Effect of rearing temperature and larval density on larval survival, age at pupation and adult size of Anopheles gambiae

The effects of temperature and larval density on survival of larvae, growth rate, age at pupation, and adult size (measured as wing length and dry weight) of laboratory-reared Anopheles gambiae (Diptera: Culicidae) were studied. Larvae were reared at three temperatures (24, 27 and 30°C) and three densities (0.5, 1 and 2 larvae/cm²). The effects of density and temperature strongly interacted to determine the mosquitoes' life-history parameters. Survival was highest at the intermediate temperature of 27°C. The differences between the temperatures increased with increasing density. At 30°C survival decreased as density increased, but at 27°C increasing density led to higher survival. Age at pupation increased as temperature decreased from 30°C to 24°C and as density decreased from 2 to 0.5 larvae/cm². Adult size also increased as temperature decreased, but showed a negative correlation with density only at 27°C. In contrast, at 24°C and 30°C a decrease in density led to a decrease in adult size. Growth rate showed a similar pattern. At 27°C growth rate decreased as density increased, but at other temperatures the opposite trend was observed.     

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.     

Genetic differentiation at the 3'UTR of PROS-Ag25: a cDNA homologue of Drosophila melanogaster proteasome subunit PROS-Dm25 in the malaria vector Anopheles gambiae

The Anopheles gambiae cDNA encoding the homologue of the Drosophila melanogaster proteasome PROS-Dm25 was identified and analysed in terms of nucleotide sequence, and chromosomal localisation. In the 3 untranslated region, a GA-rich sequence was mapped which was found to be widely polymorphic among taxa belonging to the A. gambiae complex.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

Polymorphism of esterase 11 in Mus musculus,a further esterase locus on chromosome 8

A further esterase, esterase 11, which exhibits a polymorphism detectable by electrophoresis, has been observed in the house mouse, Mus musculus. In 15 inbred strains and two outbred strains, the ES-11A phenotype has been found, composed of two bands of enzyme activity of greater anodal electrophoretic mobility than the two bands of the ES-11B phenotype found in one inbred strain, one wild stock, and 101 wild mice. In F₁ hybrids (IS/Cam×C57 BL/Gr), the phenotype shown corresponds to a mixture of the two parental phenotypes. In backcrosses, ES-11 segregates as an autosomal gene, designated Es-11, closely linked to Es-2 and Es-5 on chromosome 8.This work was supported by the Medical Research Council.     

No evidence for biased co-transmission of speciation islands in Anopheles gambiae

Genome-scale scans have revealed highly heterogeneous levels of divergence between closely related taxa in many systems. Generally, a small number of regions show high differentiation, with the rest of the genome showing no or only low levels of divergence. These patterns have been interpreted as evidence for ongoing speciation-with-gene-flow, with introgression homogenizing the whole genome except loci involved in reproductive isolation. However, as the number of selected loci increases, the probability of introgression at unselected loci decreases unless there is a transmission ratio distortion causing an over-representation of specific combinations of alleles. Here we examine the transmission of three 'speciation islands' that contain fixed differences between the M and S forms of the mosquito, Anopheles gambiae. We made reciprocal crosses between M and S parents and genotyped over 2000 F(2) individuals, developing a hierarchical likelihood model to identify specific genotypes that are under- or over-represented among the recombinant offspring. Though our overall results did not match the expected number of F(2) genotypes, we found no biased co-transmission among M or S alleles in the three islands. Our likelihood model did identify transmission ratio distortion at two of the three islands, but this distortion was small (approx. 3%) and in opposite directions for the two islands. We discuss how our results impinge on hypotheses of current gene flow between M and S and ongoing speciation-with-gene-flow in this system.     

Ecological genetic studies in the chromosomal form Mopti ofAnopheles gambiae s.str. in Mali,West Africa

Among the sibling species of the AfrotropicalAnopheles gambiae complex, the nominal taxon (An. gambiae s.str.) is the major malaria vector. Its bionomics suggest a man-dependent speciation process which involves, in West Africa, various incipient species chromosomally recognized by different combinations of 2R paracentric inversions. One of the most recent evolutionary steps of such a speciation process appears to be the chromosomal form Mopti, which is associated with dry season irrigation in arid zones, and is characterized by a remarkable ecological flexibility related to three 2R alternative arrangements, namelybc, u and +, whose expected karyotypes are found in Hardy-Weinberg equilibrium. The study of this chromosomal polymorphism in samples from a 16-locality transect in Mali shows wide variations and highly significant correlation with both temporal and spatial climatic differences. Mosquitoes homokaryotypic for 2Rbc are the actual dry season and arid areas breeders. The regular rise of 2Rbc frequency, up to fixation, during each dry season, corresponds to the South-North clinal increase of the same arrangement along the transect, from about 30% in the humid savanna to near fixation in the South-Saharan zone. This coherent ecological genetics case provides full support to the hypothesis of the adaptive nature of paracentric inversions. Moreover, the very peculiar system of combinations of contiguous 2R inversions, utilized by Mopti as well as by other chromosomal forms ofAn. gambiae, suggests a process of polygenic reorganization based on linkage disequilibria and involving the inversions as driving selection units.     

Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae

Pegasus, a novel transposable element, was discovered as a length polymorphism in the white gene of Anopheles gambiae. Sequence analysis revealed that this 535 bp element was flanked by 8 bp target site duplications and 8 bp perfect terminal inverted repeats similar to those found in many members of the Tcl family. Its small size and lack of long open reading frames preclude protein coding capacity. Southern analysis and in situ hybridization to polytene chromosomes demonstrated that Pegasus occurs in approximately 30 copies in the genomes of An. gambiae and its sibling species and is homogenous in structure but polymorphic in chromosomal location. Characterization of five additional elements by sequencing revealed nucleotide identities of 95% to 99%. Of 30 Pegasus-containing phage clones examined by PCR, only one contained an element exceeding 535 bp in length, due to the insertion of another transposable element-like sequence. Thus, the majority, if not all, extant Pegasus elements may be defective copies of a complete element whose contemporary existence in An. gambiae is uncertain. No Pegasus-hybridizing sequences were detected in nine other anophelines and three culicines examined, suggesting a very limited taxonomic distribution.     

Energy metabolism affects susceptibility of Anopheles gambiae mosquitoes to Plasmodium infection

Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthermore, when state-3 respiration was reduced in S females by silencing expression of the adenine nucleotide translocator (ANT), hydrogen peroxide generation was higher and the mRNA levels of lactate dehydrogenase increased in the midgut, while the prevalence and intensity of Plasmodium berghei infection were significantly reduced. We conclude that there are broad metabolic differences between R and S An. gambiae mosquitoes that influence their susceptibility to Plasmodium infection.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae

Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.     

Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.     

The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.     

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae

Background

The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.

Results

Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both “ping-pong” dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.

Conclusions

We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1436-1) contains supplementary material, which is available to authorized users.     

Odor-mediated flight behavior ofAnopheles gambiae gilesSensu Stricto andAn. stephensi liston in response to CO2, acetone, and 1-octen-3-ol (Diptera: Culicidae)

The flight behavior of Anopheles gambiae s.s. Giles and An. stephensi Patton exposed to different odor cues was studied in a wind tunnel. Odors consisted of CO₂, CO₂ + acetone (at two concentrations), and CO₂ + 1-octen-3-ol. Mosquitoes were released singly and their behavior was recorded on video. Parameters studied included flight velocity, percentage of time spent flying, percentage of time spent in plume, and number of turns toward the plume. Large differences in behavior toward the odors tested were observed. An. gambiaedid not respond well to CO ₂,whereas An. stephansiwas positively affected by this compound. In contrast, An. gambiaeresponded significantly to CO ₂ + acetone (at a low concentration), but the behavior of An. stephensiwas completely suppressed by this combination of odor stimuli. CO ₂ + a high concentration of acetone or CO ₂ + 1-octen-3-ol did not cause significant effects in An. gambiaecompared to no odor, while these treatments elicited strong behavioral responses in An. stephensi.The latter species responded particularly well to CO ₂ + 1-octen-3-ol. The results suggest that the observed differences may be inherent to the known differences in host preferences, where An. gambiaeis highly anthropophilic and An. stephensimore zoophilic. This would explain why the latter species responds well to CO ₂ and even better to CO ₂ + 1-octen-3-ol, a compound readily emitted by bovine ruminants.     

Responses of Anopheles gambiae complex mosquitoes to the use of untreated bednets in The Gambia

Population dynamics of the Anopheles gambiae complex of malaria vector mosquitoes were studied in four small hamlets in The Gambia. Bednets were used to reduce man/vector contact in two of the hamlets. High densities of An. gambiae, sensu lato, were present for only 3-8 weeks during the rainy season, depending on the position of the hamlet within the study area. The proportions of blood-fed mosquitoes caught indoors (83.0%) and existing from houses (11.6%) were lower in hamlets where bednets were used than in hamlets without (96.5% and 33.1% respectively). Fewer of the blood-fed mosquitoes had fed on man in houses where people slept under bednets (68.2%) than in those without (81.5%). However, the average number of infective bites received by children was still greater than one a year in hamlets where bednets were used. Consequently bednets are considered unlikely to be an effective malaria control measure so long as they are untreated with insecticide.     

Rural Development and Malaria Control in Sub-Saharan Africa

The rapidly expanding population of Sub-Saharan Africa has led to an increased demand for land in which to live and grow food. The process of rural development continues to change the physical landscape, increasing mosquito breeding and biting rates of the chief vector of malaria in Africa, Anopheles gambiae, a mosquito exquisitely adapted for exploiting people. At the same time, development alters the social environment, affecting wealth, inequality, household entitlements, and male and female workloads, which lead to changes in coping and caring strategies. Despite the fact that malaria is sensitive to changes in the physical and social environment, most control tools use only chemicals (antimalarials and insecticides), not biophysical environmental modifications nor strengthening social systems. While antimalarials and insecticides are extremely effective weapons, they are probably not sustainable in the long term due to the emergence of resistant organisms. Here we suggest that environmental and social management should be considered as part of the suite of interventions against malaria, since these are likely to be effective in specific settings and represent a sustainable approach to malaria control in rural Africa.