Feeding patterns of molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in a region of high hybridization.

BACKGROUND: Two biological forms of the mosquito Culex pipiens s.s., denoted pipiens and molestus, display behavioural differences that may affect their role as vectors of arboviruses. In this study, the feeding patterns of molestus and pipiens forms were investigated in Comporta (Portugal), where high levels of inter-form admixture have been recorded. METHODS: Indoor and outdoor mosquito collections were performed in the summer of 2010. Collected Cx. pipiens s.l. females were molecularly identified to species and form by PCR and genotyped for six microsatellites. The source of the blood meal in post-fed females was determined by ELISA and mitochondrial DNA sequencing. RESULTS: The distribution of the forms differed according to the collection method. The molestus form was present only in indoor collections, whereas pipiens and admixed individuals were sampled both indoors and outdoors. In both forms, over 90% of blood meals were made on avian hosts. These included blood meals taken from Passeriformes (Passer domesticus and Turdus merula) by females caught resting inside domestic shelters. CONCLUSION: Genetic structure and blood meal analyses suggest the presence of a bird biting molestus population in the study area. Both forms were found to rest indoors, mainly in avian shelters, but at least a proportion of females of the pipiens form may bite outdoors in sylvan habitats and then search for anthropogenic resting sites to complete their gonotrophic cycle. This behaviour may potentiate the accidental transmission of arboviruses to humans in the region.

The integrity of the glycine co-agonist binding site of N-methyl-D-aspartate receptors is a functional quality control checkpoint for cell surface delivery.

N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an approximately 3 x 10(4) decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.