Subfamily Anophelinae Research Articles

Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology

BackgroundPhylogenetic analyses provide a framework for examining the evolution of morphological and molecular diversity, interpreting patterns in biogeography, and achieving a stable classification. The generic and suprageneric relationships within mosquitoes (Diptera: Culicidae) are poorly resolved, making these subjects difficult to address.ResultsWe carried out maximum parsimony and maximum likelihood, including Bayesian, analyses on a data set consisting of six nuclear genes and 80 morphological characters to assess their ability to resolve relationships among 25 genera. We also estimated divergence times based on sequence data and fossil calibration points, using Bayesian relaxed clock methods. Strong support was recovered for the basal position and monophyly of the subfamily Anophelinae and the tribes Aedini and Sabethini of subfamily Culicinae. Divergence times for major culicid lineages date to the early Cretaceous.ConclusionsDeeper relationships within the family remain poorly resolved, suggesting the need for additional taxonomic sampling. Our results support the notion of rapid radiations early in the diversification of mosquitoes.

Review of the genus Chagasia (Diptera: Culicidae: Anophelinae)

Genus Chagasia Cruz of subfamily Anophelinae (Diptera: Culicidae) is comprised of five species in the Neotropical Region: Ch. ablusa Harbach, n. sp., Ch. bathana (Dyar), Ch. bonneae Root, Ch. fajardi (Lutz) and Ch. rozeboomi Causey, Deane & Deane. The genus is described in detail and diagnoses, keys and illustrations are provided for the identification of the adult, pupal and larval stages of each species. The larval and pupal stages of a Chagasia species (Ch. bonneae) are fully illustrated for the first time. A neotype specimen is designated for Ch. fajardi to fix its identity and distinguish it from Ch. ablusa. The species treatments also include a synonymy (where applicable), a discussion, information on distribution, a synopsis of material examined and a summary of previous literature. The work is considered to be a review rather than a revision of the genus because too few link-reared specimens were available for detailed comparative study of all life stages, and it was not possible to determine the total range of morphological variation and the actual distributions of the species.

Comparative genomics allows the discovery of cis -regulatory elements in mosquitoes

The discovery and mapping of cis-regulatory elements is important for understanding regulation of gene transcription in mosquito vectors of human diseases. Genome sequence data are available for 3 species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus (Diptera: Culicidae), representing 2 subfamilies (Culicinae and Anophelinae) that are estimated to have diverged 145 to 200 million years ago. Comparative genomics tools were used to screen genomic DNA fragments located in the 5'-end flanking regions of orthologous genes. These analyses resulted in the identification of 137 sequences, designated "mosquito motifs," 7 to 9 nucleotides in length, representing 18 families of putative cis-regulatory elements conserved significantly among the 3 species when compared to the fruit fly, Drosophila melanogaster. Forty-one of the motifs were implicated previously in experiments as sites for binding transcription factors or functioning in the regulation of mosquito gene expression. Further analyses revealed associations between specific motifs and expression profiles, particularly in those genes that show increased or decreased mRNA abundance in females following a blood meal, and those accumulating transcription products exclusively or preferentially in the midgut, fat bodies, or ovaries. These results validate the methodology and support a relationship between the discovered motifs and the conservation of hematophagy in mosquitoes.

The Culicidae (Diptera): a review of taxonomy, classification and phylogeny*

The taxonomy, classification and phylogeny of family Culicidae are reviewed. The application of explicit methods of phylogenetic analysis has revealed weaknesses in the traditional classification of mosquitoes, but little progress has been made to achieve a robust, stable classification that reflects evolutionary relationships. The current phenetic classification is discussed in view of phylogeny reconstructions based on cladistic analyses of morphological and molecular data. It is concluded that the generic and suprageneric relationships and the validity and monophyly of the generic and subgeneric groupings of Culicidae are in need of extensive reappraisal. If the classification is to reflect evolutionary history, changes to the nomenclature of mosquitoes are inevitable. There is strong morphological and molecular evidence that subfamily Anophelinae and tribes Aedini, Culicini and Sabethini of subfamily Culicinae are monophyletic, but the other taxonomic groupings are not demonstrably monophyletic or have not been subjected to phylogenetic analyses.

Morphological and molecular characterization of Anopheles (Anopheles) maculipennis Meigen, type species of the genus and nominotypical member of the Maculipennis Complex

Abstract. Adults and larvae of Anopheles maculipennis Meigen were collected in Greece in May and June 2001. Larvae and the progeny of wild‐caught females were individually reared to obtain samples of all life stages for integrated morphological and molecular study. Specimens were identified on the basis of egg morphology and correlation of their ITS2 rDNA sequences with those in GenBank. The adult, pupal, larval (fourth‐instar) and egg stages are described, and all stages except the egg are illustrated. Partial sequence data are provided for the mitochondrial cytochrome c oxidase I (COI) gene and complete sequences for the nuclear ITS2 region. Additionally, the bionomics and distribution of the species are reviewed, and its taxonomy and systematics are discussed. This study provides the first fully integrated morphological and molecular assessment of An. maculipennis, the nominotypical member of the Maculipennis Complex, and the type species of genus Anopheles, and serves as a foundation for further studies of the complex and subfamily Anophelinae.

Phylogeny of Anophelinae (Diptera: Culicidae) based on nuclear ribosomal and mitochondrial DNA sequences

Abstract Phylogenetic relationships among thirty‐two species of mosquitoes in subfamily Anophelinae are inferred from portions of the mitochondrial genes COI and COII, the nuclear 18S small subunit rRNA gene and the expansion D2 region of the nuclear large subunit 28S rRNA gene. Sequences were obtained from the genera Anopheles, Bironella and Chagasia. Representatives of all six subgenera of Anopheles were included: Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia. Using parsimony and maximum likelihood methods, various combinations of these DNA sequence data were analysed separately: 18S, 28S, combined 18S and 28S, combined COI and COII, and combined 18S, 28S, COI and COII (‘total evidence’). The combined rDNA data contain strong phylogenetic signal, moderately to strongly supporting most clades in MP and ML analyses; however, the mtDNA data (analysed as either nucleotide or amino acid sequences) contain little phylogenetic signal, except for relationships of very recently derived groups of species and, at the deepest level, for the monophyly of Anophelinae. The paraphyly of Anopheles relative to Bironella is confirmed by most analyses and statistical tests. Support for the monophyly of subgenera Anopheles, Cellia, Kerteszia and Nyssorhynchus is indicated by most analyses. Subgenus Lophopodomyia is reconstructed as the sister to Bironella, nested within a clade also containing Nyssorhynchus and Kerteszia. The most basal relationships within genus Anopheles are not well resolved by any of the data partitions, although the results of statistical analyses of the rDNA data (S‐H‐tests, likelihood ratio tests for monophyly and Bayesian MCMC analyses) suggest that the clade consisting of Bironella, Lophopodomyia, Nyssorhynchus and Kerteszia is the sister to the clade containing Cellia and Anopheles.

Occurence of larval Culicidae (Diptera) in water retained in Aquascypha hydrophora (Fungus: Stereaceae) in Central Amazônia, Brazil.

The community structure of insects, especially mosquito larvae, in water held in the fungus Aquascypha hydrophora (Berk.) Reid (Stereaceae) is reported. The study was done in the Adolpho Ducke Forest Reserve, 26 km east of Manaus, AM, Brazil, from September 1998 through November 1999. The most abundant entomofauna were immature Culicidae (n = 121) 91.7%, followed by adult Dytiscidae (n = 3) 2.3%, immature Chironomidae (n = 5) 3.8% and immature Tipulidae (n = 3) 2.3%. Culicidae associated with A. hydrophora comprised species of the subfamilies Anophelinae and Culicinae.

Toward Understanding Anophelinae (Diptera, Culicidae) Phylogeny: Insights from Nuclear Single-Copy Genes and the Weight of Evidence

A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions. Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect. In combined analyses, most of the inferred relationship were well-supported and in agreement with previous studies: monophyly of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic; a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.

