Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae.

Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. Here, we tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, we show that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.

Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology.

Haemolymph flow in mosquitoes is primarily driven by the contraction of a dorsal vessel that is subdivided into an abdominal heart and a thoracic aorta. The factors that regulate mosquito heart contractions are not understood, but in other insects heart physiology is partially controlled by several neurohormones. One of these is corazonin, a neuropeptide initially discovered because of its cardioacceleratory activity in the cockroach Periplaneta americana. In the present study, we describe the corazonin gene and transcript structure in the mosquito Anopheles gambiae, characterize its developmental expression, and test its role in modulating heart physiology. We show that the A. gambiae corazonin gene encodes the most common form of the corazonin peptide ([Arg(7) ]-corazonin) and that it is alternatively spliced, with the only difference between the transcripts occurring in the 5' untranslated region. Analysis of the developmental expression of corazonin and the corazonin receptor revealed that transcription of both follows a bimodal distribution, with highest mRNA levels in 2nd instar larvae and during the pupa to adult transition. Finally, experiments where mosquitoes were injected with various doses of corazonin and experiments where the transcription of corazonin and the corazonin receptor were reduced by RNA interference failed to detect a significant role for this neuropeptide in modulating mosquito heart physiology.

A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria.

Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites. In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL-1). The 1.27 kb cDNA clone for the AL-1 gene (AL-1) encodes a 279 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain. AL-1 is transcribed in all life stages. AL-1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge. AL-1 specifically recognizes N-acetyl-d-glucosamine and is able to bind both E. coli and M. luteus. These results suggest that AL-1 might function as a pattern recognition receptor in the immune response in Ar. subalbatus.

Pharmacological and autoradiographical characterization of serotonin transporter-like activity in sporocysts of the human blood fluke, Schistosoma mansoni.

The present study focuses on the role of the biogenic monoamine serotonin (5-hydroxytryptamine) in the biology of sporocyst stages of the human blood fluke, Schistosoma mansoni, and its importance during obligate development within its snail host Biomphalaria glabrata. Based on previous work demonstrating that snails infected with S. mansoni have reduced levels of 5-hydroxytryptamine, we hypothesized that sporocysts actively transport this molecule from the host milieu. Intact sporocysts isolated in vitro take up exogenous 5-hydroxytryptamine via a high-affinity mechanism (K(m)=1.4 micromol l(-1)), and this serotonin transporter-like activity is dependent upon extracellular Na(+) and Cl(-) and is highly sensitive to previously characterized serotonin transporter inhibitors. Autoradiography suggests that transported [(3)H]5-hydroxytryptamine localizes within the body of the sporocyst, and in many cases is found in apical gland cells. Moreover, serotonin transporter-like activity is absent in free-swimming miracidia, the infective stage for the snail host, and the increase in larval serotonin transporter-like activity after miracidium-to-sporocyst transformation is accompanied by a corresponding decrease in steady-state levels of transcripts for tryptophan hydroxylase, the rate-limiting enzyme in serotonin biosynthesis. Overall our data suggest that S. mansoni larvae express surface-exposed serotonin transporter-like molecules, and that the transition from free-living miracidium to parasitic mother sporocyst is characterized by an increased dependence upon exogenous 5-hydroxytryptamine.

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles.

The gastrointestinal uptake of micro- and nanoparticles has been the subject of recent efforts to develop effective carriers that enhance the oral uptake of drugs and vaccines. Here, we used correlative instrumental neutron activation analysis and electron microscopy to quantitatively and qualitatively study the gastrointestinal uptake and subsequent tissue/organ distribution of 4, 10, 28, and 58 nm diameter metallic colloidal gold particles following oral administration to mice. In our quantitative studies we found that colloidal gold uptake is dependent on particle size: smaller particles cross the gastrointestinal tract more readily. Electron microscopic studies showed that particle uptake occurred in the small intestine by persorption through single, degrading enterocytes in the process of being extruded from a villus. To our knowledge this is the first report, at the ultrastructural level, of gastrointestinal uptake of particulates by persorption through holes created by extruding enterocytes.

Aedes aegypti glutamine synthetase: expression and gene structure.

The peritrophic matrix (PM) is the first natural barrier a mosquito-borne parasite faces when ingested with a blood meal; consequently, understanding the biology of PM formation could provide novel transmission control strategies. Because the PM is composed of chitin (a molecule of repeating units of N-acetyl glucosamine), glycoproteins and glucose, characterizing the regulation of enzymes involved in chitin production should provide information concerning factors that influence PM formation. We previously have shown that glutamine synthetase (GS) provides the glutamine needed in the initial steps of chitin biosynthesis in the yellow fever mosquito, Aedes aegypti. In the present study we show that GS is encoded by a single 4.5 kb gene, designated mGS, containing three exons and two introns. Multiple transcripts are generated from mGS presumably by differential splicing of the introns. Sequences of two cDNAs encoding GS are identical at the protein level, but differ in their 5'-untranslated regions. GS message is constitutively expressed in all developmental stages and in most tissues, with an increase in GS transcription observed in midgut and fat body tissues of female mosquitoes following a blood meal. Transcripts are localized to the apical side of the mosquito midgut epithelium and data suggest that mGS transcription is regulated by an Oct-1 transcription factor.

A novel member of the RING-finger gene family associated with reproductive tissues of the mosquito, Aaedes aegypti.

The RING finger is a zinc-binding domain that is found in proteins from viruses, plants and animals. Here we report the characterization and tissue-specific expression of a mosquito gonadal protein gene, mgp, from the mosquito, Aedes aegypti. The putative gene product, MGP, contains two RING fingers, a B-box, and a hydrophobic core. These mosquito MGP structural motifs are highly conserved in proteins found in mouse and nematode. Northern blot analysis and in situ hybridization demonstrated the presence of multiple mgp RNA transcripts in male and female reproductive tissues. Expression of mgp in the ovary is constitutive, but an increase in message was observed in the ovaries of female mosquitoes previously exposed to a blood meal. These results suggest that MGP is a protein that might play a role(s) in mosquito gametogenesis.