Species diversity and spatial distribution of mosquitoes (Diptera: Culicidae) from La Isla Amazon Park, Napo Province, Ecuador / Diversidad de especies y distribución espacial de mosquitos (Diptera: Culicidae) del Parque Amazónico La Isla, provincia de Napo, Ecuador

Abstract Introduction: Vector-borne diseases are prevalent in the Amazon and Coastal regions of Ecuador. However, there is a scarcity of mosquito ecology studies in these areas. The most recent list of species reported for the country comprises 8 tribes, 22 genera, and 200 species. Objectives: To document the Culicidae species found in La Isla Amazon Park, Napo, Ecuador, including those with epidemiological significance; and to analyze their composition, abundance, and diversity, focusing on larval habitats during the dry and rainy periods. Methods: We evaluated different larval habitats, considering collection duration as the primary criterion. We used CDC and Shannon traps to collect adult mosquitoes during both rainy and dry periods. To assess sampling effort, we used accumulation curves and non-parametric estimators of species richness, while we employed Hill numbers to determine diversity. Additionally, we used the Berger-Parker and Pielou indices to evaluate species dominance and evenness. We conducted cluster analysis and ANOSIM tests to assess the similarity between habitats and the differences in taxonomic composition between periods. Results: We collected a total of 802 individuals from 15 species and 4 taxonomic units, 5 genera, and 4 tribes. Notably, this may be the first records of Wyeomyia felicia Dyar & Núñez Tovar and Culex derivator Dyar & Knab from Ecuador. Additionally, the presence of Culex dunni Dyar and Psorophora ferox von Humboldt (both recognized as vectors) was correlated with increased rainfall. Conclusions: The abundance of mosquitoes, including potential vector species, increased during the rainy season, indicating a higher risk of pathogen transmission. However, the relationship between rainfall amount and diversity patterns is not well-defined.

Resumen Introducción: Las enfermedades vectoriales son prevalentes en las regiones amazónica y costera de Ecuador. Sin embargo, hay una escasez de estudios de ecología de mosquitos en estas áreas. En el país se ha reportado 8 tribus, 22 géneros y 200 especies. Objetivos: Documentar las especies de Culicidae encontradas en el Parque Amazónico La Isla, Napo, Ecuador, incluyendo aquellas con importancia epidemiológica; y analizar su composición, abundancia y diversidad, enfocándose en los hábitats de las larvas durante los períodos seco y lluvioso. Métodos: Evaluamos diferentes hábitats larvarios, con la duración de la recolecta como criterio. Las trampas CDC y Shannon recolectaron mosquitos adultos durante los períodos seco y lluvioso. Evaluamos la riqueza de especies con curvas de acumulación y estimadores no paramétricos, mientras que determinamos la diversidad con los números de Hill. Además, utilizamos los índices de Berger-Parker y Pielou para evaluar la dominancia y la uniformidad de las especies. Realizamos análisis de conglomerados y la prueba ANOSIM para evaluar la similitud entre hábitats y estaciones, así como las diferencias en la composición taxonómica, respectivamente. Resultados: Recolectamos un total de 802 individuos de 15 especies y 4 unidades taxonómicas, 5 géneros y 4 tribus. Este podría ser el primer registro de Wyeomyia felicia Dyar & Núñez Tovar y Culex derivator Dyar & Knab en Ecuador. Además, la presencia de Culex dunni Dyar y Psorophora ferox von Humboldt (ambos potenciales vectores) se correlacionó con el aumento de las precipitaciones. Conclusiones: El aumento de la abundancia de mosquitos durante el periodo lluvioso indica un mayor riesgo de transmisión de patógenos. Sin embargo, la relación entre la cantidad de precipitaciones y los patrones de diversidad no está bien definida.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes

Two snapshot surveys to establish the diversity and ecological preferences of mosquitoes (Diptera: Culicidae) in the terra firme primary rain forest surrounding the Tiputini Biodiversity Station in the UNESCO Yasuní Biosphere Reserve of eastern Amazonian Ecuador were carried out in November 1998 and May 1999. The mosquito fauna of this region is poorly known; the focus of this study was to obtain high quality link-reared specimens that could be used to unequivocally confirm species level diversity through integrated systematic study of all life stages and DNA sequences. A total of 2,284 specimens were preserved; 1,671 specimens were link-reared with associated immature exuviae, all but 108 of which are slide mounted. This study identified 68 unique taxa belonging to 17 genera and 27 subgenera. Of these, 12 are new to science and 37 comprise new country records. DNA barcodes [658-bp of the mtDNA cytochrome c oxidase ( COI ) I gene] are presented for 58 individuals representing 20 species and nine genera. DNA barcoding proved useful in uncovering and confirming new species and we advocate an integrated systematics approach to biodiversity studies in future. Associated bionomics of all species collected are discussed. An updated systematic checklist of the mosquitoes of Ecuador (n = 179) is presented for the first time in 60 years.

Malaria vector species in Colombia: a review

Here we present a comprehensive review of the literature on the vectorial importance of the major Anopheles malaria vectors in Colombia. We provide basic information on the geographical distribution, altitudinal range, immature habitats, adult behaviour, feeding preferences and anthropophily, endophily and infectivity rates. We additionally review information on the life cycle, longevity and population fluctuation of Colombian Anopheles species. Emphasis was placed on the primary vectors that have been epidemiologically incriminated in malaria transmission: Anopheles darlingi, Anopheles albimanus and Anopheles nuneztovari. The role of a selection of local, regional or secondary vectors (e.g., Anopheles pseudopunctipennis and Anopheles neivai) is also discussed. We highlight the importance of combining biological, morphological and molecular data for the correct taxonomical determination of a given species, particularly for members of the species complexes. We likewise emphasise the importance of studying the bionomics of primary and secondary vectors along with an examination of the local conditions affecting the transmission of malaria. The presence and spread of the major vectors and the emergence of secondary species capable of transmitting human Plasmodia are of great interest. When selecting control measures, the anopheline diversity in the region must be considered. Variation in macroclimate conditions over a species' geographical range must be well understood and targeted to plan effective control measures based on the population dynamics of the local Anopheles species.

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 percent of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7 percent 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 comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae)

The Anopheles (Nyssorhynchus) albitarsis complex includes six species: An. albitarsis, Anopheles oryzalimnetes Wilkerson and Motoki, n. sp., Anopheles marajoara, Anopheles deaneorum, Anopheles janconnae Wilkerson and Sallum, n. sp. and An. albitarsis F. Except for An. deaneorum, species of the complex are indistinguishable when only using morphology. The problematic distinction among species of the complex has made study of malaria transmission and ecology of An. albitarsis s.l. difficult. Consequently, involvement of species of the An. albitarsis complex in human Plasmodium transmission is not clear throughout its distribution range. With the aim of clarifying the taxonomy of the above species, with the exception of An. albitarsis F, we present comparative morphological and morphometric analyses, morphological redescriptions of three species and description of two new species using individuals from populations in Brazil, Paraguay, Argentina and Venezuela. The study included characters from adult females, males, fourth-instar larvae, pupae and male genitalia of An. albitarsis, An. marajoara, An. deaneorum and An. oryzalimnetes n. sp. For An. janconnae n. sp. only characters of the female, male and male genitalia were analyzed. Fourth-instar larvae, pupae and male genitalia characteristics of all five species are illustrated. Bionomics and distribution data are given based on published literature records.

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 percent and 11.36 percent, 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 percent of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5 percent of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34 percent shared a HP/PHD ratio less than 1.50. Consequently, 1.52 percent 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 percent displayed SCP/DSD values greater than 0.50 and of these, 3.65 percent 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 percent 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.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.

Revision of the Leucosphyrus group of Anopheles (Cellia) (Diptera, Culicidae)

Revision of the Leucosphyrus Group of Anopheles (Cellia) (Diptera, Culicidae). This is a comprehensive revision of 20 species of the Leucosphyrus Group of the Neomyzomyia Series of Anopheles (Cellia). Morphological description of the adults, male and female, male genitalia, pupa and fourth-instar larva are provided for each taxon in addition to bionomics, distribution data and systematic discussion for each species, including diagnostic characters. Identification keys for females and fourth-instar larvae are provided. When possible medical importance of each species is included. Illustrations of of the adults, fourth-instar larvae and pupae are provided. Distribution maps for each species are mainly based on the material examined; however, when possible published data were also used. Tables on adult character variations, fourth-instar larval and pupal setal branching are included as appendices. A neotype for An. takasagoensis Morishita and An. sulawesi Koesoemawinangoen, and a lectotype for An. balabacensis Baisas, are designated. The authorship of An. sulawesi previously cited as Waktoedi is corrected to Koesoemawinangoen.

