Cuterebra baeri Infecting Grey-Legged Night Monkeys (Aotus griseimembra) and Red Howler Monkeys (Alouatta seniculus) in Colombia

Myiases are neglected parasitic infections, caused by the larval stages of some fly species, affecting a wide range of mammals as a part of the life cycle. In nonhuman primates (NHP) in the Americas, three bot fly species causing cutaneous myiasis have been reported: Cuterebra baeri, Cochliomyia hominivorax, and Dermatobia hominis.

Cuterebra baeri (syn. Alouattamya baeri) infected Cebus albifrons aequatorialis in Peru (Vilchez-Delgado et al. 2022), Alouatta belzebul and Aotus trivirgatus in Brazil (Guimarães 1971), Alouatta seniculus in French Guiana (de Thoisy et al. 2001) and Guiana (Stuart et al. 1998), and Alouatta palliata in Costa Rica (Zeledón-Araya et al. 1957; Calderón-Arguedas et al. 2004), Guiana (Shannon and Greene 1926), Panama (Milton 1996; Colwell and Milton 1998), and Mexico (Cristobal-Azkarate et al. 2012). Cochliomyia hominivorax infected Alouatta seniculus and Pithecia pithecia in French Guiana (Vie and Richard-Hansen 1997). Dermatobia hominis infected Sapajus macrocephalus and Saguinus mystax in Peru (Tirado Herrera and Heymann 1998; Vilchez-Delgado et al. 2022), and Alouatta palliata in Panama (Smith 1977), along with Dermatobia sp. parasitizing Aotus sp. in Peru (Tantalean et al. 1990).

The epidemiology and the effect on the host may vary significantly, depending on the parasite species (Cristobal-Azkarate et al. 2012). Reports of myiases often state that these infections tend to cause little or no harm to the hosts; however, harmful or fatal effects have been observed under certain circumstances (Tuten et al. 2011).

Studies on myiasis in NHP are scarce and mainly based on morphologic identification of larvae; molecular approaches have been barely used. Although morphologic identification has been the traditional approach in ectoparasites, there are species for which no information on the larval stage is available. For instance, although details of larval and pupal morphology have been established in a few species (e.g., Cuterebra ruficrus and Cuterebra tenebrosa), some species of Cuterebra from South America are known only from a few adult specimens (Colwell and Otranto 2021). Therefore, the molecular approach is an important tool addressing reliable parasite identification when no morphologic diagnostic characters are available.

A short DNA sequence used as a marker called a DNA barcode provides a widely used technology for accurate and rapid species identification (Yu et al. 2021). The lack of reference sequences obtained from properly morphologically identified individuals is a limitation when using this technique for parasite identification. Molecular studies on myiasis have included the sequencing of a region of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene from larvae of Cuterebra baeri infesting NHP in Peru and Panama (Otranto et al. 2003; Vilchez-Delgado et al. 2022) and the development of microsatellite markers to characterize the genetic structure within populations of Cuterebra baeri parasitizing howler monkeys in Panama (Milton et al. 2011).

In Colombia, Cuterebra sp. has been morphologically identified in Aotus vociferans and Aotus nancymaae in the Amazon region (Roncancio et al. 2018). However, no additional records on bot flies from other NHP species are available in the region. Using molecular tools, our study aimed to identify bot flies parasitizing two free-ranging primate species living sympatrically in central Colombia.

