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PubMed2000

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae.

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.

Amino Acid Sequence↗
PubMed2000

The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response.

Although Drosophila possesses potent immune responses, little is known about the microbial pathogens that infect Drosophila. We have identified members of the bacterial genus Erwinia that induce the systemic expression of genes encoding antimicrobial peptides in Drosophila larvae after ingestion. These Erwinia strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts. Erwinia infections induce an antimicrobial response in Drosophila larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after Erwinia infection is reduced in two Drosophila mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating Drosophila antimicrobial responses and also illustrating that this Drosophila-Erwinia interaction provides a powerful model for dissecting host-pathogen relationships.

Animals↗
PubMed1995

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.

Aedes↗
PubMed1986

Regulation of expression of the wound tumor virus genome in persistently infected vector cells is related to change in translational activity of viral transcripts.

The interaction between a plant virus and its insect vector was studied at the molecular level by examining wound tumor virus (WTV) gene expression in cultured cells derived from its leafhopper vector. Infection of vector cells by WTV is noncytopathic and results in an acute phase (through day 5), followed by persistence beginning with the first cell passage. Viral-specific polypeptide synthesis and viral genome RNA accumulation increased to a maximum level during the first 5 days following inoculation and then decreased as infected cells were passaged (to 5 to 20% of the level observed during the acute phase by passages 10 to 15). In contrast, viral-specific mRNAs were present at approximately the same level in the acute phase and in the early stage (passage 10) of the persistent phase of infection. Although viral transcripts isolated at different times after inoculation exhibited identical electrophoretic migration patterns, they had different functional activities in cell-free translation systems. Transcripts isolated from persistently infected cells were inefficiently translated in vitro, reflecting the situation in infected cells. These results indicate that the decline in the level of viral polypeptide synthesis associated with the persistent phase of WTV infection is related to a change in the translational activity of viral transcripts.

Cells, Cultured↗
PubMed1995

Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.

Amino Acid Sequence↗
PubMed1992

Can transposable elements be used to drive disease refractoriness genes into vector populations?

A number of biological procedures are currently being considered as alternatives to insecticide-based methods for the control of insect vectors of disease. Among these are the adaptation of various genetic mechanisms to drive genes of interest, such as refractoriness to malaria in mosquitoes, into natural populations, for vector control purposes. Here, Margaret Kidwell and Jose Ribeiro develop a rationale for the possible use of transposable genetic elements, one of these potential drive mechanisms, and some of the problems being faced in seeking to determine the feasibility of such a strategy are described.

Journal Article↗
PubMed2006

Detection and titration of bluetongue virus in Culicoides insect cell culture by an antigen-capture enzyme-linked immunosorbent assay.

Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. sonorensis may be useful for virus isolation and studies to better understand BTV replication in the insect vector. However, their use is hampered because BTV infection does not produce significant cytopathic effects in these insect cell cultures. Detection of virus replication in these cells typically requires co-cultivation with susceptible vertebrate cell culture. This report describes the use of an antigen-capture enzyme-linked immunosorbent assay (Ag-Cap ELISA) for direct detection and titration of BTV in cultures of a Culicoides cell line. This assay should facilitate use of this cell line for virus isolation, titration and studies of BTV replication.

Animals↗
PubMed2005

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine.

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.

Animals↗
PubMed1993

Patterns of erythrocyte digestion by bloodsucking insects: constraints on vector competence.

Two general patterns of erythrocyte digestion were observed in representative species from four insect orders. Ingested erythrocytes were hemolyzed rapidly, and blood meals remained liquefied within body lice, Pediculus humanus L. and the fleas Ctenocephalides felis (Bouché) and Xenopyslla cheopis (Rothschild). Peritrophic membrane was absent. In contrast, there was a lag time of 6-18 h before substantial degradation of erythrocytes within the blood meals of bed bugs, Cimex lectularius L.; the sand fly Phlebotomus papatasi Scopoli; and the mosquitoes Anopheles stephensi Liston and Culex pipiens L. Blood meals of sand flies and mosquitoes were clotted and surrounded by peritrophic membrane at 18-24 h after feeding. Clotting and peritrophic membrane were less pronounced in bed bugs. It is proposed that acquisition and maintenance of pathogen types (i.e., prokaryotic versus eukaryotic) within insects are constrained by the general pattern of bloodmeal processing.