Revisão do Grupo Leucosphyrus de Anopheles (Cellia) (Diptera, Culicidae). Apresenta-se a revisão taxonômica de 20 espécies do Grupo Leucosphyrus da Série Neomyzomyia de Anopheles (Cellia). Apresentam-se as descrições morfológicas dos adultos, machos e fêmeas, genitália masculina, pupa e larva de quarto estádio, bem como dados de bionomia, distribuição e discussão sistemática para cada espécie, incluindo caracteres diagnósticos. Foram acrescentadas chaves dicotômicas de identificação para as espécies, utilizando caracteres dos adultos fêmeas e larvas de quarto estádio. Quando possível, foram incluídos dados sobre a importância médica de cada espécie. Foram também apresentadas ilustrações dos adultos, das larvas de quarto estádio e das pupas. Os mapas de distribuição das espécies foram elaborados com base no material examinado, no entanto, quando possível utilizou-se dados adicionais publicados na literatura. Fornecem-se tabelas com as variações dos caracteres dos adultos, das cerdas das larvas de quarto estádio das pupas. São designados os neótipos de An. takasagoensis Morishita e An. sulawesi Koesoemawinangoen, e o lectótipo de An. balabacensis Baisas. O nome do autor de An. sulawesi previamente citado como sendo Waktoedi foi corrigido para Koesoemawinangoen.

Description of the immature stages of Anopheles (Nyssorhynchus) rondoni (Neiva & Pinto) (Diptera: Culicidae)

The larval and pupal stages of Anopheles (Nyssorhynchus) rondoni (Neiva and Pinto) (Albimanus Section) are fully described and illustrated for the first time. The larval stage is similar to An. (Nys.) strodei Root. It can be recognized by the following combination of characteres: clypeal index, 1.6-2.9; single, aciculate setae 2,3-C; seta 3-C 0.5-0.7 the lenght of 2-C; setae 1,2-P rarely sharing a common tubercle; seta 1-P with narrow leaflets. The pupal stage is distinguished from other Nyssorhynchus by having setae 1-IV-VII and 5-V-VII branched. Only minor variation was found in setal counts between specimens from Peixoto de Azevedo, State of Mato Grosso and Bocaina, State of Sao Paulo, Brazil.

Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae)

Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR) markers were used to identify four species related to Anopheles (Nyssorhynchus) albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995), which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys.) albitarsis, species B is undescribed, species C is An. (Nys) marajoara Galväo and Damasceno and species D is An. (Nys.) deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of Säo Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.

The identification of Anopheles (Nyssorhynchus) rondoni (Diptera: Culicidae) in Mato Grosso, Brazil: an analysis of key character variability

A morphological study was made of a population of Anopheles (Nyssorhynchus) rondoni (Neiva and Pinto) from northern Mato Grosso, Brazil. This population usually lacked the primary key character of a dark basal band on hindtarsomere 3, i.e., hindtarsomere 3 was all white as in most other members of the subgenus. It was determined that this species can be recognized instead by the presence of a dark spot on the thorax made up of a large dark prescutellar space that is contiguous with a concolorous central area on the scutellum. A secondary character of a dark area on the costa created by the fusion of the humeral dark, presector dark and sector dark proximal spots is also usually reliable. Regression analyses comparing the lengths and ratios of the dark bands on hindtarsomeres 2 to those on 3 describe a straight line relationship. This suggests that the "atypical" population is at one end of a character gradient. We propose that in the subgenus Nyssorhynchus individuals that have a long basal band on hindtarsomere 2 are more likely to also have a basal band on hindtarsomere 3. The pupal stage of this species has not been previously described. Reared-associated specimens from this study show that the pupa can be easily differentiated from all other Nyssorhynchus by the relatively stout, usually 2 or 3 branched (1-5), setae 1 and 5 on segments IV-VII.