Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi

<p>Abstract</p> <p>Background</p> <p><it>Brugia malayi </it>and <it>B. pahangi </it>are two closely related nematodes that cause filariasis in humans and animals. However, <it>B. pahangi </it>microfilariae are able to develop in and be transmitted by the mosquito, <it>Armigeres subalbatus</it>, whereas most <it>B. malayi </it>are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the <it>B. malayi </it>V2 array was carried out to determine the transcriptional differences between <it>B. malayi </it>and <it>B. pahangi </it>microfilariae with similar age distribution.</p> <p>Results</p> <p>Following microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in <it>B. pahangi </it>microfilariae and 159 probes, representing 107 genes, in <it>B. malayi </it>microfilariae. In <it>B. pahangi</it>, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in <it>B. malayi </it>microfilariae. The remaining 47 (38%) transcripts in <it>B. pahangi </it>and 46 (43%) transcripts in <it>B. malayi </it>microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in <it>B. pahangi </it>microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in <it>B. malayi </it>microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible.</p> <p>Conclusion</p> <p>In this study, we identified gene transcripts that were preferentially expressed in the microfilariae of <it>B. pahangi </it>and <it>B. malayi</it>, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to researchers keen on understanding the inherent differences, at the molecular level, between <it>B. malayi </it>and <it>B. pahangi </it>microfilariae especially because these microfilariae are capable of surviving in the same vertebrate host but elicit different immune response outcomes in the mosquito, <it>Ar. subalbatus</it>.</p

Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development.</p> <p>Methods</p> <p>The <it>P. falciparum </it>gene, <it>PF11_0394</it>, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR) and green fluorescent protein (GFP)-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined.</p> <p>Results</p> <p>The PF11_0394 protein has orthologs in other <it>Plasmodium </it>species and Apicomplexans, but none outside of the group Apicomplexa. <it>PF11_0394 </it>transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites.</p> <p>Conclusions</p> <p>PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in <it>P. falciparum </it>biology, it could be another candidate for a new vaccine.</p

Tabulation, bibliography, and structure of binary intermetallic compounds. II. Compounds of berylium, magnesium, and calcium

This is a tabulation, bibliography, and structure of binary intermetallic compounds, including those of berylium, magnesium, and calcium. This report is the second in a series. ISC-795 ~ the first in this series~ listed the compounds of lithium, sodium, potassium, and rubidium

Global identification of bursicon-regulated genes in Drosophila melanogaster

Abstract Background Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation. Results We used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster) that received recombinant bursicon (r-bursicon). Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes). Analysis of these genes by inference from the fly database http://flybase.bio.indiana.edu revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR) which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data. Conclusion Using r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. The data obtained here will form the basis for future studies aimed at elucidating the exact mechanisms upstream from the secretion of bursicon and its binding to target cells

Phenoloxidase activity acts as a mosquito innate immune response against infection with semliki forest virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses

Mosquito Infection Responses to Developing Filarial Worms

Human lymphatic filariasis is a mosquito-vectored disease caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. These are relatively large roundworms that can cause considerable damage in compatible mosquito vectors. In order to assess how mosquitoes respond to infection in compatible mosquito-filarial worm associations, microarray analysis was used to evaluate transcriptome changes in Aedes aegypti at various times during B. malayi development. Changes in transcript abundance in response to the different stages of B. malayi infection were diverse. At the early stages of midgut and thoracic muscle cell penetration, a greater number of genes were repressed compared to those that were induced (20 vs. 8). The non-feeding, intracellular first-stage larvae elicited few differences, with 4 transcripts showing an increased and 9 a decreased abundance relative to controls. Several cecropin transcripts increased in abundance after parasites molted to second-stage larvae. However, the greatest number of transcripts changed in abundance after larvae molted to third-stage larvae and migrated to the head and proboscis (120 induced, 38 repressed), including a large number of putative, immunity-related genes (∼13% of genes with predicted functions). To test whether the innate immune system of mosquitoes was capable of modulating permissiveness to the parasite, we activated the Toll and Imd pathway controlled rel family transcription factors Rel1 and Rel2 (by RNA interference knockdown of the pathway's negative regulators Cactus and Caspar) during the early stages of infection with B. malayi. The activation of either of these immune signaling pathways, or knockdown of the Toll pathway, did not affect B. malayi in Ae. aegypti. The possibility of LF parasites evading mosquito immune responses during successful development is discussed

A resting box for outdoor sampling of adult Anopheles arabiensis in rice irrigation schemes of lower Moshi, northern Tanzania

Malaria vector sampling is the best method for understanding the vector dynamics and infectivity; thus, disease transmission seasonality can be established. There is a need to protecting humans involved in the sampling of disease vectors during surveillance or in control programmes. In this study, human landing catch, two cow odour baited resting boxes and an unbaited resting box were evaluated as vector sampling tools in an area with a high proportion of Anopheles arabiensis, as the major malaria vector. Three resting boxes were evaluated against human landing catch. Two were baited with cow odour, while the third was unbaited. The inner parts of the boxes were covered with black cloth materials. Experiments were arranged in latin-square design. Boxes were set in the evening and left undisturbed; mosquitoes were collected at 06:00 am the next morning, while human landing catch was done overnight. A total of 9,558 An. arabiensis mosquitoes were collected. 17.5% (N = 1668) were collected in resting box baited with cow body odour, 42.5% (N = 4060) in resting box baited with cow urine, 15.1% (N = 1444) in unbaited resting box and 24.9% (N = 2386) were collected by human landing catch technique. In analysis, the house positions had no effect on the density of mosquitoes caught (DF = 3, F = 0.753, P = 0.387); the sampling technique had significant impact on the caught mosquitoes densities (DF = 3, F 37. 944, P < 0.001). Odour-baited resting boxes have shown the possibility of replacing the existing traditional method (human landing catch) for sampling malaria vectors in areas with a high proportion of An. arabiensis as malaria vectors. Further evaluations of fermented urine and longevity of the urine odour still need to be investigated

Evidence of two lineages of the dengue vector Aedes aegypti in the Brazilian Amazon, based on mitochondrial DNA ND4 gene sequences

Genetic variation was estimated in ten samples populations of Aedes aegypti from the Brazilian Amazon, by using a 380 bp fragment of the mitochocondrial NADH dehydrogenase subunit 4 (ND4) gene. A total of 123 individuals were analyzed, whereby 13 haplotypes were found. Mean genetic diversity was slightly high (h = 0.666 ± 0.029; π = 0.0115 ± 0.0010). Two AMOVA analyses indicated that most of the variation (~70%-72%) occurred within populations. The variation found among and between populations within the groups disclosed lower, but even so, highly significant values. FST values were not significant in most of the comparisons, except for the samples from Pacaraima and Rio Branco. The isolation by distance (IBD) model was not significant (r = 0.2880; p = 0.097) when the samples from Pacaraima and Rio Branco were excluded from the analyses, this indicating that genetic distance is not related to geographic distance. This result may be explained either by passive dispersal patterns (via human migrations and commercial exchange) or be due to the recent expansion of this mosquito in the Brazilian Amazon. Phylogenetic relationship analysis showed two genetically distinct groups (lineages) within the Brazilian Amazon, each sharing haplotypes with populations from West Africa and Asia

Midgut Barrier Imparts Selective Resistance to Filarial Worm Infection in Culex pipiens pipiens

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro