Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA.

Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1%+/- 1.8%; injected: 37.9% +/- 3.6% (Mean +/- SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal.

Differential expression of fat body genes in Glossina morsitans morsitans following infection with Trypanosoma brucei brucei.

To determine which fat body genes were differentially expressed following infection of Glossina morsitans morsitans with Trypanosoma brucei brucei we generated four suppression subtractive hybridisation (SSH) libraries. We obtained 52 unique gene fragments (SSH clones) of which 30 had a known orthologue at E-05 or less. Overall the characteristics of the orthologues suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly; (ii) that self-cured flies are mounting an oxidative stress response; and (iii) that self-cured flies are displaying increased energy usage. The three most consistently differentially expressed genes were further analysed by gene knockdown (RNAi). Knockdown of Glossina transferrin transcripts, which are upregulated in self-cured flies compared with flies infected with trypanosomes, results in a significant increase in the number of trypanosome infections establishing in the fly midgut, suggesting transferrin plays a role in the protection of tsetse flies from trypanosome infection.

Antioxidant gene expression in the blood-feeding fly Glossina morsitans morsitans.

We report the characterization of 11 antioxidant genes from the tsetse fly Glossina m. morsitans. Through similarity searches which detected homology we suggest that these genes consist of two superoxide dismutases (one with a putative signal peptide), three thioredoxin peroxidases (one with a putative signal peptide), three peroxiredoxins, one further signal peptide-containing peroxidase with its closest similarity to a glutathione peroxidase, one catalase and one thioredoxin reductase. We describe the changes occurring in the expression levels of these genes during fly development, in different adult tissues, in the adult midgut through the digestive cycle and following trypanosome infection. Overall, nine of the 11 genes studied showed responses to changes in physiological circumstance, with the peroxiredoxin group showing the smallest variations throughout.

Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae).

Haematophagous insects produce pharmacological substances in their saliva to counteract vertebrate host haemostasis events such as coagulation, vasoconstriction and platelet aggregation. To investigate the bioactive salivary molecules of the triatomine bug Triatoma brasiliensis, we produced subtraction-enriched cDNAs of salivary-gland specific genes using suppression subtractive hybridization. Six full-length differentially expressed cDNAs (Tb113, Tb125, Tb152, Tb169, Tb180 and Tb198) were selected, cloned and sequenced. Sequence similarity searches of the databases using the putative amino acid sequence of our clones gave the following results: Tb152 - Triabin, an antithrombin induced platelet aggregation factor found in salivary gland extracts of T. pallidipennis. Tb169 - Pallidipin, an anticollagen induced platelet aggregation factor also found in T. pallidipennis salivary homogenates. Tb180 - Procalin, the major allergen of T. protracta saliva. The other three salivary-gland specific cDNAs produced no obvious homologies. Comparison of these salivary gland-specific cDNAs of with those of other triatomines combined with functional studies using recombinant proteins will allow a better understanding of the co-evolutionary process occurring between these insects and their vertebrate hosts, and may also lead to the discovery of novel antihaemostatic agents.

Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans.

Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

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.

Grouping of trypanosome species in mixed infections in Glossina pallidipes.

Trypanosomes in the dissection-positive proboscis of Glossina pallidipes were identified by PCR using species-specific primers. Of the 3741 flies dissected 643 were proboscis positive. PCR was performed on 406 dissection-positive probosces giving positive identifications in 352 (86.7%) and infection rates of 14.8% for congolense-type infections, 2.8% for vivax-type infections and 1.4% for the unidentified group. Of the 352 PCR identified infections 225 were single, 111 were double, 13 were triple infections and there were 3 quadruple infections. Statistical analysis suggests that mixed infections group into 3 largely separate divisions among the tsetse population (i) Trypanosoma congolense savannah and T. congolense Kenya coast, (ii) T. simiae, T. congolense Tsavo and T. godfreyi and (iii) T. vivax. We conclude that either differing feeding patterns among members of the fly population or the ability of the trypanosomes in each of the infection categories to significantly influence the maturation of trypanosomes in the other categories are the most likely causes of the groupings noted. Chi-squared analysis of dissection and PCR methods of trypanosome identification revealed profound differences (chi 2 = 19.1; D.F. = 1; P > 0.05). If confirmed in other studies these findings have serious implications for our understanding of trypanosome epidemiology in tsetse flies, much of which is founded on data from dissection-based trypanosome identifications.

Cloning, sequencing, temporal expression and tissue-specificity of two serine proteases from the midgut of the blood-feeding fly Stomoxys calcitrans.

Using highly degenerate, serine-protease-specific PCR primers on a midgut-specific cDNA library it was estimated that a minimum of 24 independent serine proteases were expressed in the midgut of Stomoxys calcitrans. The relative abundance of these 24 independent serine proteases has been estimated by restriction analysis of PCR products, showing that 69% fall into six almost equally abundant groups. Two highly abundant serine protease cDNAs (Ssp1 and Ssp2) were isolated and sequenced. They encode preproenzymes of 272 amino acids (Mr 28521) and 255 amino acids (Mr 27097) with putative signal peptides of 17 amino acids and 16 amino acids, putative activation peptides of 15 amino acids and 10 amino acids and mature enzymes of 239 amino acids (Mr 25322; pI 4.89) and 228 amino acids (Mr 24182; pI 7.59), respectively. Both deduced amino acid sequences contain the Asp/His/Ser catalytic triad and the highly conserved sequences surrounding it. Ssp2 also has the aspartate and two glycine residues in the specificity pocket, marking this as a typical trypsin. The positioning of the residues in the specificity pocket of Sspl is unusual; aspartate and glycine residues are present, which is typical of trypsin, but both are separated from surrounding conserved residues by additional amino acids; the second glycine found in the specificity pocket of trypsin is replaced by a serine, which is typical of chymotrypsin. Although a serine protease, the precise substrate specificity of Sspl remains to be determined. Northern analysis shows that both serine proteases are expressed constitutively with only a 20% change in the levels of expression of Ssp1 and Ssp2 through the digestive cycle, and that expression occurs predominantly in the opaque region of the midgut, the region responsible for secretion of digestive enzymes.

Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans.

We have cloned and sequenced two defensins, Smd1 and Smd2, from anterior midgut tissue of the blood-sucking fly Stomoxys calcitrans. The DNA and N-terminal protein sequences suggest both are produced as prepropeptides. Smd1 differs from the classic defensin pattern in having an unusual six-amino acid-long N-terminal sequence. Both Smd1 and Smd2 have lower pI points and charge than insect defensins derived from fat body/hemocytes. Northern analysis shows both of these defensin molecules are tissue specific; both are produced by the anterior midgut tissue and, unlike the other insect defensins reported to date, neither appears to be expressed in fat body or hemocytes. Northern analysis also shows that mRNAs for both defensins are constitutively produced in the anterior midgut tissues and that these transcripts are up-regulated in response to sterile as well as a lipopolysaccharide-containing blood meal. However, anti-Gram-negative biological activity in the midgut is substantially enhanced by lipopolysaccharide. These findings suggest that the insect midgut has its own tissue-specific immune mechanisms and that this invertebrate epithelium is, like several vertebrate epithelia, protected by specific antibacterial peptides.