill pathogens via phagocytosis and via the production of soluble antimicrobial factors. Mosquito hemocytes are circulated throughout the hemocoel (body cavity) by the swift flow of hemolymph (blood), and data show that some hemocytes also exist as sessile cells that are attached to tissues. The purpose of this study was to create a quantitative physical map of hemocyte distribution in the mosquito, Anopheles gambiae, and to describe the cellular immune response in an organismal context. RESULTS: Using correlative imaging methods we found that the number of hemocytes in a mosquito decreases with age, but that regardless of age, approximately 75% of the hemocytes occur in circulation and 25% occur as sessile cells. Infection induces an increase in the number of hemocytes, and tubulin and nuclear staining showed that this increase is primarily due to mitosis and, more specifically, autonomous cell division, by circulating granulocytes. The majority of sessile hemocytes are present on the abdominal wall, although significant numbers of hemocytes are also present in the thorax, head, and several of the appendages. Within the abdominal wall, the areas of highest hemocyte density are the periostial regions (regions surrounding the valves of the heart, or ostia), which are ideal locations for pathogen capture as these are areas of high hemolymph flow. CONCLUSIONS: These data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel.