Sequence analysis of the ribosomal DNA internal transcribed spacer 2 from populations of Anopheles nuneztovari (Diptera: Culicidae)

Sequence variation of the ribosomal DNA internal transcribed spacer 2 (ITS2) was examined for populations of the malaria vector Anopheles nuneztovari collected in Colombia, Venezuela, Bolivia, Suriname, and Brazil. Mosquitoes from Colombia and Venezuela had identical ITS2 sequences and were distinguished from sequences in other populations by three insertion/deletion events (indels) and by one transversion. The length of the ITS2 was 363-369 bp, and it had a G+C content of 55.3%- 55.7%. Variation in the length of the ITS2 between and within populations was due to indels in simple repeats. ITS2 consensus sequences were similar or identical for samples from the following three groups: (1) Colombia, Bolivia, and Venezuela; (2) Suriname and northern Brazil; and (3) eastern and central Brazil. The presence of two different consensus sequences from a single location near Manaus, Brazil, suggests that populations from eastern Brazil and those from Suriname converge in this region of the Amazon Basin. These data show that putative cryptic species of An. nuneztovari are distinguished by very minor differences in DNA sequence of the ITS2 region.      

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.     

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.     

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F(ST)) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.     

Population analysis using the nuclear white gene detects Pliocene/Pleistocene lineage divergence within Anopheles nuneztovari in South America

Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae), a locally important malaria vector in some regions of South America, has been hypothesized to consist of at least two cryptic incipient species. We investigated its phylogeographic structure in several South American localities to determine the number of lineages and levels of divergence using the nuclear white gene, a marker that detected two recently diverged genotypes in the primary Neotropical malaria vector Anopheles darlingi Root. In An. nuneztovari, five distinct lineages (1-5) were elucidated: (1) populations from northeastern and central Amazonia; (2) populations from Venezuela east and west of the Andes; (3) populations from Colombia and Venezuela west of the Andes; (4) southeastern and western Amazonian Brazil populations, and (5) southeastern and western Amazonian Brazil and Bolivian populations. There was a large amount of genetic differentiation among these lineages. The deepest and earliest divergence was found between lineage 3 and lineages 1, 2 and 4, which probably accounts for the detection of lineage 3 in some earlier studies. The multiple lineages within Amazonia are partially congruent with previous mtDNA and ITS2 data, but were undetected in many earlier studies, probably because of their recent (Pleistocene) divergence and the differential mutation rates of the markers. The estimates for the five lineages, interpreted as recently evolved or incipient species, date to the Pleistocene and Pliocene. We hypothesize that the diversification in An. nuneztovari is the result of an interaction between the Miocene/Pliocene marine incursion and Pleistocene climatic changes leading to refugial isolation. The identification of cryptic lineages in An. nuneztovari could have a significant impact on local vector control measures.     

Molecular Phylogeny of Neotropical Anopheles (Nyssorhynchus) Albitarsis Species Complex (Diptera: Culicidae)

A phylogeny was reconstructed for four species belonging to the Neotropical Anopheles (Nyssorhynchus) albitarsis complex using partial sequences from the mitochondrial cytochrome oxidase I (COI) and NADH dehydrogenase 4 (ND4) genes and the ribosomal DNA ITS2 and D2 expansion region of the 28S subunit. The basis for initial characterization of each member of the complex was by correlated random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Analyses were carried out with and without an outgroup (An.(Nys.) argyritarsis Robineau-Desvoidy) by using maximum parsimony, maximum likelihood, and Bayesian methods. A total evidence approach without the outgroup, using separate models for "fast" (COI and ND4 position 3) and "slow" (rDNA ITS2 and D2, and COI and ND4 position 1) partitions, gave the best supported topology, showing close relationships of An. albitarsis Lynch-Arribálzaga to An. albitarsis B and An. marajoara Galv?o & Damasceno to An. deaneorum Rosa-Freitas. Analyses with the outgroup included showed poorer support, possibly because of a long branch attraction effect caused by a divergent outgroup, which caused one of the An. marajoara specimens to cluster with An. deaneorum in some analyses. The relationship of the above-mentioned result to a separately proposed hypothesis suggesting a fifth species in the complex is discussed.     

Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F₁-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.     

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.     

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 Galv?o and Damasceno and An. forattinii Wilkerson and Sallum are registered for the first time in the state of Amapá.     

Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.     

Evidence for a conspecific relationship between two morphologically and cytologically different forms of Korean Anopheles pullus mosquito

Despite the medical importance of anopheline mosquitoes as vectors of Korean vivax malaria, differentiation between Korean anopheline mosquitoes by traditional morphological taxonomic criteria is difficult. An. yatsushiroensis is the second most common Anopheles mosquito species in Korea and a possible vector of Korean vivax malaria together with An. sinensis, the predominant anopheline species. Recently, An. yatsushiroensis has been declared a synonym of An. pullus, based on comparisons of egg morphology and adult progeny, although they differ in ecology and morphology. To verify the species status of these two ambiguous forms, we established isofemale lines of Korean An. pullus and An. yatsushiroensis (An. pullus form yatsushiroensis) mosquitoes and investigated their genetic relationship by metaphase karyotype analysis, comparing the DNA sequences of rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunits I (COI) and II (COII), and by hybridization experiments. Two isofemale lines had differently shaped X and Y chromosomes. However reciprocal crosses between them yielded viable progeny with completely synaptic salivary gland polytene chromosomes. DNA analyses also strongly supported their conspecificity. The two strains also showed great sequence similarity in the ITS2, COI and COII regions (variation rate = 0.0 to 0.8%). Based on these findings, we conclude that the two forms, though differing distinctly in morphological, cytological and ecological traits, remain interfertile.     

Molecular systematics of the Philippine malaria vector Anopheles flavirostris

Allozyme and molecular sequence data from the malaria vector Anopheles flavirostris (Ludlow) (Diptera: Culicidae) were analysed from 34 sites throughout the Philippines, including the type locality, to test the hypothesis that this taxon is a single panmictic species. A finer-scaled allozyme study, of mainly Luzon samples, revealed no fixed genetic differences in sympatric sites and only low levels of variation. We obtained data from partial sequences for the internal transcribed spacer 2 (ITS2) (483 bp), the third domain (D3) (330 bp) of the 28S ribosomal DNA subunit and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (261 bp). No sequence variation was observed for ITS2, only a one base pair difference was observed between Philippine and Indonesian D3 sequences and An. flavirostris sequences were unique, confirming their diagnostic value for this taxon. Sixteen COI haplotypes were identified, giving 25 parsimony informative sites. Neighbour-Joining, Maximum Parsimony, Maximum Likelihood and Bayesian phylogenetic analysis of COI sequences for An. flavirostris and outgroup taxa revealed strong branch support for the monophyly of An. flavirostris, thus confirming that Philippine populations of this taxon comprise a single separate species within the Minimus Subgroup of the Funestus Group. Variation in the behaviour of An. flavirostris is likely to be intraspecific rather than interspecific in origin.     

Anthropophilic Anopheles species composition and malaria in Tierradentro,Córdoba,Colombia

Malaria is still a primary health problem in Colombia. The locality of Tierradentro is situated in the municipality of Montelíbano, Córdoba, in the northwest of Colombia, and has one of the highest annual parasite index of malaria nationwide. However, the vectors involved in malaria transmission in this locality have not yet been identified. In this study, the local anthropophilic Anopheles composition and natural infectivity with Plasmodium were investigated. In August 2009, 927 female Anopheles mosquitoes were collected in eight localities using the human landing catch method and identified based on their morphology. Cryptic species were determined by restriction fragment length polymorphism-internal transcribed spacer (ITS)2 molecular analysis. Eight species [Anopheles nuneztovari s.l. (92.8%), Anopheles darlingi (5.1%), Anopheles triannulatus s.l. (1.8%), Anopheles pseudopunctipennis s.l. (0.2%), Anopheles punctimacula s.l. (0.2%), Anopheles apicimacula (0.1%), Anopheles albimanus (0.1%) and Anopheles rangeli (0.1%)] were identified and species identity was confirmed by ITS2 sequencing. This is the first report of An. albimanus, An. rangeli and An. apicimacula in Tierradentro. Natural infectivity with Plasmodium was determined by ELISA. None of the mosquitoes was infectious for Plasmodium. An. nuneztovari s.l. was the predominant species and is considered the primary malaria vector; An. darlingi and An. triannulatus s.l. could serve as secondary vectors.     

