Anopheles nuneztovari

Anopheles nuneztovari is a Neotropical cryptic species complex in the Albimanus Section of subgenus Nyssorhynchus within the genus Anopheles, distributed across eastern Panama, Colombia, Venezuela, the Guianas, Ecuador, Peru, Bolivia, and the Brazilian Amazon. Originally described by Arnoldo Gabaldón in 1940 from specimens collected in San Carlos, Cojedes State, western Venezuela, and named in honor of the Venezuelan entomologist Manuel Núñez Tovar, An. nuneztovari sensu lato has long served as a classic model in medical entomology for studying geographic divergence in vector behavior and chromosome evolution. Whereas trans-Andean and foothill populations of An. nuneztovari sensu stricto in western Venezuela and northern/Pacific Colombia are anthropophilic, late-night indoor biters that act as primary vectors of human malaria (Plasmodium vivax and Plasmodium falciparum), cis-Andean populations across the central Amazon Basin—comprising the resurrected sibling species An. goeldii and An. dunhami—exhibit predominantly exophagic, crepuscular, and zoophilic behavior with localized vectorial importance along disturbed river margins.

Morphological Placement in the Albimanus Section

Within the Albimanus Section of Nyssorhynchus, An. nuneztovari s.l. belongs to the Oswaldoi Group. Adult females (wing length 3.0–3.9 mm) possess snow-white hind tarsomeres 3 and 4 (Ta-III_3–4), a basal dark ring on hind tarsomere 5 (Ta-III₅), and dark basal scaling covering 20% to 35% of hind tarsomere 2 (Ta-III₂). On the wing costa, the humeral pale spot (HP) is large—typically exceeding 1.5 to 2.0 times the length of the prehumeral dark spot (PHD)—while the subcostal pale spot is well developed, helping differentiate An. nuneztovari s.l. from sympatric Anopheles aquasalis and An. oswaldoi. However, overlapping wing and tarsal ratios across the Oswaldoi Group mean that definitive identification requires examination of the male phallosome (specifically the prominent, well-sclerotized subapical leaflets of the aedeagus in An. nuneztovari s.s. versus reduced or absent leaflets in An. goeldii and An. dunhami) or molecular sequencing.

Chromosome X Inversion Polymorphism (Cytotypes A, B, and C)

In pioneering polytene chromosome studies during the 1970s and 1980s, J. B. Kitzmiller, George Frizzi, and Jan Conn discovered that An. nuneztovari s.l. comprises three distinct cytogenetic forms defined by fixed paracentric inversions on the X chromosome (designated inversions X_a, X_b, and X_c) as well as autosomal inversion polymorphisms on chromosomes 2 and 3:

• Cytotype A: Prevalent across the central and eastern Brazilian Amazon, carrying the standard X_a arrangement.
• Cytotype B: Restricted to western Venezuela (Barinas, Apure, Táchira, and Zulia states along the eastern and western flanks of the Mérida Andes), fixed for a complex homozygous inversion on the X chromosome.
• Cytotype C: Distributed across northern and western Colombia (the Magdalena, Cauca, Sinú, and Atrato river valleys and Chocó Pacific lowlands) and eastern Panama, differentiated by chromocenter structure and accessory X-chromosome banding.

Taxonomic Resurrection of Anopheles goeldii and Anopheles dunhami

Subsequent integrative analyses combining nuclear white and CAD gene sequences, ribosomal ITS2 haplotypes, mitochondrial COI barcoding, and male genitalia morphometrics by Calado, Sallum, Scarpassa, and Conn demonstrated that nominal An. nuneztovari s.l. encompasses at least three evolutionary species:

Although Cytotypes B (western Venezuela) and C (Colombia) exhibit slight chromosomal and microsatellite differentiation across the Serranía del Perijá and Cordillera Oriental, cross-mating experiments and nuclear gene trees indicate that they remain conspecific geographic populations of An. nuneztovari sensu stricto.

Turbid, Sunlit, and Peridomestic Breeding Sites

Across both its Andean-foothill and Amazonian distributions, An. nuneztovari s.l. is an ecological generalist that colonizes sunlit or partially shaded freshwater bodies ranging from permanent lakes and vegetated river backwaters to temporary, turbid, human-created excavations. Unlike Anopheles darlingi, which requires deep, stable, clear water margins, larvae of An. nuneztovari s.l. tolerate suspended clay turbidity, fluctuating water levels, and higher water temperatures (up to 31–33 °C). Common larval habitats include:

• Livestock pastures and wheel ruts: Rain-filled cattle hoofprints, tractor tire tracks, and muddy pasture ponds bordered by Brachiaria and Paspalum grasses in the Colombian Urabá–Bajo Cauca region and Venezuelan Barinas foothills.
• Fishponds, brick pits, and roadside borrow ditches: Excavated clay pits and earthen aquaculture ponds in peri-urban settlements.
• Hydroelectric reservoir margins: Following the impoundment of major Amazonian and Suriname rivers (such as the Tucuruí Reservoir in Pará, the Balbina Reservoir in Amazonas, and the Brokopondo Reservoir in Suriname), floating mats of Salvinia, Pistia, and Eichhornia triggered massive population explosions of An. goeldii / An. nuneztovari s.l.

