Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiae

The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

Aedes aegypti transferrin. Gene structure, expression pattern, and regulation

Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.     

A gut-specific serine protease from the malaria vector Aanopheles gambiae is downregulated after blood ingestion

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.     

Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

In haematophagous insects digestion of the blood meal provides nutrients for survival and essential components for egg production. We have isolated and partially characterized two gut-specific genes from the black fly Simulium vittatum. Sequence analysis revealed that both are highly similar to digestive proteases, one to trypsins and the other to carboxypeptidases. RNA blot analysis indicates that the expression of these two genes is regulated in a sexspecific manner; when fed the same sucrose-based diet, expression in males is substantially lower than in females. In females, expression of both genes is strongly induced by a blood meal. At 6 h after the blood meal the trypsin-like gene product was immunolocalized to the midgut epithelium and to the outer layers of the peritrophic matrix.     

Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine-rich domains, two of which flank a proline/serine/threonine-rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal-responsive gene from an aquatic insect.     

Isolation, endocrine regulation and mRNA distribution of the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae)

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.     

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.     

Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements

The Anopheles gambiae adult peritrophic matrix protein 1 (AgAper1) regulatory elements were used to drive the expression of phospholipase A2 (PLA2), a protein known to disrupt malaria parasite development in mosquitoes. These AgAper1 regulatory elements were sufficient to promote the accumulation of PLA2 in midgut epithelial cells before a blood meal and its release into the lumen upon blood ingestion. Plasmodium berghei oocyst formation was reduced by approximately 80% (74-91% range) in transgenic mosquitoes. Blood-seeking behaviour and survival of AgAper1-PLA2 transgenic mosquitoes were comparable to sibling wild-type mosquitoes, while fertility was substantially lower. Ultrastructural studies suggest that decreased fitness is a consequence of internal damage to midgut epithelial cells.     

Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.