A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Euplocania (Psocodea: Ptiloneuridae) in Peru, description of six new species.

Based on a review of specimens from Peru, six new species of the Euplocania (Ptiloneuridae) were found and assigned to species groups. The new species, here described and illustrated are: Euplocania alfonnerii n. sp., E. amarui n. sp., E. garcialdretei n. sp., E. katarii n. sp., E. matsiguenka n. sp. and E. wchipere n. sp. An identification key for Euplocania from Peru is included.

New species of Euplocania Enderlein (Psocodea: 'Psocoptera': Psocomorpha) from Colombia and Ecuador.

Four species of Euplocania from Colombia (Euplocania huitota n. sp., E. kakwa n. sp., E. nukak n. sp. and E. ticuna n. sp.) and four species from Ecuador (E. napensis n. sp., E. shuar n. sp., E. teslai n. sp. and E. waorani n. sp.), in four species groups, are described and illustrated. The new species group ticuna, from Colombia and waorani, from Ecuador are diagnosed. The number of species in Euplocania is raised to 61, in 17 species groups. A key to the males of the known species of Euplocania is presented.

Diversity and vertical distribution of psocids (Psocodea: Psocoptera) in two forests of the Colombian Amazon

Introduction: The knowledge on the diversity of psocids in Colombia has increased, following the global trend, but the main interest of most of the studies has been oriented to solve taxonomic aspects, leaving aside other biological aspects, of which little is known, such as the ecological interactions of these organisms in tropical forests. Objective: To describe the psocid diversity in two Colombian Amazonian localities and provide the first data on their vertical distribution in tropical forests. Methods: Monthly samplings of Psocoptera were conducted during new moon periods, from July 2014 to May 2015. Surveys were carried out in two localities of the Putumayo department, Colombia (Puerto Asís and Mocoa), using LED light traps. Results: During the sampling period, 152 species, in 23 genera and nine families were collected. The richest family was Psocidae, followed by Epipsocidae and Lachesillidae. More than 80 % of the species found are still undescribed, and among the described ones, nine represent new records for Colombia, and are shared with neighboring countries in the Amazon basin. Fourteen (9.2 %) species are shared by the two localities surveyed. Puerto Asís is considerably richer than Mocoa. Vertical stratification of Psocoptera was found in both localities, with the canopy being the most species rich stratum. Conclusion: The relatively high diversity of psocidos found, as well as the high number of new species, is an indicator of the need to continue the exploration of these organisms in the rest of the Colombian territory and Andean countries, especially in the Amazon foothills. Stratification of the studied psocoptera community was evident, with the canopy stratum being the richest in species, a trend that continues throughout the year.

Introducción: El conocimiento que se tiene acerca de la diversidad de psócidos en Colombia ha ido en aumento, siguiendo la tendencia mundial, pero el interés principal de la mayoría de estudios se ha centrado en la resolución de aspectos taxonómicos dejando de lado otros aspectos de su biología tales como las interacciones ecológicas de estos organismos en bosques tropicales. Objetivo: Describir la diversidad de psócidos en dos localidades de la Amazonía colombiana y proporcionar los primeros datos sobre su distribución vertical en bosques tropicales. Métodos: Se realizaron muestreos mensuales de psócidos durante los periodos de luna nueva entre julio 2014 y mayo 2015, en dos localidades del departamento de Putumayo, Colombia, utilizando trampas de luz LED. Resultados: Durante el periodo de muestreo, se recolectaron 152 especies, 23 géneros y nueve familias. La familia más rica en especies fue Psocidae, seguida por Epipsocidae y Lachesillidae. Más del 80 % de las especies encontradas no están descritas y entre las descritas, nueve representan nuevos registros para Colombia y son compartidas con países vecinos de la cuenca del Amazonas. Catorce especies (9.2 %) son compartidas por las dos localidades de este estudio; Puerto Asís presentó mayores valores de diversidad que Mocoa. En ambas localidades se encontró una estratificación en la distribución vertical de Psocoptera, siendo el dosel el estrato más rico en especies. Conclusión: La relativamente alta diversidad de psócidos encontrada, así como el alto número de especies nuevas, es un indicador de la necesidad de continuar las exploraciones de estos organismos en el resto del territorio colombiano y países andinos, especialmente en el piedemonte amazónico. Fue evidente una estratificación de la comunidad de psocópteros estudiada, siendo el estrato dosel el más rico en especies, una tendencia que se mantiene a lo largo del año.

Saving DNA from museum specimens: The success of DNA mini-barcodes in haplotype reconstruction in the genus Anastrepha (Diptera: Tephritidae).

The fragmentation of DNA in historical specimens is very common, so obtaining sequences that allow molecular identification and the study of diversity is quite challenging. In this study, we used preserved and fresh specimens of the fruit fly genus Anastrepha, a genus of economic impact of fruit crops of the Neotropic. From these specimens, we evaluated: (1) the success PCR amplification rates of mini-barcodes fragments of the cytochrome c oxidase subunit I (COI) gene, and (2) the usefulness of mini-barcodes in the reconstruction of haplotypes for the identification of species and the diversity analysis. We used 93 specimens from 12 species, which had been preserved in 70% ethanol for more than 20 years. Internal primers were designed in the COI region and primers available in the literature were also evaluated. We obtained amplifications for 62.36% of the samples processed, and reconstructed haplotypes between 171 bp and 632 bp. Variable amplification rates between combinations of primers and between species were obtained, and molecular identification of some museum specimens was achieved. It was also possible to compare the haplotypes obtained in four species from which both fresh and museum samples were available. Our results also show the importance of the adjustment of the primers for the amplification, allowing to amplify fragments of up to 400 bp. The use available resources in biological collections is key to increasing knowledge of species of interest, and by means of the amplification of mini-barcodes, short sequences can be obtained that allow the molecular identification of specimens and the reconstruction of haplotypes with multiple purposes.

The species group Amabilis of the genus Euplocania Enderlein (Psocodea: Psocomorpha: Ptiloneuridae).

We here describe and illustrate 11 species of Euplocania in species group Amabilis from Brazil, Colombia and Ecuador. They raise to 33 the number of species described in the genus. An identification key to the males in the species group is included.

A new genus of Ptiloneuridae, its position within the family, and descriptions of five species (Psocodea, 'Psocoptera').

Upon examination of ptiloneurid specimens recently collected in forests of natural areas and Natural National Parks in Colombia, ten males and seven females were found that represent five species of an undescribed ptiloneurid genus. It differs from the other genera in the family by characters of the wings, hypandrium, phallosome, epiproct, female gonapophyses, and sternum IX. An identification key to the males of the genera of Ptiloneuridae, and a key to the species of the new genus are presented. A test on the validity and monophyly of the new genus, and its position within the family was also conducted.

Morphometric variations of two populations of Anopheles albitarsis F (Diptera: Culicidae) in the Orinoquia region, Colombia / Variaciones morfométricas de poblaciones de Anopheles albitarsis F (Diptera: Culicidae) en la Orinoquía colombiana

Abstract Introduction: Anopheles albitarsis F has been reported with natural infection by Plasmodium falciparum. In the Orinoquía, this species is in sympatry with Anopheles darlingi, a major malaria vector. Objective: To determine morphometric variations in diagnostic characters of wings and hind legs in adult mosquitoes of two populations of the Orinoquia region, where malaria is a major public health issue. Materials and methods: Using linear and geometric morphometry approaches, wing patterns of Costa veins from wild females, females obtained from entomological series and isofamilies were analyzed. Shape and wing size were estimated. The second hindtarsomere was analyzed by means of linear morphometry. Results: Two patterns of spots on the Costa vein of the wings were observed. Significant statistical differences (p<0.05) were found in wing size and shape between wild females and females obtained under laboratory conditions. In the populations studied, a PHD/HP ratio of 1.17-1.19 in the Costa vein and a 0.38-0.54 for the DSIII2/TaIII2 ratio in the second posterior tarsomere of the hind legs, proposed as diagnostic characters. Conclusion. The studied populations of An. albitarsis F showed significant differences between wild females and those obtained under laboratory conditions, not only in wing size, but also in shape. New ranges presented for the diagnostic characters of the species.

Resumen Introducción. Anopheles albitarsis F ha sido registrada con infección natural por Plasmodium falciparum. En la Orinoquía, se encuentra en simpatría con Anopheles darlingi, un vector primario de malaria. Objetivo. Determinar variaciones morfométricas en caracteres diagnósticos de ala y pata posterior de mosquitos adultos procedentes de dos poblaciones naturales de la Orinoquía, donde la malaria constituye un problema de salud pública. Materiales y métodos. Se analizaron, mediante morfometría lineal y geométrica, los patrones de manchas de la vena Costa del ala de hembras silvestres, de hembras obtenidas de series entomológicas y de isofamilias. Se estimó la forma y el tamaño alar. El segundo tarsómero posterior se analizó por morfometría lineal. Resultados. Se observaron dos patrones de manchas de la vena Costa; se encontraron diferencias estadísticamente significativas en el tamaño y forma alar (p<0.05) entre las hembras silvestres y las obtenidas en condiciones de laboratorio. Para las poblaciones estudiadas, se proponen como caracteres diagnósticos la proporción PHD/HP de 1.17-1.19 en la vena Costa y 0.38-0.54 para la proporción DSIII2/ TaIII2 del segundo tarsómero posterior de la pata posterior. Conclusión. Las poblaciones estudiadas de An. albitarsis F presentaron diferencias significativas entre las hembras silvestres y las obtenidas en condiciones de laboratorio, no solo en el tamaño de las alas, sino también en la forma. Se presentan nuevos rangos para los caracteres diagnósticos de la especie.