Late-Night Endophagy in the Andes vs. Crepuscular Zoophily in Amazonia

The hallmark of the An. nuneztovari complex is the sharp behavioral dichotomy between trans-Andean/Andean-foothill An. nuneztovari s.s. and cis-Andean Amazonian An. goeldii:

1. Andean Foothills and Colombian/Venezuelan Valleys (An. nuneztovari s.s.): In the Catatumbo basin, Barinas foothills, Bajo Cauca, Córdoba, and Chocó, An. nuneztovari s.s. is strongly anthropophilic (Q_h = 0.35–0.75) and feeds avidly on humans both indoors (endophagy) and in peridomestic courtyards throughout the night, reaching a pronounced biting peak between 22:00 and 02:00 h when residents are asleep. However, even after feeding indoors, over 80% of engorged females exit houses before dawn (exophily) to rest on outdoor vegetation 50 to 200 meters away.
2. Amazon Basin (An. goeldii and An. dunhami): Throughout most of the Brazilian Amazon, An. goeldii exhibits a sharp crepuscular biting peak at sunset (18:00–19:30 h), feeds predominantly outdoors on cattle, pigs, and wild mammals (Q_h < 0.15), and rarely enters houses after 20:00 h. Nevertheless, when human settlements encroach directly upon hydroelectric lakes or deforested river margins lacking livestock, high biting densities (>100 bites per person per evening) enable An. goeldii to sustain focal transmission.

Sporozoite Infections and Insecticide Resistance Challenges

Natural sporozoite infections with Plasmodium vivax (both VK210 and VK247), Plasmodium falciparum, and Plasmodium malariae have been repeatedly confirmed by ELISA and PCR in An. nuneztovari s.s. across Venezuela and Colombia (where it accounts for a major share of transmission alongside An. darlingi and Anopheles albimanus) as well as in An. goeldii in Pará and Amapá, Brazil.

During the Global Malaria Eradication campaign in the 1960s and 1970s, Arnoldo Gabaldón documented "refractory malaria" in western Venezuela caused by An. nuneztovari s.s.: because females entered DDT-sprayed houses late at night, bit sleeping occupants, and exited rapidly without resting on sprayed walls, indoor residual spraying failed to interrupt transmission. In addition, intensive agricultural pesticide applications in northern Colombia (Córdoba and Antioquia) and western Venezuela have selected for organophosphate resistance (insensitive acetylcholinesterase ace-1) and metabolic pyrethroid/DDT resistance mediated by elevated glutathione S-transferases and mixed-function oxidases. Control strategies in An. nuneztovari s.s. endemic foci therefore rely on long-lasting insecticidal nets (which intercept late-night indoor biters at the moment of host contact), peri-domestic larval source management with Bacillus thuringiensis israelensis, and radical cure of P. vivax carriers.

Why did indoor DDT spraying fail to eliminate malaria transmitted by Anopheles nuneztovari in western Venezuela?

Although An. nuneztovari sensu stricto enters houses late at night (22:00–02:00 h) to bite sleeping humans, it exhibits exophilic resting behavior and irritability to DDT, flying out of the dwelling soon after taking a blood meal to rest on outdoor vegetation rather than remaining on sprayed interior walls long enough to absorb a lethal insecticide dose.

How do Amazonian populations of the Anopheles nuneztovari complex differ behaviorally from Colombian and Venezuelan populations?

Populations in western Venezuela and Colombia (An. nuneztovari sensu stricto) bite humans indoors and outdoors late at night and serve as primary malaria vectors, whereas Amazonian populations (now classified primarily as the sibling species An. goeldii and An. dunhami) bite mostly outdoors at dusk (18:00–19:30 h) and feed preferentially on livestock and wild mammals.

How can Anopheles dunhami be distinguished from Anopheles nuneztovari sensu stricto?

In the male genitalia, An. nuneztovari s.s. possesses prominent, sclerotized subapical leaflets at the tip of the aedeagus, whereas An. dunhami lacks aedeagal leaflets and displays a distinct subapical plate on the ventral claspette; molecularly, the two species are cleanly separated by ITS2 sequences and mitochondrial COI barcodes.

1. Gabaldón, A. (1940). Estudios sobre Anofelinos. Serie I. 1. Descripción de Anopheles (Nyssorhynchus) nuñez-tovari n. sp. y consideraciones sobre una sub-división del grupo Nyssorhynchus (Diptera, Culicidae). Publicaciones de la División de Malariología, Caracas, 5, 3–7.
2. Conn, J., Puertas, Y. R., & Seawright, J. A. (1993). A new cytotype of Anopheles nuneztovari from western Venezuela and Colombia. Journal of the American Mosquito Control Association, 9(3), 294–301. https://www.biodiversitylibrary.org/page/16244864
3. Calado, D. C., Foster, P. G., Bergo, E. S., dos Santos, C. L. S., Galardo, A. K. R., & Sallum, M. A. M. (2008). Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and appraisal of An. nuneztovari molecular variability. Memórias do Instituto Oswaldo Cruz, 103(8), 791–799. doi:10.1590/S0074-02762008000800009
4. Scarpassa, V. M., & Conn, J. E. (2011). Mitochondrial DNA detects a complex evolutionary history with Pleistocene epoch divergence for the neotropical malaria vector Anopheles nuneztovari sensu lato. American Journal of Tropical Medicine and Hygiene, 85(5), 857–867. doi:10.4269/ajtmh.2011.11-0150
5. Naranjo-Díaz, N., Rosero, D. A., Rua-Uribe, G., Luckhart, S., & Correa, M. M. (2013). Abundance, behavior and entomological inoculation rates of anthropophilic anophelines from a primary Colombian malaria endemic area. Parasites & Vectors, 6, 61. doi:10.1186/1756-3305-6-61