Evolution of Mitochondrial and Ribosomal Gene Sequences in Anophelinae (Diptera: Culicidae): Implications for Phylogeny Reconstruction

In this study, two mitochondrial genes, cyt b and ND5, and the D2 expansion segment of the 28S nuclear ribosomal gene were used to reconstruct a phylogeny of the mosquito subfamily Anophelinae. The ingroup consisted of all three genera of Anophelinae and five of six subgenera of Anopheles. Six genera of Culicinae were used as the outgroup. Extreme conservation at the protein level coupled with rapid saturation of synonymous positions probably accounted for the lack of meaningful phylogenetic signal in the cyt b gene. In contrast, abundant variation at all codon positions of the ND5 gene allowed recovery of the basal and most of the recent relationships. Phylogenetic analysis of D2 produced results consistent with those of ND5. Combined analysis indicated well-supported monophyletic Anophelinae (with Chagasia basal), Anopheles + Bironella, and subgeneric clades within the genus Anopheles. Moreover, subgenera Nyssorhynchus and Kerteszia were supported as a monophyletic lineage. The Kishino–Hasegawa test could not reject the monophyly of Anopheles, whereas the recently proposed hypothesis of close affinity of Bironella to the subgenus Anopheles was rejected by the analyses of ND5 and combined data sets. The lack of resolution of Bironella and Anopheles clades, or basal relationships among subgeneric clades within Anopheles, suggests their rapid diversification. Recovery of relationships consistent with morphology and previous molecular studies provides evidence of substantial phylogenetic signal in D2 and ND5 genes at levels of divergence from closely related species to subfamily in mosquitoes.

Phylogeny of Anophelinae (Diptera Culicidae) Based on Morphological Characters

Phylogenetic relationships of mosquitoes in the subfamily Anophelinae are presented based on a cladistic analysis of 163 morphological characters from females, males, fourth-instar larvae, and pupae of 64 species. Species examined include one Chagasia Cruz, three Bironella Theobald, and 60 species representing all six subgenera of Anopheles Meigen. Uranotaenia lowii Theobald and Aedeomyia squamipennis (Lynch Arribalzaga) are used as outgroups. This analysis indicates that Anophelinae is monophyletic, that Chagasia is the earliest-diverged lineage within Anophelinae, and that the genus Anopheles, as currently defined, is paraphyletic because it excludes Bironella. Four nonhomoplastic synapomorphies support the monophyly of the clade composed of the genera Bironella and Anopheles. Three major lineages are recognized within this clade. The basal lineage (lineage 1) contains An. (Nyssorhynchus), An. (Kerteszia), and An. implexus (Theobald). This analysis supports the monophyly of the sister groups Nyssorhynchus Blanchard and Kerteszia Theobald, but finds the Albimanus, Argyritarsis, and Myzorhynchella sections of the subgenus Nyssorhynchus to be paraphyletic. The second lineage (lineage 2) contains species of the subgenus Cellia Theobald, which is monophyletic. Except for the Cellia series, however, all other series of the subgenus Cellia are paraphyletic. The third lineage (lineage 3) contains species of the subgenera Anopheles Meigen, Stethomyia Theobald, Lophopodomyia Antunes, and of the genus Bironella. Bironella and Stethomyia are monophyletic sister groups. The Lophoscelomyia and Arribalzagia series are monophyletic. The Arribalzagia and Cycloleppteron series are sister groups nested within the Myzorhynchus series. We conclude that there is no support for the generic recognition of Bironella, nor the subgeneric rank of Lophopodomyia and Stethomyia.

