Culicoides species adults were collected in light traps located on the coast of Elota, Sinaloa, Mexico, in February 2022. All specimens were females, and it was determined based on their morphology that they belonged to the Culicoides variipennis complex. The identification of the species was carried out by means of a comparative analysis of genetic sequences of the cytochrome c oxidase subunit I gene, which resulted in C. occidentalis, this being its first report in Sinaloa and its third nationwide.
The Culicoides variipennis are classified within the subgenus Monoculicoides, with 6 distinct species currently recognized within the group (C. albertensis Wirth and Jones, C. australis Wirth and Jones, C. sonorensis Wirth and Jones, C. occidentalis Wirth and Jones, C. variipennis Coquillett, and C. mullensi Shults and Borkent (Shults 2021). Morphological characterization of adults, exclusively C. occidentalis, represents a challenge for researchers because of their similarities with female C. sonorensis and male C. variipennis; however, the heterogeneity of developmental habitats and historical geographic distributions of the species may separate them ecologically and thus be important factors in directing efforts to identify the taxa (Holbrook et al. 2000).
Historical records of the C. variipennis complex indicate that they are primarily Nearctic species with distributions limited to North America (Wirth and Jones 1957, Holbrook et al. 2000, Huerta et al. 2012). In the USA, C. sonorensis has been listed as a competent vector for bluetongue virus (BTV) transmission, being primarily associated with livestock production, particularly cattle (McGregor et al. 2022). The BTV infection rates in field-collected populations of C. variipennis and C. occidentalis from different US sites were less than 3%, indicating that they are species with low probability of transmitting the virus to the animal host (Tabachnick 1996); however, studies, such as in vitro infections, that test for vector competence between the species are lacking (McGregor et al. 2022).
In Mexico the complex is distributed in 13 states, with C. sonorensis being the most widely reported (Table 1; Wirth and Jones 1957; Borkent and Grogan 2009; Holbrook et al. 2000; Huerta et al. 2012, 2020). Culicoides occidentalis has been recorded in northwestern Mexico in the state of Baja California, Baja California Sur, as well as in central Mexico in Puebla (Table 1; Wirth and Jones 1957, Alcocer et al. 2001, Huerta et al. 2012). At present, these species have not been related to the BTV transmission, although the virus is widely established in the national territory, affecting sheep and cattle (Suzan et al. 1983, Vilchis et al. 1986, Rojas-Anaya et al. 2023).
Culicoides adults were collected with light traps located on the coast of Elota, Sinaloa (trap 1: 23°54ʹ18″N, 106°57ʹ29″W, elevation 5 miles above sea level [masl)] trap 2: 23°54ʹ20″N, 106°57ʹ26″W, elevation 3 masl; trap 3: 23°54ʹ32.7″N, 106°57ʹ38″W, elevation 3 masl), at twilight hours (5:30 to 7:30 p.m.) in February 2022. The bags collected from the traps holding the specimens were placed in 200-ml plastic vessels containing 100 ml of 70% ethanol and transferred to the entomological collection of the Faculty of Biology, Autonomous University of Sinaloa, for the corresponding studies.
Forty-seven Culicoides adults were isolated from the collection bags (from light traps 1, 2, and 3, 14, 20, and 13 specimens were collected, respectively). Specimens were identified by morphology according to the dichotomous keys proposed by Holbrook et al. (2000). The total number of Culicoides collected corresponded to females characterized by a wide third palpal segment and a clearly pronounced sensory pit (Fig. 1A, 1B). Since these characteristics are similar in C. sonorensis and C. occidentalis (Holbrook et al. 2000), and both species have been recorded from states near and adjacent to Sinaloa (Table 1; Wirth and Jones 1957, Holbrook et al. 2000, Borkent and Grogan 2009), morphology in this case is unreliable for species identification. In this regard the specimens were processed by molecular techniques for taxonomic discrimination.
The molecular analysis was carried out with mitochondrial DNA because it has been reported to be reliable for the identification of C. occidentalis from the rest of the species of the C. variipennis complex (Shults 2021, Shults et al. 2022). Fifteen specimens were placed in 1.5-ml plastic tubes and macerated with a sterile pistil. Subsequently, total DNA extraction from adults was performed with the Wizard SV Genomic DNA Purification System kit (Promega, Madison, WI) according to the manufacturer’s instructions. A partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) as a molecular marker. The GoTaq Green Master Mix kit (Promega) was used under the manufacturer’s conditions, as well as the oligonucleotides LCO sense (5ʹ-ggtcaacaaatcataaagatattgg-3ʹ) and HCO antisense (5ʹ-taaacttcagggtgaccaaaaaaaatca-3ʹ; Folmer et al. 1994). The final reaction was 12.5 μl (6.25 μl of PCR mix, 0.5 μl of each oligonucleotide [10 pMol], 2 μl of DNA, and 3.25 nuclease-free water) and amplified with the following conditions: 4 min at 95°C, followed by 34 cycles at 95°C for 40 sec, 55°C for 40 sec, and 72°C for 50 sec, and a final extension of 72°C for 5 min. The amplicon pools of Culicoides were sequenced at Macrogen (Seoul, Korea) and compared with C. occidentalis sequences previously reported in GenBank (Shults et al. 2022) with the BioEdit program (version 7.2.5) (Hall 2011).
Specimens were collected within a few meters of the temporary salt marsh at Ceuta (range approximately 50–100 m), which is consistent with the developmental habitats of immature stages (Schmidtmann et al. 2000). The results of PCR and sequence analysis verified that the species collected at the study sites corresponded to C. occidentalis, this by observing a >99% similarity with sequences previously reported in GenBank (accession numbers OL604770.1 and OL604771.1) for this species. This is its first record in Sinaloa. The comparison of sequences with other species of the C. variipennis complex historically reported in Mexico was approximately 97% (Fig. 1C).
In Sinaloa the Culicoides genus is poorly known, and, according to historical records, 4 species, C. blantoni, C. phlebotomus, C. cancer, and C. furens, are distributed (Huerta et al. 2012, Mendez-Andrade and Ibáñez-Bernal 2023). The dipteran C. occidentalis is 1 of the 3 species of the C. variipennis complex that has been least studied in Mexico, and its record in Sinaloa opens the possibility of understanding its ecology through population comparisons in different geographical areas, as well as elucidating its status as a vector of etiological agents with medical and veterinary importance, which is still uncertain (McGregor et al. 2022). In this study we report for the first time the presence of C. occidentalis in the state of Sinaloa, extending its distribution to 3 states of the Mexican Republic.
Posgrado en Ciencias Biológicas, Facultad de Biología, Universidad Autónoma de Sinaloa, Avenida de las Américas y Boulevard Universitarios S/N, Culiacán, Sinaloa, Mexico 80013.
Laboratorio de Conservación de la Fauna Silvestre, Facultad de Biología, Avenida de Las Américas y Boulevard Universitarios S/N, Culiacán, Sinaloa, Mexico 80013.