Sampling was conducted in a lowland rainforest fragment in San Juan del Carare, Santander Department, Colombia (06°43′N, 74°90′W; 150–200 m above sea level), in a region within a matrix of pastures for cattle ranching and agroindustrial monocultures. The area has a mean annual humidity of 80%, an annual median rainfall of 3,496 mm, and a mean annual temperature of 28 C. At the study site, NHP are habituated. The individuals sampled included one adult male and one adult female Aotus griseimembra, belonging to different social groups, and one adult female Alouatta seniculus. Primates were anesthetized using tiletamine-zolazepam (Zoletil, Virbac, Fort Worth, Texas, USA) for Aotus griseimembra 0.3 mL of Zoletil 50 (25 mg/mL tiletamine plus 25 mg/mL zolazepam, i.e., 7.5 of mg tiletamine plus 7.5 of mg zolazepam) and for the larger Alouatta seniculus 0.9 mL of Zoletil 100 (50 mg/mL tiletamine plus 50 mg/mL zolazepam, i.e., 45 mg of tiletamine plus 45 mg of zolazepam), using 1- and 1.5-mL darts (Pneu-Dart, Williamsport, Pennsylvania, USA), respectively, and delivered remotely with a CO₂ injection rifle (JM model, Dan-Inject, Austin, Texas, USA). Scalpel dissection was performed to remove the larvae. Four bot fly larvae were collected from grey-legged night monkeys and one from the howler monkey. The capture and handling of the animals was approved by Universidad de los Andes and the National Environmental Licensing Authority of Colombia (permit nos.: 2017025578-1-000; 2017043863-1-000; 2017065795-1-000; 2017013727-1-000; 2017052943-1-000; 2017081458-1-000; and 2017108650-1-000).

Larvae were measured (length 23.3–24.8 mm; width 12.3–15.2 mm), photographed (Fig. 1), and stored in 96% ethanol. From each larva, DNA was isolated using the Isolate II Genomic DNA Kit (Meridian Bioscience, London, UK); PCR amplification of a partial region of the mitochondrial cox1 gene was performed according to Cavallero et al. (2017). All PCR products were visualized on a 1% agarose gel, and good quality amplicons were purified using Sure Clean (Bioline, London, UK) and shipped to an external company for sequencing (Eurofins Genomics, Ebersberg, Germany). Sequences were manually edited with Trace implemented in MEGA7 (Kumar et al. 2016) and used as input for the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information, 2021).

All specimens were identified as Cuterebra baeri, according to the best match in BLAST, which showed 98% identity with bot flies identified in monkeys from Panama (accession no. AF497777; Otranto et al. 2003). The five partial cox1 sequences that we obtained (two deposited in GenBank: accession nos. OK393818 and OK396016) were all identical and were compared with the following available homologous sequences for phylogenetic inference: Cuterebra baeri (GQ409320 and AF497777), Cuterebra jellisoni (AF497778), and Dermatobia hominis (AY463155) used as the outgroup. The best evolutionary model explaining the dataset was T92, according to the lowest Bayesian information criterion in Modeltest (Kumar et al. 2016), and the maximum likelihood method was used to obtain a phylogenetic tree. A dataset of nine partial cox1 sequences of 650 base pairs was used. The maximum likelihood tree with the highest log likelihood is shown in Figure 2. Partial cox1 sequences obtained here for Cuterebra baeri formed a cluster with 99% statistical bootstrap support with the other Cuterebra baeri specimens from GenBank, while Cuterebra jellisoni was placed as a sister branch. This strongly supports the identity of our specimens as belonging to Cuterebra baeri.

Bot flies infesting NHP in Colombia have not previously been molecularly identified. Our molecular identification enabled confirmation of Cuterebra baeri in Alouatta seniculus and Aotus griseimembra in Colombia.

According to the International Union for Conservation of Nature Red List, Aotus griseimembra is classified as vulnerable, while Alouatta seniculus is least concern; however, the trend of both species populations is decreasing (Link, Palacios, Cortés-Ortiz et al. 2021; Link, Urbani, and Mittermeier 2021). In this context, note that some species of ectoparasites may cause severe damage to the hosts (Solórzano-García and Pérez-Ponce de León 2018). For instance, it has been observed that parasitism by Cuterebra baeri may compromise Alouatta palliata populations through primary and secondary effects, such as nutritional stress, mortality, and lack of growth of the primate population (Milton 1996).

To better understand the ecologic dynamics of Cuterebra baeri, we encourage the monitoring of its occurrence in NHP. Remaining questions include why some sympatric NHP species are likely to be more prone to be parasitized by bot flies than others, any seasonal patterns of infection, and the influence of ecologic variables and anthropogenic disturbance on bot fly prevalence in wild NHP populations.

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