Sandra Sayuri Nagaki

Phylogenetic analysis and DNA-based species confirmation in Anopheles (Nyssorhynchus).

Specimens of neotropical Anopheles (Nyssorhynchus) were collected and identified morphologically. We amplified three genes for phylogenetic analysis–the single copy nuclear white and CAD genes, and the COI barcode region. Since we had multiple specimens for most species we were able to test how well the single or combined genes were able to corroborate morphologically defined species by placing the species into exclusive groups. We found that single genes, including the COI barcode region, were poor at confirming species, but that the three genes combined were able to do so much better. This has implications for species identification, species delimitation, and species discovery, and we caution that single genes are not enough. Higher level groupings were partially resolved with some well-supported groupings, whereas others were found to be either polyphyletic or paraphyletic. There were examples of known groups, such as the Myzorhynchella Section, which were poorly supported with single genes but were well supported with combined genes. From this we can infer that more sequence data will be needed in order to show more higher-level groupings with good support. We got unambiguously good support (0.94–1.0 Bayesian posterior probability) from all DNA-based analyses for a grouping of An. dunhami with An. nuneztovari and An. goeldii, and because of this and because of morphological similarities we propose that An. dunhami be included in the Nuneztovari Complex. We obtained phylogenetic corroboration for new species which had been recognised by morphological differences; these will need to be formally described and named.

Redescription of Anopheles (Nyssorhynchus) antunesi Galvao & Amaral and description of a new species of the Myzorhynchella Section (Diptera: Culicidae) from Serra da Mantiqueira, Brazil

Anopheles (Nyssorhynchus) pristinus Nagaki & Sallum, n. sp. of the Myzorhynchella Section is described based on morphological characters of adult females, males, fourth-instar larvae, pupae and male genitalia. Anopheles (Nyssorhynchus) antunesi Galvão & Amaral is characterized to fix its identity and distinguish it from An. pristinus. The eggs of An. antunesi are described for the first time. Molecular characterization employing sequences of the COI mitochondrial gene and the ITS2 region of ribosomal DNA are provided for each species. An. antunesi and An. pristinus are compared with morphologically similar species of the Myzorhynchella Section. The results of the present study suggest that the new species has been misidentified as both An. antunesi and Anopheles lutzii Cruz. An. antunesi and An. pristinus are sympatric, occurring at high altitudes in Serra da Mantiqueira, Southeastern Brazil.

Systematic notes on Anopheles Meigen (Diptera: Culicidae) species in the state of Amapá, Brazil

Identification of Anopheles nuneztovari Gabaldón and An. goeldii Rozeboom and Gabaldón based on the male genitalia traits is discussed. An. goeldii is in the synonymy of An. nuneztovari, however, characters of the aedeagus of male genitalia distinguish both species. We hypothesize that An. goeldii may be a valid species, however, further studies using molecular characters, especially ITS2 rDNA sequences will be necessary to elucidate the taxonomic status of the species. An. konderi Galvão and Damasceno and An. forattinii Wilkerson and Sallum are registered for the first time in the state of Amapá.

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.

Brazilian mosquito (Diptera: Culicidae) fauna: I. Anopheles species from Porto Velho, Rondônia state, western Amazon, Brazil

This study contributes to knowledge of Anopheles species, including vectors of Plasmodium from the western Brazilian Amazon in Porto Velho, Rondônia State. The sampling area has undergone substantial environmental changes as a consequence of agricultural and hydroelectric projects, which have caused intensive deforestation and favored habitats for some mosquito species. The purpose of this study was to diagnose the occurrence of anopheline species from collections in three locations along an electric-power transmission line. Each locality was sampled three times from 2010 to 2011. The principal adult mosquitoes captured in Shannon trap were Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An.gilesi and An. costai. In addition, larvae were collected in ground breeding sites for Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. and An. oswaldoi-konderi. Anopheles darlingi was the most common mosquito in the region. We discuss Culicidae systematics, fauna distribution, and aspects of malaria in altered habitats of the western Amazon.

Redescription of Anopheles (Nyssorhynchus) lutzii, and Resurrection of Anopheles guarani from Synonymy with an. lutzii (Diptera: Culicidae)

ABSTRACT Anopheles lutzii Cruz (Diptera: Culicidae) is redescribed using specimens collected in Pariquera-Açu, Vale do Ribeira, state of Säo Paulo, southeastern Mata Atlântica, Brazil. Specimens of An. lutzii from Vale do Ribeira and two females from Nova Friburgo, Rio de Janeiro, are compared with three syntypes of An. lutzii, deposited in the Instituto Oswaldo Cruz, Rio de Janeiro. Comparisons of external morphology of specimens from the type locality of Anopheles guarani Shannon demonstrate it is a valid species, and that Anopheles niger Theobald is conspecific with Anopheles guarani stat. rev. The adult male, male terminalia, fourth-instar larva, and pupa of An. guarani stat. rev. are described for the first time. Diagnostic characters of the male and female, male terminalia, fourth-instar larva and pupa of An. lutzii and An. guarani stat. rev. are illustrated. An. guarani stat. rev. is herein resurrected from the synonymy with An. lutzii, and Anopheles niger comb. nov. is transferred from the synonymy with An. lutzii to the synonymy of An. guarani stat. rev.

Phylogeny of Anophelinae using mitochondrial protein coding genes

Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.

Mitochondrial Genomes of Anopheles (Kerteszia) (Diptera: Culicidae) From the Atlantic Forest, Brazil

Abstract Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, such as the Anopheles albitarsis complex. Except for a few studies that employed a limited number of nuclear or mitochondrial loci to address the genetic structure and species status of Anopheles cruzii, Anopheles bellator, and Anopheles homunculus, little is known about genetic markers that can be employed in studies focusing on Kerteszia species. The complete mitochondrial genomes of seven specimens of An. bellator, An. cruzii, An. homunculus, and Anopheles laneanus were sequenced using long-range polymerase chain reaction and Illumina sequencing. The mitochondrial genomes varied from 15,446 to 15,738 bp in length and contained 37 genes (13 protein-encoding genes, 2 rRNA genes [12S rRNA and 16S rRNA] and 22 tRNA genes), and the AT-rich control region, as all do other Anopheles mitochondrial genomes sequenced to date. Specimens from four populations of An. cruzii showed differences in codon composition.