Mosquitoes of the Anopheles maculipennis group (Diptera: Culicidae) in Romania, with the discovery and formal recognition of a new species based on molecular and morphological evidence

Mosquitoes of the Anopheles maculipennis group were collected in five districts of Romania (Constant,a, Giurgiu, Ilfov, Mehedint,i and Suceava) between March 2000 and June 2003. Two hundred and ninety-seven specimens were identified by molecular methods. Nuclear rDNA ITS2 sequences of 178 specimens were compared with GenBank sequences for nine known Palaearctic species of the group, and 119 specimens were identified using an ITS2 PCR-RFLP assay developed during the study. Five genetically distinct species of the group were identified: A. atroparvus van Thiel, A. maculipennis Meigen, A. melanoon Hackett and A. messeae Falleroni and a previously unrecognized species. The new species, herein formally described and named A. daciae sp. n., was collected in the Black Sea coastal region and plains adjacent to the Danube River in southern Romania. Anopheles daciae is most similar to and sympatric with A. messeae. It is contrasted with A. messeae and characterized on the basis of unique nuclear ITS2 and mitochondrial COI DNA sequences and morphological characters of the eggs. The larval, pupal and adults stages of the two species were also compared, but no reliable characters were found to distinguish them. It seems likely that A. daciae is more widespread in eastern Europe and the Balkan States, and could be responsible for malaria transmission in these regions that is currently attributed to A. messeae. Anopheles melanoon is reported from Romania for the first time.     

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phi ST 0.1131, P < 0.001). The variation among individuals within populations (phi ST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.     

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.     

A molecular phylogeny of Anopheles annulipes (Diptera: Culicidae) sensu lato: the most species-rich anopheline complex

The Australasian Annulipes Complex is the most species-rich among Anopheles mosquitoes, with at least 15 sibling species suspected. Members of this complex are the most likely vectors of malaria in the past in southern Australia and are involved in the spread of myxomatosis among rabbits. In this, the first comprehensive molecular study of the Annulipes Complex, 23 ITS2 rDNA variants were detected from collections throughout Australia and Papua New Guinea, including diagnostic variants for the previously identified An. annulipes species A-G. Specimens of each ITS2 variant were sequenced for portions of the mitochondrial COI, COII and nuclear EF-1alpha genes. Partitioned Bayesian and Maximum Parsimony analyses confirmed the monophyly of the Annulipes Complex and revealed at least 17 clades that we designate species A-Q. These species belong to two major clades, one in the north and one mainly in the south, suggesting that climate was a driver of species radiation. We found that 65% (11) of the 17 sibling species recorded here had unique COI sequences, suggesting that DNA barcoding will be useful for diagnosing species within the Annulipes Complex. A comparison of the taxa revealed morphological characters that may be diagnostic for some species. Our results substantially increase the size of the subgenus Cellia in Australasia, and will assist species-level studies of the Annulipes Complex.     

基于rDNA-ITS2序列的中国按蚊属塞蚊亚属部分种类的系统发育研究(双翅目:蚊科)

应用rDNA-ITS2序列,采用邻接法(NJ)、最大简约法(MP)和最大似然法(ML),以按蚊亚属Anopheles的中华按蚊An.(An.)sinensis和赫坎按蚊An.(An.)hyrcanus为外群,对采自中国的按蚊属Anopheles塞蚊亚属Cellia 21种蚊进行了系统发育分析.结果表明:rDNA-ITS...     

Molecular characterization of the Anopheles maculipennis complex during surveillance for the 2004 Olympic Games in Athens

Specimens belonging to the Anopheles maculipennis complex were collected as larvae or resting adults from May 2003 to November 2004 in the area of the Athens 2004 Olympic Rowing Centre in Schinias, Attiki, Greece, and identified by morphological and molecular analyses. Of the 201 specimens collected, 199 were found to be Anopheles sacharovi Favre and two were An. maculipennis Meigen s.s. on the basis of similarity to published sequence data for the rDNA internal transcribed spacer (ITS2) region and the mitochondrial cytochrome c oxidase I gene (COI). Sequence data from a number of specimens were obtained for both genes and compared with corresponding GenBank data derived from diverse geographical areas. A high degree of homology in ITS2 sequences was found in both species, ranging from 99.5% to 100% in An. sacharovi and 99.4% to 100% in An. Maculipennis, with no intraspecific variation in either of the two species in our study. The degree of homology in the COI sequences was 94.8-99.8% in An. sacharovi and 95.0-99.8% in An. maculipennis. The 522-bp fragment produced a rather high degree of intrapopulation polymorphism for An. sacharovi, generating nine different haplotypes, five of which were singletons. Intraspecific variation for these sequences ranged from 0.2% to 1.4%, but was much lower (0.77%) for the two An. maculipennis sequences. These findings represent the first characterization of the An. maculipennis complex in the area of Schinias.