Systematics and biogeography of Spinoctenus, a new genus of wandering spider from Colombia (Ctenidae)

Among ctenid spiders, ctenines comprise the most diverse subfamily. In this study, a new genus of Cteninae, Spinoctenus, is proposed to include the type species S. yotoco, sp. nov. Ten new species are also described: S. escalerete, S. pericos, S. eberhardi, S. spinosus, S. stephaniae, S. nambi, S. florezi, S. tequendama, S. chocoensis and S. flammigerus. Results of the parsimony and Bayesian phylogenetic analyses using morphological and behavioural characters indicate the monophyly of this genus, closely related to Phoneutria Perty, 1883 and Ctenus Walckenaer, 1805. This genus can be distinguished from the remaining Ctenidae by three unambiguous synapomorphies: embolus with folded process, tegulum with median process, and RTA curved internally close to the cymbium. A dispersal-vicariance biogeographical analysis of the genus in the Andean and Choco regions indicates the origin of Spinoctenus in the Western and Central Andean Cordilleras. From this region, three events of dispersal occurred to the other regions (one to the Choco and two to the Eastern Cordillera), which were subsequently followed by three events of vicariance, suggesting that dispersal and vicariance were equally important in shaping the current distribution patterns of Spinoctenus species. The discovery of this new genus containing a large number of new species in the Andean and Choco regions highlights the current poor knowledge of the Colombian biodiversity.

Systematics and biogeography of Spinoctenus, a new genus of wandering spider from Colombia (Ctenidae)

Among ctenid spiders, ctenines comprise the most diverse subfamily. In this study, a new genus of Cteninae, Spinoctenus, is proposed to include the type species S. yotoco, sp. nov. Ten new species are also described: S. escalerete, S. pericos, S. eberhardi, S. spinosus, S. stephaniae, S. nambi, S. florezi, S. tequendama, S. chocoensis and S. flammigerus. Results of the parsimony and Bayesian phylogenetic analyses using morphological and behavioural characters indicate the monophyly of this genus, closely related to Phoneutria Perty, 1883 and Ctenus Walckenaer, 1805. This genus can be distinguished from the remaining Ctenidae by three unambiguous synapomorphies: embolus with folded process, tegulum with median process, and RTA curved internally close to the cymbium. A dispersal-vicariance biogeographical analysis of the genus in the Andean and Choco regions indicates the origin of Spinoctenus in the Western and Central Andean Cordilleras. From this region, three events of dispersal occurred to the other regions (one to the Choco and two to the Eastern Cordillera), which were subsequently followed by three events of vicariance, suggesting that dispersal and vicariance were equally important in shaping the current distribution patterns of Spinoctenus species. The discovery of this new genus containing a large number of new species in the Andean and Choco regions highlights the current poor knowledge of the Colombian biodiversity.

New species of Colombian Triplocania Roesler (Psocodea: 'Psocoptera': Ptiloneuridae).

Based on a survey of the genus Triplocania (Psocodea: 'Psocoptera') in forests of natural areas and Natural Parks in Colombia, 46 species were found, 42 of which are new to science and are here described and illustrated. Added to T. colombiana García Aldrete, the number of species of Triplocania now known in Colombia is raised to 47. To date, 40 species described in this paper are recorded only in Colombia, and seven species are shared with neighboring countries, three with Brazil, one with Venezuela, two with Ecuador, and one with Ecuador and Brazil. Females of three previously known species are also described. An identification key to the species of Triplocania present in Colombia is included.

Five new species of the genus Euplocania Enderlein (Psocodea, 'Psocoptera', Psocomorpha, Ptiloneuridae) from Colombia.

Five new species of Euplocania from Colombia belonging in four species groups are here described and illustrated. They increase to 22 the number of species described in the genus, thirteen of which are endemic to Colombia, with one species shared with Brazil and Peru. Three new species groups of Euplocania are here diagnosed. A key to the identification of males of Colombian Euplocania is included.

