Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

Isozyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia

Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on UPGMA methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright's F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.     

Characterization of putative hydrophobic substrate binding site residues of a Delta class glutathione transferase from Anopheles dirus

To date, investigations of the hydrophobic substrate site of the insect Delta class glutathione transferase are limited in number. In the present study, putative hydrophobic site residues of AdGSTD4-4 have been proposed and characterized. These residues are Gln-112, Thr-174, Phe-212, Arg-214, Tyr-215 and Phe-216. It was found that Gln-112 does not contribute significantly to the catalytic properties of AdGSTD4-4. Arg-214, Tyr-215 and Phe-216 made contributions to catalytic properties and the rate-limiting step. Thr-174 and Phe-212 appeared to be important in enzymatic catalysis by stabilizing the active site β1-α1 loop on which the critical catalytic residue Ser-9 is located. The aromatic Phe-212 pi cloud appears to be important for interactions with its hydrophobic size representing an almost equally important factor. The data suggests that these residues are not directly involved in catalysis but exert their influence through secondary interactions. In addition, active site rearrangements occur to bring different residues into play even for conjugation through the same mechanisms. Therefore, due to the conformational rearrangements topologically equivalent residues observed in crystal structures may not perform equivalent roles in catalysis in different GST classes.     

Toxin-binding proteins from midgut epithelium membranes of Anopheles stephensi larvae

Proteins of 65 and 57 kD were isolated from the apical membranes of midgut epithelium of Anopheles stephensi larvae by affinity chromatography. These proteins can specifically bind endotoxin Cry11A and activate toxin Cry4B (Cry4B-tox) under conditions of ligand blotting, and both Cry proteins compete for this binding. At least in the case of Cry4B-tox, the binding with 65 and 57 kD proteins is reversible. The ability of the products of limited proteolysis of Cry11A and Cry4B to bind the 65 and 57 kD proteins correlates with their toxicity to A. stephensi larva. The N-terminal amino acid sequence of the 57 kD protein is unique and absent in the NCBI GenBank. The proteins of 65 and 57 kD share most of the properties studied with Aedes aegypti toxin-binding proteins. It is possible that they altogether represent a novel class (or classes) of delta-endotoxin receptors.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.     

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.     

大劣按蚊吸血活动与交配受精的关系

大劣按蚊Anophelos dirus是我国及东南亚地区的重要传疟媒介。研究表明成蚊羽化后,在未喂血的情况下,可有较高的交配受精率。在同一蚊笼,吸血机会完全相同的条件下,大蚊笼受精雌蚊吸血率为59.88%,未受精雌蚊的吸血率为35.38%;小蚊笼受精雌蚊的吸血率为74.13%,未受精雌蚊吸血率仅为43.56%。正常交配雌蚊的吸血率为49.71%,未进行交配的处女雌蚊吸血率为28.42%。同为正常交配蚊群,喂血组雌蚊受精率55.85%,不喂血组的受精率为44.51%。初步认为大劣按蚊的吸血活动与交配受精有一定的关系。     

Mass treatment with ivermectin for filariasis control in Papua New Guinea: impact on mosquito survival

Field studies were carried out to determine the impact of mass human treatment with ivermectin on the survival of anthropophagic mosquitoes of the Anopheles punctulatus complex (Diptera: Culicidae), the vectors of lymphatic filariasis and malaria in Papua New Guinea. In a village where mass treatment had been given, using 400 microg/kg ivermectin plus 6 mg/kg diethylcarbamazine citrate (DEC), we performed pre- and post-treatment collections of freshly blood-engorged mosquitoes from the same nine bedrooms. All blood-fed mosquitoes collected less than 4 days after mass treatment died within 9 days, whereas 67% of those collected before treatment survived for >9 days. Comparison (using the log-rank test) of the survival curves for mosquitoes collected (i) before treatment, (ii)<4 days after treatment, and (iii) 28 days after treatment, showed the survival rate of group (ii) to be significantly lower than the other two (chi2=176, df=2, P<0.0001). Pre- and post-treatment all-night landing catches showed no reduction in human biting rates in the experimental village. In another village, where people were mass treated with ivermectin (400 microg/kg) only, the survival rates of freshly blood-engorged An. punctulatus collected from bedroom resting-sites less than 1 day after treatment, were compared to similar collections carried out at the same time in a nearby village where people were not treated with ivermectin. The 48-h survival rate for the ivermectin-treated village was 31% compared to 94% for the other; this difference was highly significant (chi2=32.42, df=1, P<0.0001). Mosquitoes fed 2 months post-treatment with DEC or collected 38 days post-treatment with ivermectin had normal survival rates. We conclude that the duration of the systemic lethal effect of ivermectin on mosquitoes is insufficient to be of epidemiological significance in filariasis control programmes that are based on biannual and annual single-dose treatments, but might reduce vectorial capacity sufficiently to block epidemics of dengue or even malaria.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.