Animals↗
PubMed1987

Trypanosoma musculi: influence of uric acid on in vitro formation of metacyclic trypomastigotes.

Uric acid is the most important constituent in the urine of insects. Infective metacyclic forms of the stercorarian trypanosomes are produced in the rectum of their insect vector and are thus in contact with uric acid. Using a culture system which permitted the growth of the insect stages of Trypanosoma musculi, we studied the influence of uric acid on the metacyclogenesis of this parasite. When added to the culture, uric acid enhanced the production of metacyclic forms. Furthermore, it rendered these trypanosomes highly infective by the oral route. It is suggested that uric acid may play an important role on the metacyclogenesis of T. musculi.

Animals↗
PubMed2002

Vaccination against Leishmania infections.

Leishmaniasis, that affects millions of people worldwide, is an infectious disease caused by the protozoan parasite Leishmania. Incidence of the condition appears to be increasing in several parts of the world. Of the three main presentations of the disease, i.e. cutaneous, mucocutaneous and visceral, only the first one tends to heal spontaneously, while the other two are considered fatal if left to run their natural course. Recovery from leishmaniasis, whether spontaneous or drug-induced, is usually accompanied by solid immunity against reinfection, which provides a rationale for attempting to design vaccines against the disease. This review presents an outline of the main immunological features of Leishmania infections and of the mechanisms thought to operate in recovery from the disease. It describes various experimental approaches to vaccination in man and animal models, including the use of virulent and avirulent organisms, of dead parasites and extracts thereof, and of purified parasite proteins. Assays using novel technologies, such as the direct injection of DNAs encoding parasite proteins, or the inoculation of viral or bacterial vectors expressing such molecules, as well as recent experiments aimed at inducing an immune response against saliva of the insect vector, are also reviewed. Observations made during the course of these studies have reinforced the notion that vaccination against leishmaniasis is indeed feasible. However, in spite of intensive efforts by many groups and many reports of success in man and in animal models, a consensus is yet to emerge as to what constitutes the best approach to vaccination against leishmaniasis.

Animals↗
PubMed1992

Surface-associated antigens of Brugia malayi L2 and L3 parasites during vector-stage development.

Surface and metabolic labeling procedures were used to characterize the composition and the time of expression of Brugia malayi L2 and L3 surface-associated molecules as the larvae develop within the mosquito vector. Larvae were harvested from mosquito tissues at 5 (early L2), 8 (late L2) and 11 (L3) days post-infection and labeled with 125I-Iodo-Gen. The results of one-dimensional analysis showed that there is a progressive increase in the complexity of peptides associated with the surface of developing larvae, culminating in the expression of 7 major labeled components on L3s. Both L2 and L3 parasites have surface-associated components of 42, 35, 33, 19 and 17 kDa. Between days 8 and 11 of development in the insect vector, Brugia malayi undergoes the L2 to L3 molt and acquires additional major immunogenic peptides of 40 and 22 kDa. Two-dimensional analyses of extracts from 125I-labeled L2s and L3s revealed that the major 35-, 33-, 19- and 17-kDa molecules are part of a peptide complex that forms a 'ladder' between 17 and 150 kDa. To gain information on the times during which the major surface-associated molecules are produced by the parasite, larvae were labeled with [35S]methionine either in situ as they developed within the mosquito or during culture after exiting the vector. For in situ labeling, [35S]methionine was introduced into the hemolymph of infected mosquitoes by micro-injection at days 2, 5 and 8 post-infection and the larvae were allowed to develop for an additional 3 days. The results of 1- and 2-dimensional analyses of [35S]methionine-labeled extracts from vector-stage or post-vector-stage larvae indicate that the molecules associated with the surface of B. malayi L3s are synthesized between day 5 and day 11 of development in the insect host. Immediately after the larvae exit the vector, the synthesis of the 40 and 22-kDa peptides is drastically reduced or terminated.