Genetic diversity and population structure of Anastrepha striata (Diptera: Tephritidae) in three natural regions of southwestern Colombia using mitochondrial sequences.

Anastrepha striata is widely distributed across the Americas and is a pest of economically important crops, especially crops of the Myrtaceae family. Insect population structures can be influenced by the presence of physical barriers or characteristics associated with habitat differences. This study evaluated the effect of the Western Andes on the population structure of A. striata. Individuals were collected from Psidium guajava fruits from three natural regions of southwestern Colombia (Pacific Coast, mountainous region and the inter-Andean valley of the Cauca River). Based on a 1318 bp concatenated of the genes Cytochrome Oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), 14 haplotypes with few changes among them (between 1 and 3) were found. There was only one dominant haplotype in all three regions. No genetic structure associated with the three eco-geographical regions of the study was found. Moreover, the Western Andes are not an effective barrier for the genetic isolation of the populations from the Pacific Coast compared with the inter-Andean valley populations. This genetic homogeneity could be partially due to anthropogenic intervention, which acts as a dispersal agent of infested fruits. Another hypothesis to explain the lack of structure would be the relatively recent arrival of A. striata to the region, as indicated by an analysis of the demographic history, which reveals a process of population expansion. This study represents the first attempt to understand the population genetics of A. striata in Colombia and could contribute to the integral management of this pest.

Two new species of Edmockfordia García Aldrete (Psocodea, 'Psocoptera', Epipsocidae), from Valle del Cauca, Colombia, and description of the female E.chiquibulensis García Aldrete.

Two new species of Edmockfordia García Aldrete, from Valle del Cauca, Colombia, and the female of Edmockfordiachiquibulensis García Aldrete, are described and illustrated. A key to the species of Edmockfordia is included; the genus was previously known only from Belize. The genus is re-diagnosed to include female characters. The distribution of the genus is considerably widened, from Belize to northeastern South America.

Descriptions and records of Cladiopsocidae and Dolabellopsocidae (Insecta: Psocodea: 'Psocoptera') from Valle del Cauca and National Natural Park Gorgona, Colombia.

The results of a survey on the species diversity of the families Cladiopsocidae and Dolabellopsocidae (Psocodea: 'Psocoptera': Psocomorpha: Epipsocetae) in Valle del Cauca and in the National Natural Park (NNP) Gorgona, Colombia, are presented. The specimens studied were collected in the context of two scientific projects, in which 12 species in the two families were identified, five in Cladiopsocidae and seven in Dolabellopsocidae. In the first family, Cladiopsocus presented a new record in the country and four new species; in the latter, two genera were identified, Dolabellopsocus, with three new species and two new records, and Isthmopsocus, with two new species. This study presents the description of the nine new species from Valle del Cauca and NNP Gorgona, the new records for Colombia and identification keys to the neotropical species of both families.

New genera of Lachesillidae (Psocodea: 'Psocoptera': Eolachesillinae: Graphocaeciliini) from Valle del Cauca, Colombia.

Two new related Colombian genera of Lachesillidae, including three new species, from the Department of Valle del Cauca, are here described and illustrated. They belong in the tribe Graphocaeciliini (Eolachesillinae). The new genera differ from the other genera in the tribe lacking ocelli, by characters of the male clunium, phallosome, endophallus, paraprocts, and female subgenital plate, gonapophyses and IX sternum. The types are deposited in the Entomological Museum of the Universidad del Valle (MUSENUV), in Santiago de Cali, Colombia.

Genetic variability of five Periplaneta americana L. (Dyctioptera: Blattidae) populations in southwestern Colombia using the AFLP molecular marker technique.

The genetic structure of five Periplaneta americana (L.) populations from three cities (Cali, Popayán and Buenaventura) located in southwestern Colombia was estimated using the AFLP molecular marker technique. A set of 174 loci were analyzed, 120 of which were polymorphic. Genetic diversity was estimated at different geographic levels for the total population and between cities. Genetic diversity averaged 0.32. The largest gene flow was detected between two population from Cali (F ST=0.088; Nm=2.6) and the lowest from the third population in this city (Univalle) and Popayán and Buenaventura (F ST=0.13; Nm=1.6 and F ST=0.12; Nm=1.8, respectively). The individuals' low mobility, the population' geographic separation, and possibly genetic drift have resulted in the population of this cockroach species having a significant degree of structuring, both between cities (Phist=0.13; P<0.001) and within them (Phist=0.87; P<0.001). These results suggest a high degree of subdivision within the P. americana population studied.

