ABSTRACT
In July 2022, adult female Culicoides were collected from San Buenaventura (54 specimens) and Urique (3 specimens) in Chihuahua, Mexico. Culicoides reevesi and Culicoides debilipalpis were new records for the state, with C. reevesi also being a first for Mexico. The study highlights DNA barcode identification challenges and emphasizes the need for ongoing surveillance along the Mexico-USA border.
Culicoides is a widespread genus with 1,347 species globally, 84 in Mexico, but none previously recorded in Chihuahua (Mendez-Andrade and Ibáñez-Bernal, 2023). Accurate identification is key for understanding species distribution and vector surveillance. Deoxyribonucleic acid (DNA) barcoding, especially with cytochrome c oxidase subunit I (COX1) gene, is effective for species identification, as shown with Culicoides occidentalis (Wirth and Jones) in Sinaloa (Cazares-Martínez et al. 2023). However, in cases like C. sonorensis (Wirth and Jones) and C. variipennis (Coquillett) in Ontario (Jewiss-Gaines et al. 2017), the COX1 alone was insufficient, requiring the eukaryotic elongation factor 1a (EF1α) and internal transcribed spacer 1 (ITS1) markers. This study uses taxonomic methods and DNA barcoding to identify Culicoides in Chihuahua, compares their DNA barcodes within Mexico, and stresses the need for ongoing border surveillance.
In July 2020 and 2021, during the rainy season, Culicoides were collected in Chihuahua using human landing catches near the Santa María River in Buenaventura (29°50′37″N, 107°28′19″W; 1553 masl) and the Urique River in Urique (27°13′06″N, 107°54′48″W; 550 masl) from 4 to 8 pm, during peak biting activity. Collected specimens were preserved in 96% ethanol and stored at –20°C for morphological identification and DNA barcoding. Morphospecies were identified using Huerta et al. (2020) keys. Wing photos were taken with a Carl Zeiss microscope and Nikon DS-Ri2 camera, with some specimens deposited at the Instituto de Diagnóstico y Referencia Epidemiológicos, Mexico. The amplification of the 658-bp barcode region of the mitochondrial ccox1) gene was performed following the methodology of Ramos-Lagunes et al. (2024). The polymerase chain reaction (PCR) products were visualized, purified, and then bidirectionally sequenced using Sanger sequencing. A GenBank search retrieved Culicoides spp. sequences using keywords like Mexico, COX1, Culicoides, and C. reevesi (Wirth). Then, the sequences were aligned, and genetic distances were calculated using Tamura’s 3-parameter model. A phylogenetic Bayesian tree was constructed using C. reevesi sequences from California and other Culicoides sequences from Mexico, applying the protocol of Ramos-Lagunes et al. (2024).
More than 200 specimens of Culicoides were collected in San Buenaventura, Chihuahua, but only 54 were used for this study. The remainer were used for viral metagenomics analysis. Corresponding to Urique location, only 3 specimens were collected. Culicoides reevesi was identified in San Buenaventura, whereas C. (Haematomyidium) debilipalpis (Lutz) was collected in Urique, Chihuahua (Fig. 1). Culicoides reevesi is a typical member of C. leoni species-group; their sub-genus is unplaced (Mendez-Andrade and Ibáñez-Bernal 2023). Three species in this species-group are known from Mexico namely C. gabaldoni (Ortiz), C. glabellus (Wirth) and C. leoni (Barbosa). Culicoides reevesi is mainly distinguished from these related species by the antennal flagella with flagellomeres 9–10 distinctly shorter than flagellomeres 8 or 11; sensilla coeloconica are present on flagellomeres 1 and 6 to 8. The male genitalia have well-defined parameres in the ventral lobe and a fine fringe of denticles on the distal portions (Grogan et al. 2004). Both species are new records from the state of Chihuahua, and C. reevesi is a new national record for Mexico.
Females of Culicoides reevesi (above) and Culicoides debilipalpis (below); dorsal view of full body (center), antennae and maxillary palps (left) and wings showing taxonomical characters (right).
Females of Culicoides reevesi (above) and Culicoides debilipalpis (below); dorsal view of full body (center), antennae and maxillary palps (left) and wings showing taxonomical characters (right).
Culicoides debilipalpis belongs to the subgenus Haematomyidium (Goeldi). In Mexico, there are 5 species included to this subgenus: Culicoides eadsi (Wirth), C. ginesi (Ortiz), C. kettlei (Breidenbaugh and Mullens), and C. paraensis (Goeldi). Culicoides debilipalpis is closely related to C. eadsi. Morphometric characteristics, such as distinct features at the base of the mediocubital fork of the wing and the aedeagus of the male genitalia, can distinguish females of both species.
