Simulium damnosum s.l.: isolation and identification of prophenoloxidase following an infection with Onchocerca spp. using targeted differential display.

Phenoloxidase (PO) is the key enzyme for melanin synthesis and plays an important role in the defense and recognition of pathogens in insects and other arthropods. We now report the upregulated transcription of the gene encoding the precursor of PO, prophenoloxidase, in Onchocerca-infected Simulium damnosum s.l., the main vector of human and bovine onchocerciasis in subsaharan Africa. Using homology-based generic primers in a polymerase chain reaction-based targeted differential display, the gene itself was identified and partially sequenced.

The effects of protease inhibitors and sugars on the survival and development of the parasite Onchocerca ochengi in its natural intermediate host Simulium damnosum s.l.

A range of protease inhibitors and carbohydrates were administered to the haemolymph of the vector Simulium damnosum s.l. to test for their effects on the success of an Onchocerca ochengi infection in vivo. We found that serine protease inhibitors led to a significant increase of parasite survival. Two sugars, D(+)-galactose and methyl-alpha-D-mannopyranoside, had the same effect. These effects are possibly due to the successful in vivo blocking of the two respective types of inducible immune molecules, the serine protease and the carbohydrate binding lectins, both of which have been identified in simuliids.

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge.

The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E. coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M.luteus infection since anti-E.coli activity was similar in haemolymph from both E.coli or M.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4 kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.

Phenotypic segregation of Aedes aegypti for immune antibacterial activity and resistance to filariae.

We report the phenotypic selection of two lines of Aedes aegypti, from a filariae susceptible parental stock (Refm). This selection was based upon the level of inducible anti-gram negative Escherichia coli activity within the haemolymph following E. coli infection. These lines, denoted 'high' and 'low', demonstrated significant differences in anti-E. coli responses throughout. However no difference was observed in the anti-gram positive Micrococcus luteus response following E. coli challenge. F4, F6 and F9 mosquitoes were experimentally infected with Brugia pahangi microfilariae. Reductions of between 53 and 82% in the mean number of larvae completing development in the 'high' compared with the 'low' responding line were observed. Corresponding reductions of between 30 and 50% in the mosquito infection rates also occurred between these lines. These reductions were significant for trials on the F6 and F9 generations. We have therefore selected for Ae. aegypti possessing significant refractoriness to filaria infection. Importantly, this 'high' line has not been exposed to selection pressure using filariae development as a phenotypic selective marker.

Simulium damnosum s.l.: identification of inducible serine proteases following an Onchocerca infection by differential display reverse transcription PCR.

Vector-derived proteases are thought to be key to the regulation of filarial infections in Simulium damnosum s.I. To identify proteases of S. damnosum s.I. induced by infection with Onchocerca ochengi, a PCR-based differential display technique was used. By combining this method with homology-based serine protease primers transcripts can be detected from S. damnosum s.I. RNA.

A simple field method for the purification of Onchocerca ochengi microfilariae from a mixed Onchocerca infection in cattle.

Onchocerca ochengi, a bovine parasite, is a suitable model for research on human River Blindness. However, the microfilariae are normally found concomitantly with at least one of the other three bovine Onchocerca species O. dukei, O. gutturosa and O. armillata causing difficulties for the work on the microfilariae. We describe a simple and field applicable method for the separation of living O. ochengi microfilariae from the other Onchocerca species using Sephadex G-25 columns. Elution of mixed populations resulted in the passage of O. gutturosa and/or O. dukei in the initial 1 ml fraction with O. ochengi eluting as an almost 100% pure species in the 4th and 5th fractions.

Full sequence and characterization of two insect defensins: immune peptides from the mosquito Aedes aegypti.

We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection. Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects. These mosquito peptides, designated Ae. aegypti defensins A and B, are isoforms. Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character. Apparent convergence between isoforms from Ae. aegypti and the fleshfly Phormia terranovae is discussed. The synergistic activity previously described between Ae. aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae. aegypti.

Brugia pahangi: the effects of cecropins on microfilariae in vitro and in Aedes aegypti.

Synthetic cecropins, antibacterial peptides from insect haemolymph, have been tested for their ability to attenuate the motility of microfilariae of the filarial nematode Brugia pahangi in an in vitro assay. Fifty micromolar concentrations of these peptides, equivalent to physiological concentrations in immune-stimulated insects, cause significant attenuation of motility compared with untreated microfilariae. Similar results were obtained with cecropins A and B. This is the lowest concentration for which cecropin has been reported to be active against eukaryote organisms. Antiserum to the cecropin homologue sarcotoxin 1A successfully blocked the observed activity. When the same concentration of cecropin B was coinjected with B. pahangi microfilariae into adult females of the mosquito, Aedes aegypti, a significant reduction in the numbers of developing larvae was observed.

The in vitro culture of Onchocerca spp.: II. The excretory/secretory products of the first and third stage larvae of Onchocerca lienalis and O. ochengi.