Genetic variability of Five Periplaneta americana L. (Dyctioptera: Blattidae) populations in southwestern Colombia using the AFLP molecular marker technique

The genetic structure of five Periplaneta americana (L.) populations from three cities (Cali, Popay¨¢n and Buenaventura) located in southwestern Colombia was estimated using the AFLP molecular marker technique. A set of 174 loci were analyzed, 120 of which were polymorphic. Genetic diversity was estimated at different geographic levels for the total population and between cities. Genetic diversity averaged 0.32. The largest gene flow was detected between two population from Cali (F ST = 0.088; Nm = 2.6) and the lowest from the third population in this city (Univalle) and Popay¨¢n and Buenaventura (F ST = 0.13; Nm = 1.6 and F ST = 0.12; Nm = 1.8, respectively). The individuals' low mobility, the population' geographic separation, and possibly genetic drift have resulted in the population of this cockroach species having a significant degree of structuring, both between cities (¦µst = 0.13; P < 0.001) and within them (¦µst = 0.87; P < 0.001). These results suggest a high degree of subdivision within the P. americana population studied.

Resistencia a deltametrina de cepas de Blattella germanica (Dictyoptera: Blattellidae) en la ciudad de Cali, Colombia / Deltamethrin resistance in Blattella germanica (Dyctioptera: Blattellidae) strains from Cali city, Colombia

estatus de plaga se debe a su gran capacidad de infestación y al desarrollo de resistencia a insecticidas en corto plazo. Como no se han realizado estudios locales previos es necesario determinar el estado de la resistencia a diferentes insecticidas, datos que servirán de referencia para establecer un sistema de vigilancia que mejorará las medidas de control. OBJETIVO: establecer el estado de la resistencia al piretroide deltametrina en poblaciones de B. germanica de la ciudad de Cali (Colombia). MÉTODOS: se colectaron 5 cepas de B. germanica en la ciudad de Cali, en lugares con altos niveles de infestación. Se evaluaron adultos machos y hembras de esta especie expuestos al piretroide deltametrina mediante bioensayos con botellas. RESULTADOS: en machos y hembras de B. germanica de la ciudad de Cali, se determinó la CK50 a deltametrina. Los machos fueron más susceptibles que las hembras. Dos de las cepas evaluadas presentaron un elevado factor de resistencia (Club deportivo: FR= 22 para hembras y 13 para machos; Plaza de mercado 2: FR= 14,33 para hembras y 13,5 para machos) y corresponden a áreas con una historia de mayor frecuencia de aplicación de insecticidas. La cepa más susceptible (Hospital: CK50= 0,03 mg/mL y 0,02 mg/mL para hembras y machos, respectivamente) no ha estado sometida a una presión de selección constante con insecticidas, debido a los riesgos que implican estas aplicaciones en lugares donde se encuentran pacientes con tratamientos médicos delicados. CONCLUSIONES: estos resultados constituyen el primer registro de resistencia a deltametrina en poblaciones de B. germanica en Cali (Colombia). En las poblaciones susceptibles se puede continuar la aplicación de deltametrina dentro de un programa de vigilancia y control permanente.

INTRODUCTION: Blattella germanica is one of the most important pests worldwide. The pest status is due to their great infestation capacity and ability to develop relative short-term insecticide resistance. Since local studies about resistance on B. germanica to any insecticide have not been made, it is necessary to find out the state of resistance of this species to insecticides. The collected data will allow establishing an effective monitoring system that will improve some control measures already in place. OBJECTIVE: to ascertain the level of resistance to deltamethrin in some populations of B. germanica from Cali (Colombia). METHODS: five different strains of B. germanica were collected in Cali (Colombia), in areas with high levels of infestation. Adult males and females were exposed and tested to deltamethrin using bottled bioassays. RESULTS: KC50 to pyrethroid deltametrhin was determined in adult males and females of B. germanica from Cali city. Males were more susceptible than females. Two evaluated strains showed a high resistance factor (Sports Club: RT=22 for females and 13 for males; Market 2: RT=14.33 for females and 13.50 for males) representing areas with history of highly frequent insecticide application. The most susceptible strain (Hospital: KC50=0.03 mg/mL and 0.02 mg/mL for females and males respectively) has not been submitted to constant selection pressure with insecticides because of the likely risks of this type of application for human health in places where critically-ill patients are treated.. CONCLUSIONS: this study contributed the first evidence of deltamethrin resistance in populations of B. germanica from Cali (Colombia). It is possible to carry on using deltamethrin in susceptible populations within an ongoing monitoring and control program.

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.