Identification of Rhipicephalus microplus genes that modulate the infection rate of the rickettsia Anaplasma marginale.

Arthropod vectors transmit a diversity of animal and human pathogens, ranging from RNA viruses to protozoal parasites. Chemotherapeutic control of pathogens has classically focused either on insecticides that kill the vector itself or antimicrobials for infected patients. The limitation of the former is that it targets both infected and uninfected vectors and selects for resistant populations while the latter requires prompt and accurate diagnosis. An alternative strategy is to target vector molecules that permit the pathogen to establish itself, replicate, and/or develop within the vector. Using the rickettsial pathogen Anaplasma marginale and its tropical tick vector, Rhipicephalus microplus, as a model, we tested whether silencing specific gene targets would affect tick infection rates (the % of fed ticks that are infected with the pathogen) and pathogen levels within infected ticks. Silencing of three R. microplus genes, CK187220, CV437619 and TC18492, significantly decreased the A. marginale infection rate in salivary glands, whereas gene silencing of TC22382, TC17129 and TC16059 significantly increased the infection rate in salivary glands. However in all cases of significant difference in the infection rate, the pathogen levels in the ticks that did become infected, were not significantly different. These results are consistent with the targeted genes affecting the pathogen at early steps in infection of the vector rather than in replication efficiency. Identifying vector genes and subsequent determination of the encoded functions are initial steps in discovery of new targets for inhibiting pathogen development and subsequent transmission.

Gene flow pattern among Aedes aegypti populations in Mexico.

Patterns of gene flow vary greatly among Aedes aegypti populations throughout Mexico. The populations are panmictic along the Pacific coast, isolated by distance in northeast Mexico, and exhibit moderate gene flow across the Yucatan peninsula. Nine Ae. aegypti collections from 6 cities in Oaxaca, Mexico, were taken to examine the local patterns of gene flow. Genetic variation was examined in a 387-bp region of the nicotinamide adenine dinucleotide dehydrogenase subunit 4 mitochondrial gene (ND4) using single-strand conformation polymorphism analysis, and 3 haplotypes were detected. Cluster analysis on the linearized FST genetic distances failed to group collections in geographic proximity. Regression analysis of linear or road distances on linearized F(ST) indicated that proximal collections were as diverse as distant collections across an approximately 800-km range. The geographical distribution of the Mexican mosquito haplotype frequencies was determined for the ND4 sequences from 524 individuals from Oaxaca (this study) and 2,043 individuals from our previous studies. Herein, we report on yet another pattern dominated by genetic drift among 9 Ae. aegypti collections from 6 cities in Oaxaca, Mexico, and compare it to those reported in other regions of Mexico. Molecular analysis of variance showed that there was as much genetic variation among collections 4 km apart as there was among all collections. The numbers of haplotypes and the amount of genetic diversity among the collections from Oaxaca were much lower than detected in previous studies in other regions of Mexico and may reflect the effects of control efforts or adaptations to the altitudinal limits (1,500 m) of the species in Mexico. The geographical distribution of mosquito haplotypes in Mexico is also reported. Furthermore, based on the distribution of the mosquito haplotypes in America, we suggest that mosquito dispersion is very efficient, most likely due to commercial transportation.

Temporal characterisation of the organ-specific Rhipicephalus microplus transcriptional response to Anaplasma marginale infection.

Arthropods transmit important infectious diseases of humans and animals. Importantly, replication and the development of pathogen infectivity are tightly linked to vector feeding on the mammalian host; thus analysis of the transcriptomes of both vector and pathogen during feeding is fundamental to understanding transmission. Using Anaplasma marginale infection of Rhipicephalus microplus as the experimental model, we tested three hypotheses exploring the temporal and organ-specific nature of the tick midgut and salivary gland transcriptomes during feeding and in response to infection. Numerous R. microplus genes were regulated in response to feeding and were differentially regulated between the midgut and salivary gland; additionally, there was a progression in regulated gene expression in the salivary gland over time. In contrast, relatively few tick genes were specifically regulated in response to A. marginale infection and these genes were predominantly annotated as hypothetical or were of unknown function. Notable among the genes with informative annotation was that several ribosomal proteins were down-regulated, suggesting that there may be a corresponding decrease in translation. The hypotheses that R. microplus midgut and salivary gland genes are differentially regulated and that the salivary gland transcriptome is dynamic over time were accepted. This is consistent with, and important for understanding the roles of, the two organs, the midgut serving as an initial site of uptake and replication while the salivary gland serves as the final site of replication and secretion. The nominal effect of A. marginale on the tick transcriptome in terms of numbers of regulated genes and fold of regulation supports the view that the vector-pathogen relationship is well established with minimal deleterious effect on the tick. The small set of predominantly hypothetical genes regulated by infection suggests that A. marginale is affecting a novel set of tick genes and may provide new opportunities for blocking transmission from the tick.

A dengue receptor as possible genetic marker of vector competence in Aedes aegypti.

BACKGROUND: Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells. RESULTS: (1) We determined the MG proteins that bind DENV by an overlay protein binding assay (VOPBA) in Ae. aegypti mosquitoes of the DS3, DMEB and IBO-11 strains. The main protein identified had an apparent molecular weight of 67 kDa, although the protein identified in the IBO-11 strain showed a lower mass (64 kDa). (2) The midgut proteins recognized by DENV were also determined by VOPBA after two-dimensional gel electrophoresis. (3) To determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against R67 and R64 and tested them against the three strains by immunoblotting; both antibodies recognized the 67 and 64 kDa proteins, corroborating the VOPBA results. (4) Specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the MG, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut (pPMG) in the neighborhood of the hindgut. (5) Quantitative analysis showed more intense fluorescence in DS3 and DMEB than in IBO-11. (6) The viral envelope antigen was not homogeneously distributed during MG infection but correlated with MG density and the distribution of R67/R64. CONCLUSION: In this paper we provide evidence that the 67 kDa protein (R67/R64), described previously as a DENV receptor, is related to vector competence in Ae. aegypti. Consequently, our results strongly suggest that this protein may be a marker of vector competence for DENV in Ae. aegypti mosquitoes.

The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells.

BACKGROUND: Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. RESULTS: (1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. CONCLUSION: Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.