The developing first and third larval stages of two bovine Onchocerca species were maintained in vitro in the presence of 35S-methionine, following their initial development in the vector species Simulium ornatum s.1. to characterise the expression and secretion of their metabolites. The first larval stages of O. lienalis and O. ochengi did not release any E/S products. In contrast the supernatants of the third stage larvae of both species contained a double band of 21 and 20 kDa for O. ochengi and 23 and 22 kDa bands for O. lienalis when kept at room temperature. A temperature shift to 37 degrees C led to the increased expression of the 23 kDa protein with the infective larvae of O. lienalis. The possible role of these molecules is discussed.

The in vitro culture of Onchocerca spp.: I. Preliminary observations on the development of the second to the third stage larvae.

Second stage larvae of Onchocerca lienalis and O. gutturosa were induced to moult successfully to the third stage in vitro. Following development in their vectors Simulium ornatum s.l. and Culicoides nubeculosus the second stage larvae were kept in a cell free culture system. At least 25% of the second stage larvae of either Onchocerca species completed the moult successfully although no feeder layers had been used. In the absence of CO2 (5% in air) the number of the second stage larvae moulting successfully to the infective stage dropped to 6 and 9% respectively.

Mechanisms of resistance to Onchocerca infection in blackflies.

In this article Peter Ham, Hans Hagen, Andrea Baxter and Jorg Grunewald focus on the susceptibility of blackflies to parasitic filarial infection (particularly Onchocera spp, most of the vectors of which belong to the genus Simulium). They outline what is known about, as well as speculating on, the various defence mechanisms of these insects. Investigations have involved the use of natural and surrogate vectors of bovine onchocerciasis as models for the human vector-parasite relationship.

Concomitant malaria (Plasmodium gallinaceum) and filaria (Brugia pahangi) infections in Aedes aegypti: effect on parasite development.

Mixed infections with malarial (Plasmodium gallinaceum) and filarial (Brugia pahangi) parasites were carried out in 8 trials with filaria susceptible (REFM) and filaria refractory (REP-RR) Aedes aegypti strains. A secondary infection with B. pahangi microfilariae (mff) by intrathoracic inoculation, reduced the development rate of a pre-existing P. gallinaceum infection. The level of reduction ranged from 9.5 to 49% in REFM and from 50 to 90% in REP-RR. An immune response against oocysts was seen as melanization in mosquitoes with a double infection in the strain refractory to B. pahangi (REP-RR) and a reduction in oocyst size in both mosquito strains. Melanization was not observed in mosquitoes infected only with P. gallinaceum. This may indicate that activation of the prophenoloxidase (PPO) cascade in response to mff in the haemolymph can also be addressed against oocysts in the midgut. No significant difference in the number of filarial parasites recovered was observed when comparing groups with a single or double infection. Retardation in development of filaria larvae was observed in mosquitoes with double infection (REFM strain), together with melanization and a higher rate of abnormal development. Nutritional deficiency caused by superinfection might also be responsible for the delay in filarial development and reduced oocyst size.

Induction of the prophenoloxidase-activating system of Simulium (Diptera: Simuliidae) following Onchocerca (Nematoda: Filarioidea) infection.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

Purification of an insect defensin from the mosquito, Aedes aegypti.

Using a new, sensitive assay of bacterial growth inhibition, inducible antibacterial activity has been identified in the haemolymph of the mosquito, Aedes aegypti following inoculation with bacteria or with microfilariae of the filarial nematode Brugia pahangi, but not after inoculation with sterile culture medium. A lower level of antibacterial activity has also been observed in untreated individual mosquitoes. Following bacterial inoculation, a basic, inducible antibacterial peptide has been detected using native PAGE at pH 4, which corresponds with a 4.5 kDa peptide detected by tricine SDS-PAGE followed by silver staining. A peptide has been purified from immune haemolymph by ultrafiltration, followed by reversed-phase HPLC, yielding a single major peak with antibacterial activity. Partial amino acid sequence analysis of this fraction has revealed substantial homology with insect defensins. The data are consistent with the peptide being another member of this family, and we propose the name Aedes aegypti defensin.

Differential lectin binding of Onchocerca lienalis and Onchocerca ochengi infective larvae following their development in Simulium ornatum s.l.

Lectins have been used to investigate species specific differences in carbohydrate moieties on the surface of the infective larvae of two Onchocerca species following their development in Simulium ornatum. Of the seven FITC-labelled lectins used in this study only two, Arachis hypogea (PNA) and Helix pomatia (HPA), bound to the surface of O. lienalis, whereas no lectin binding could be detected on the surface of O. ochengi infective larvae. This indicates that in principal lectins might be a potential tool for the differentiation of more closely related Onchocerca species.

Approaches to vector control: new and trusted. 1. Humoral immune responses in blackfly and mosquito vectors of filariae.

The vectors of filariasis, mosquitoes and blackflies, are capable of mounting a defence response to the infection. This selective review describes the molecules that are involved in these immune systems. Several antibacterial peptides are known to be induced and secreted into the haemolymph by the fat body and the circulating haemocytes. In addition, haemagglutinating lectins with carbohydrate specificities to the surface of the developing filarial larvae appear. Activation of a range of proteases occurs rapidly as does activation of the prophenoloxidase pathway. The possible roles of these and other molecules is discussed, together with mention of a working hypothesis as to how these molecules may be regulated.