Nineteen C. reevesi COX1 DNA barcodes were obtained and deposited in GenBank (accession numbers PQ198017–PQ198035). Culicoides debilipalpis was identified morphologically, but it was not possible to obtain DNA from the single remaining specimen collected. The C. reevesi COX1 sequences from Mexico showed 90.36–98.94% identity in GenBank nucleotide basic local alignment search tool (nBLAST). A Bayesian tree (Fig. 2), including 7 US and 19 Mexican C. reevesi sequences, confirmed species identification. An additional C. reevesi sequence from the USA formed a highly divergent group, possibly indicating a separate group within the species. However, misidentification is also a possibility. To avoid compromising the statistical robustness of the genetic analysis, it was excluded, as there was insufficient evidence to classify it as a distinct evolutionary group. The overall genetic variation in C. reevesi from Mexico and the USA was 0.01 ± 0.00 SE. Bayesian analysis grouped other Mexican species according to nBLAST identification, with C. hylas (Macfie) and C. jamanciensis/paolae forming distinct groups. However, C. insignis (Lutz) showed 2 groups, suggesting a possible species complex, though this conclusion is provisional, as incomplete taxonomic identification in the barcode of life data (BOLD) system may lead to its rejection.
Phylogenetic tree of Bayesian inference for Culicoides reevesi from US and Mexico based on the COX1 DNA barcoding region. The tree was rooted using Aedes aegypti as an outgroup. Numbers in selected nodes are labelled with posterior probability values (P = 0 – 1). *Culicoides species undetermined. ** In this group, the COX1 sequences are labeled as C. jamaiciensis/C. paolae. This ambiguity likely stems from misidentifications in the taxonomic classification.
Phylogenetic tree of Bayesian inference for Culicoides reevesi from US and Mexico based on the COX1 DNA barcoding region. The tree was rooted using Aedes aegypti as an outgroup. Numbers in selected nodes are labelled with posterior probability values (P = 0 – 1). *Culicoides species undetermined. ** In this group, the COX1 sequences are labeled as C. jamaiciensis/C. paolae. This ambiguity likely stems from misidentifications in the taxonomic classification.
This study reports the first records of 2 Culicoides species in Chihuahua, including C. reevesi, marking its first record in Mexico and expanding the known Culicoides fauna in the country. Culicoides reevesi was originally described in California by Wirth (1952). This species is classified in the catalog of the New World North of Mexico in the C. leoni group (Borkent and Grogan, 2009). This species is present in the southwestern United States; it is known from Arizona, California, New Mexico, and Utah (Grogan et al. 2004). There were no previous records of C. reevesi in Mexico (Mendez-Andrade and Ibáñez-Bernal 2023); however, based on its known current distribution, it is plausible to find this species in the northern region of Mexico, particularly in Chihuahua. Culicoides reevesi is known as a significant human-biting pest (Grogan et al. 2004), with black-tailed deer, Odocoileus hemionus (Rafinesque), as its only other reported host (Hopken et al. 2017). Although no pathogen transmission to humans or animals has been recorded, further studies on these arthropods are needed. Culicoides debilipalpis is widely distributed from the USA to Mexico, Central America, and Argentina (Borkent and Spinelli, 2007). First recorded in Mexico by Macfie (1948) based on specimens from Chiapas, it was later confirmed in Veracruz, Yucatan, and Oaxaca (Huerta et al. 2020). There were no previous records in Chihuahua, making this the first documented presence of C. debilipalpis in the state. This species is a known vector of arboviruses affecting livestock and wild ruminants, such as Epizootic Hemorrhagic Disease virus (EHDV) (Jimiénez-Cabello et al. 2023) and Bluetongue virus (BTV) (McGregor et al. 2022). Given its role in pathogen transmission, further studies on pathogen screening and Culicoides ecology are crucial.
Phylogenetic analysis for Culicoides species identification using COX1 DNA barcoding has been widely discussed (Cazares-Martínez et al. 2023, Jewiss-Gaines et al. 2017). In Mexico, DNA barcoding was used to identify the C. variipennis complex (Cazares-Martínez 2023). Our analysis showed no significant genetic variation within C. reevesi, indicating no differentiation between specimens from California, USA, and Chihuahua, Mexico (Fig. 1). Identifying Culicoides species in Mexico is challenging because of incomplete taxonomic data and lack of voucher specimens in GenBank. Although DNA barcoding aids species identification, morphometric taxonomy remains crucial, as genetic distances can be misleading, sometimes under 2% between species or over 2% within species (Cazares-Martínez 2023).
In conclusion, this study improves the understanding of the distribution of C. reevesi and C. debilipalpis in Mexico, reporting their first records in Chihuahua and the first national record of C. reevesi.
Rodolfo González-Peña thanks CONAHCYT, Mexico, for his doctoral scholarships (No. 842817), which supported the completion of this study.
REFERENCES CITED
Author notes
Laboratorio de Arbovirología, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi” Universidad Autónoma de Yucatán, Mérida, México
Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua, México
Laboratorio de Entomología, Instituto de Diagnóstico y Referencia Epidemiológicos, Ciudad de México, México
Animal and Plant Health Agency, Virology Department, Rabies and Wildlife Zoonoses Research Group, Addlestone, United Kingdom
Laboratorio Medicina de la Conservación, Centro de Biotecnología Genómica del Instituto Politécnico Nacional, Reynosa, Tamaulipas, México
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chihuahua, México