Larval growth and biosynthesis of reserves in mosquitoes

Developmental instars of four species of mosquitoes have been analyzed for growth and synthesis of biomass with respect to their caloric content of protein, lipids, and carbohydrates for each instar of Aedes aegypti and Culex pipiens of the subfamily Culicinae, and Anopheles albimanus, and An. gambiae of the subfamily Anophelinae. The diameter of the thorax grows during the intermolt, reflecting continuous increase in biomass because it correlates significantly with the larval synthesis of total protein, lipids, and carbohydrates. For Ae. aegypti the fourth instar was sexed to disclose the sex-specific synthetic potential. In Ae. aegypti the protein increased in linear proportion with larval body size, whereas lipid synthesis followed a significant, exponential regression, which was clearly steeper in male larvae and most pronounced in the last instar. When normalized for size, the size-specific protein and lipid contents showed minimal levels of 0.25 and 0.1, respectively, regardless of standard or crowded rearing conditions. The rate of lipid synthesis in Ae. aegypti was determined by incubating fourth instar larvae with 14C-acetate and estimating the lipids. The rate was highest in the early larvae and decreased towards the end shortly before pupation; in male larvae incorporation was twice the rate of female larvae. Cx. pipiens reached the largest body sizes of all species tested, with protein and lipids increasing linearly with size. Their minimal levels of size-specific caloric contents were around 0.35 for protein and 0.25 for lipids. Anopheles also showed a linear relationship between larval size and caloric protein and lipid contents. Their minimal threshold levels in size-specific contents were 0.35 for protein and 0.2 for total lipids, similar to Culex, but slightly higher than in Aedes. Starvation of Ae. aegypti larvae and subsequent feeding partially improved their lipid contents, but never to the levels of non-starving, optimal controls. Conversely, well-fed final instars exposed to complete starvation showed a tremendous reduction of the protein and lipids contents in the surviving imagines, accompanied by 73% mortality. These results demonstrate the biosynthetic plasticity and the significance of the phagoperiod in Ae. aegypti during the final fourth instar for growth. The characteristic differences between these two subfamilies in their larval physiology are discussed in relation to ecological factors as encountered in the field under natural conditions, and in relation to our earlier findings on the reproductive physiology.

Phylogeny and classification of the Culicidae (Diptera)

The generic relationships and higher classification of the family Culicidae are examined on the basis of a phylogenetic analysis. New and traditional morphological characters studied and compared throughout the Culicidae resulted in the acquisition of character data relative to the majority of species within each genus. Polymorphisms and morphological observations are discussed and additional information and illustrations are provided for the majority of characters and their character states. The analysis of seventy‐three adult, pupal and fourth‐instar larval characters coded for the thirty‐eight currently recognized genera of mosquitoes resulted in relationships and groupings which differ significantly from traditional hypotheses. The analysis supports the monophyly of the subfamily Anophelinae and the tribes Culicini and Sabethini. The Anophelinae form the most basal clade of the family. The results indicate that Aedini is a paraphyletic assemblage with respect to the Mansoniini, each of which is monophyletic in itself. The Aedini + Mansoniini form a sister group to the Culicini + Sabethini, with the Aedini and the Culicini placed in ancestral relationships to the Mansoniini and the Sabethini, respectively. Based on the topography of generic relationships among more ‘generalized’ mosquitoes, the boundaries and relationships of the tribes Aedeomyiini, Uranotaeniini, Ficalbiini, Hodgesiini, Orthopodomyiini and Culisetini appear to be problematic. Relationships between genera of the tribe Aedini are generally poorly resolved due to a significant amount of polymorphism, especially within the genus Aedes as currently defined. There is no support for separate subfamily recognition for the genus Toxorhynchites, which is downgraded to tribal status as a result of the analysis. The results are discussed in relation to previous hypotheses based on subjective inference and cladistic analyses.

Similarities to a LINE element shared by Anopheline and Culicine mosquitoes map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells

To extend our understanding of amplicon structure in methotrexate-resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3′-end of the Type I dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an ∼0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae).

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes-Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae-Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.

The type specimens of mosquitoes (Diptera, Culicidae) deposited in the entomological collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil

A list of type specimens of 100 mosquito species deposited in the Entomological collection of the Instituto Oswaldo Cruz is presented. It includes five holotypes belonging to the subfamily Anophelinae; 56 holotypes of Culicinae and two of Toxorhynchitinae. A lectotype is designated for Toxorhynchites fluminensis. The holotypes of six nominal species - Psorophora chiquitana, Psorophora circunflava, Psorophora melanota, Psorophora lanei, Wyeomyia brucei and Uranotaenia noctivaga - previously considered non existent or of unknown location were found in the collection.