Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Resisting infection by Plasmodium berghei increases the sensitivity of the malaria vector Anopheles gambiae to DDT

BACKGROUND The evolution of insecticide resistance threatens current malaria control methods, which rely heavily on chemical insecticides. The magnitude of the threat will be determined by the phenotypic expression of resistance in those mosquitoes that can transmit malaria. These differ from the majority of the mosquito population in two main ways; they carry sporozoites (the infectious stage of the Plasmodium parasite) and they are relatively old, as they need to survive the development period of the malaria parasite. This study examines the effects of infection by Plasmodium berghei and of mosquito age on the sensitivity to DDT in a DDT-resistant strain of Anopheles gambiae. METHODS DDT-resistant Anopheles gambiae (ZANU) mosquitoes received a blood meal from either a mouse infected with Plasmodium berghei or an uninfected mouse. 10 and 19 days post blood meal the mosquitoes were exposed to 2%, 1% or 0% DDT using WHO test kits. 24 hrs after exposure, mortality and Plasmodium infection status of the mosquitoes were recorded. RESULTS Sensitivity to DDT increased with the mosquitoes' age and was higher in mosquitoes that had fed on Plasmodium-infected mice than in those that had not been exposed to the parasite. The latter effect was mainly due to the high sensitivity of mosquitoes that had fed on an infected mouse but were not themselves infected, while the sensitivity to DDT was only slightly higher in mosquitoes infected by Plasmodium than in those that had fed on an uninfected mouse. CONCLUSIONS The observed pattern indicates a cost of parasite-resistance. It suggests that, in addition to the detrimental effect of insecticide-resistance on control, the continued use of insecticides in a population of insecticide-resistant mosquitoes could select mosquitoes to be more susceptible to Plasmodium infection, thus further decreasing the efficacy of the control.

Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd′ of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd′ suggests that the inversion may be the result of the transposable element’s activity. Characteristics of Odysseus’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites

Immune responses of the malaria vector mosquito Anopheles gambiae were monitored systematically by the induced expression of five RNA markers after infection challenge. One newly isolated marker encodes a homologue of the moth Gram-negative bacteria-binding protein (GNBP), and another corresponds to a serine protease-like molecule. Additional previously described markers that respond to immune challenge encode the antimicrobial peptide defensin, a putative galactose lectin, and a putative serine protease. Specificity of the immune responses was indicated by differing temporal patterns of induction of specific markers in bacteria-challenged larvae and adults, and by variations in the effectiveness of different microorganisms and their components for marker induction in an immune-responsive cell line. The markers exhibit spatially distinct patterns of expression in the adult female mosquito. Two of them are highly expressed in different regions of the midgut, one in the anterior and the other in the posterior midgut. Marker induction indicates a significant role of the midgut in insect innate immunity. Immune responses to the penetration of the midgut epithelium by a malaria parasite occur both within the midgut itself and elsewhere in the body, suggesting an immune-related signaling process.

Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae

The isolation and study of Anopheles gambiae genes that are differentially expressed in development, notably in tissues associated with the maturation and transmission of the malaria parasite, is important for the elucidation of basic molecular mechanisms underlying vector–parasite interactions. We have used the differential display technique to screen for mRNAs specifically expressed in adult males, females, and midgut tissues of blood-fed and unfed females. We also screened for mRNAs specifically induced upon bacterial infection of larval stage mosquitoes. We have characterized 19 distinct cDNAs, most of which show developmentally regulated expression specificity during the mosquito life cycle. The most interesting are six new sequences that are midgut-specific in the adult, three of which are also modulated by blood-feeding. The gut-specific sequences encode a maltase, a V-ATPase subunit, a GTP binding protein, two different lectins, and a nontrypsin serine protease. The latter sequence is also induced in larvae subjected to bacterial challenge. With the exception of a mitochondrial DNA fragment, the other 18 sequences constitute expressed genomic sequence tags, 4 of which have been mapped cytogenetically.

Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis

Chromosomal forms of Anopheles gambiae, given the informal designations Bamako, Mopti, and Savannah, have been recognized by the presence or absence of four paracentric inversions on chromosome 2. Studies of karyotype frequencies at sites where the forms occur in sympatry have led to the suggestion that these forms represent species. We conducted a study of the genetic structure of populations of An. gambiae from two villages in Mali, west Africa. Populations at each site were composed of the Bamako and Mopti forms and the sibling species, Anopheles arabiensis. Karyotypes were determined for each individual mosquito and genotypes at 21 microsatellite loci determined. A number of the microsatellites have been physically mapped to polytene chromosomes, making it possible to select loci based on their position relative to the inversions used to define forms. We found that the chromosomal forms differ at all loci on chromosome 2, but there were few differences for loci on other chromosomes. Geographic variation was small. Gene flow appears to vary among different regions within the genome, being lowest on chromosome 2, probably due to hitchhiking with the inversions. We conclude that the majority of observed genetic divergence between chromosomal forms can be explained by forces that need not involve reproductive isolation, although reproductive isolation is not ruled out. We found low levels of gene flow between the sibling species Anopheles gambiae and Anopheles arabiensis, similar to estimates based on observed frequencies of hybrid karyotypes in natural populations.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5′ flanking sequence (−855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae

Eight novel families of miniature inverted repeat transposable elements (MITEs) were discovered in the African malaria mosquito, Anopheles gambiae, by using new software designed to rapidly identify MITE-like sequences based on their structural characteristics. Divergent subfamilies have been found in two families. Past mobility was demonstrated by evidence of MITE insertions that resulted in the duplication of specific TA, TAA, or 8-bp targets. Some of these MITEs share the same target duplications and similar terminal sequences with MITEs and other DNA transposons in human and other organisms. MITEs in A. gambiae range from 40 to 1340 copies per genome, much less abundant than MITEs in the yellow fever mosquito, Aedes aegypti. Statistical analyses suggest that most A. gambiae MITEs are in highly AT-rich regions, many of which are closely associated with each other. The analyses of these novel MITEs underscored interesting questions regarding their diversity, origin, evolution, and relationships to the host genomes. The discovery of diverse families of MITEs in A. gambiae has important practical implications in light of current efforts to control malaria by replacing vector mosquitoes with genetically modified refractory mosquitoes. Finally, the systematic approach to rapidly identify novel MITEs should have broad applications for the analysis of the ever-growing sequence databases of a wide range of organisms.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Revisão taxonômica e filogenia da Seção Myzorhynchella de Anopheles (Nyssorhynchus) (Diptera: Culicidae)

Anopheles é o gênero da família Culicidae mais estudado devido sua importância médica. Atualmente o gênero Anopheles compreende 472 espécies válidas que estão divididas em sete subgêneros. Os principais vetores de plasmódio da Malária no Brasil pertencem ao subgênero Nyssorhynchus, que inclui 39 espécies oficialmente reconhecidas e um número crescente de complexos de espécies crípticas que estão distribuídas em três Seções: Myzorhynchella, Albimanus e Argyritarsis. Atualmente a Seção Myzorhynchella é formada por seis espécies: An. lutzii, An. parvus, An. nigritarsis, An. guarani, An. antunesi e An. pristinus. Para o desenvolvimento da análise morfológica, observou-se material-tipo depositado em diferentes coleções, espécimes depositados na coleção entomológica da FSP/USP, além de outros obtidos em coletas realizadas durante o presente estudo em diferentes localidades do Brasil. As análises moleculares foram desenvolvidas a partir de espécimes obtidos nas coletas. Revisão taxonômica da Seção Myzorhynchella é apresentada, incluindo-se descrições de quatro novas espécies e redescrições das demais, informações sobre bionomia, importância médica, caracterização molecular, distribuição geográfica, estado de preservação do material-tipo, além de chaves de identificação de adultos, larva de quarto estádio e genitália masculina. Os resultados das análises filogenéticas utilizando sequências de ITS2, COI e Catalase indicam a existência de pelo menos doze espécies dentro da Seção Myzorhynchella, os espécimes que vêm sendo identificados como An. antunesi constitui um complexo formado por possíveis cinco espécies e aqueles de An. parvus e An. pristinus também podem representar complexos de espécies. As sequências de ITS2 podem ser utilizadas como marcador diagnóstico para espécies da Seção Myzorhynchella. Contudo, o estudo ainda demonstra que pouco se conhece sobre a diversidade de espécies de Anopheles que ocorrem em ambientes onde a malária ocorre em baixa endemicidade. Pelo número de espécies novas encontradas e pela escassez de trabalhos com espécies da Seção, fica evidente a necessidade de mais estudos.

A morphological study of the Anopheles punctulatus group (Diptera: Culicidae) in the Solomon Islands, with a description of Anopheles (Cellia) irenicus Schmidt, sp.n.

A description of Anopheles (Cellia) irenicus Schmidt, sp.n. (formerly A. farauti No. 7) is provided. This species is one of six recorded from the Solomon Islands within the A. punctulatus group, which contains the major vectors of the causative agents of malaria and lymphatic filariasis in the southwest Pacific. Morphological markers are described for adult females, fourth-instar larvae and pupae that identify most specimens of A. irenicus. Keys are presented to distinguish members of the A. punctulatus group in the Solomon Islands.

Efficacy of a new self-supporting low-profile bednet for personal protection against Anopheles farauti (Diptera : Culicidae) in a village in Papua New Guinea

A new United States (U.S.) self-supporting low-profile bednet was designed by Walter Reed Army Institute of Research in collaboration with Breakthrough Technologies. The bednet incorporated permethrin-impregnated screening into a frame that erected automatically when removed from its bag. The new U.S. bednet was compared with the current Australian Defense Force (ADF) mosquito bednet at Buka Island, North Solomons Province, Papua New Guinea, in March 1999. At the time of the test, Anopheles farauti Laveran was the most abundant biting mosquito. Both bednet types provided > 97.8% protection compared with an unprotected collector. The untreated U.S. Army prototype bednet provided better protection than the untreated ADF bednet against mosquitoes entering the bednet during the night.