Animals↗
PubMed2002

Differentially expressed midgut transcripts in Culicoides sonorensis (Diptera: ceratopogonidae) following Orbivirus (reoviridae) oral feeding.

Understanding the vector insect's gene expression response to a virus infection may aid design of control measures for arbovirus diseases. Culicoides sonorensis is a vector of several agriculturally important pathogens, such as epizootic haemorrhagic disease virus (EHDV) that causes disease in ruminants. Two approaches, differential display and suppression subtractive hybridization, were used to identify 400+ Culicoides transcripts that were more abundant in midguts 1 day following an oral meal containing EHDV. Of these, quantitative PCR confirmed seven to be more abundant in virus-fed midguts than controls. One such transcript encodes a putative RNA editase, CsRED1, induced by dsRNA. Transcripts encoding putative receptors involved in cell differentiation included CsLAR, a protein tyrosine phosphatase, and CsFZ2, homologous to the wingless receptor in D. melanogaster. Transcripts encoding putative translation machinery components included CseIF3, CseIF5A and CsRPS6. Overall, the cDNA fragments identified in this study increased in the midgut at one day postfeeding; by 2 days postfeeding, increases in transcript levels shifted from the midgut to the remainder of the infected midge.

Animal Feed↗
PubMed1996

Extensive chromosome aberrations in Spiroplasma citri Strain BR3.

Genetic variations in the plant pathogen, Spiroplasma citri strain BR3, were characterized through physical genome mapping of the original isolate, BR3-3X, and two derivatives, BR3-T and BR3-G, obtained after several years of different maintenance conditions. BR3-T was transmitted from plant to plant via its natural insect vector, the leafhopper Circulifer tenellus, while BR3-G was maintained only in plants by periodic grafting and has lost its ability to be insect transmitted. By pulsed field gel electrophoresis (PFGE) analysis and DNA hybridization, extensive changes in chromosomal DNA restriction patterns relative to the parent, BR3-3X, were observed in both BR3-T and BR3-G, each of which also had a larger genome size than the parent line. Genetic organization was relatively conserved between BR3-T and BR3-3X. In contrast, a large chromosomal inversion and deletions of approximately 10 kb near each of the inversion borders were observed in BR3-G. One of the deletions, which included several possibly functional genes, was closely linked to a SpV1-related transposase gene. The locations of the deletion borders were also determined. The results of this study demonstrated remarkable genome instability of spiroplasmas.

Amino Acid Sequence↗
PubMed1993

Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms.

A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.

Adenylyl Cyclases↗
PubMed2004

Selection pressures in the capsid genes of plant RNA viruses reflect mode of transmission.

To determine the selection pressures faced by RNA viruses of plants, patterns of nonsynonymous (dN) and synonymous (dS) substitution in the capsid genes of 36 viruses with differing modes of transmission were analysed. This analysis provided strong evidence that the capsid proteins of vector-borne plant viruses are subject to greater purifying selection on amino acid change than those viruses transmitted by other routes and that virus-vector interactions impose greater selective constraints than those between virus and plant host. This could be explained by specific interactions between capsid proteins and cellular receptors in the insect vectors that are necessary for successful transmission. However, contrary to initial expectations based on phylogenetic relatedness, vector-borne plant viruses are subject to weaker selective constraints than vector-borne animal viruses. The results suggest that the greater complexity involved in the transmission of circulative animal viruses compared with non-circulative plant viruses results in more intense purifying selection.

Animals↗