Browsing by Author "Masiga, Daniel K"

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Aquaporins Are Critical for Provision of Water during Lactation and Intrauterine Progeny Hydration to Maintain Tsetse Fly Reproductive Success
(2014) Benoit, Joshua B; Hansen, Immo A; Attardo, Geoffrey M; Michalková, Veronika; Mireji, Paul O; Bargul, Joel L; Drake, Lisa L; Masiga, Daniel K; Aksoy, Serap
Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4–6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20–25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.
Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans
(2014) Obiero, George FO; Mireji, Paul O; Nyanjom, Steven RG; Christoffels, Alan; Robertson, Hugh M; Masiga, Daniel K
Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO2 were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.
RESEARCH Open Access High-resolution melting analysis reveals low Plasmodium parasitaemia infections among microscopically negative febrile patients in western Kenya
(2014) Kipanga, Purity N; Omondi, David; Mireji, Paul O; Sawa, Patrick; Masiga, Daniel K; Villinger, Jandouwe
Background: Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting. Methods: Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections. Results: The coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials. Conclusions: The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions
RESEARCH Open Access Sex-specific induction of CYP6 cytochrome P450 genes in cadmium and lead tolerant Anopheles gambiae
(2013) Musasia, Fauzia K; Isaac, Alfred O; Masiga, Daniel K; Omedo, Irene A; Ochieng, Richard; Mireji, Paul O; Mwakubambanya, Ramadhan
Background: Anopheles gambiae, one of the main Afro-tropical mosquito vector of malaria, has adapted to heavy metals in its natural habitat, and developed resistance to most conventional insecticides. Investigations were conducted to establish an association between tolerance to cadmium or lead-heavy metals, and expression of specific genes for cytochrome p450 enzymes associated with pyrethroid resistance in the mosquito. Methods: Juvenile aquatic stages of the mosquito were selected for tolerance to cadmiun or lead through chronic exposure of the stages to maximum acceptable toxicant concentrations (MATCs) of the metals. Using real-time quantitative polymerase chain reaction (qPCR), three replicates each of male or female cadmium or lead-tolerant individuals and relevant controls were separately screened for expression of CYP6M2 , CYP6P3 and CYP6Z1 genes. The variance in expression levels of the genes amongst the treatments was compared by ANOVA statistical tool. Results: Expressions of all the genes were significantly lower (P <0.05) in females than in males. Within gender, there 1.3 - 2.3 or 3.1-4.2-fold reduction in expression of the genes in cadmium or lead selected than respective control populations. Expression of all the classes of gene was elevated in cadmium selected female populations relative to their respective controls. Conclusion: These findings suggest that tolerance to cadmium or lead in the mosquito can influence response in cytochrome p450 genes associated with metabolism of pyrethroids in the mosquito in a sex-specific manner. This can, in turn, affect sensitivity of the mosquito to pyrethroids and other xenobiotics associated with these genes, with potential implications in mosquito vector control operations.