Rhinella alata is a small terrestrial bufonid that occurs in Ecuador, Colombia, and Panama. Between January 2014 and October 2015, we inspected 339 R. alata from Panama and report myiasis in eight of these toads. All infested toads were male and presented with mobile dark fly larvae visible beneath the ventral skin. At necropsy, we identified the larvae as belonging to the family Sarcophagidae (flesh flies). Flesh flies have been variously considered as predators, parasitoids, and parasites of anurans. There are at least four species of flesh flies that infect adult amphibians in the Neotropics, with the most common and widespread being Lepidodexia bufonivora. Myiasis has only rarely been reported in Panamanian anurans. Anuran cases of sarcophagid myiasis are usually fatal and we suspect myiasis as the cause of death for the R. alata that died in the current study.
Rhinella alata is a small toad of the Rhinella margaritifera complex that occurs in Ecuador, Colombia, and Panama (dos Santos et al. 2015). This common species is primarily diurnal and terrestrial, inhabiting forest leaf litter and feeding on ants (Jaeger and Hailman 1981; Rand and Myers 1990). In Panama, R. alata breeds explosively and sporadically in permanent streams and pools throughout the wet season and the beginning of the dry season (June to January; Ibáñez et al. 1999).
During the course of field research on the ecology of R. alata in Colón and Panamá, Republic of Panama, from January 2014 to October 2015 we examined 339 toads from water-body margins in three areas (Fig. 1 and Table 1): Gamboa (n=5), Masambi (n=256), and Pipeline Road (n=78). Eight male toads (five from Gamboa, three from Masambi; Fig. 1 and Table 1) had dark mobile larvae visible through their translucent ventral skin. In addition, toads exhibited one or more of the following clinical signs: swollen vocal sac and venter area and ventral skin redness. These toads were alive and active when collected (seven were found in or near a breeding chorus). We brought the eight toads to the lab and monitored them for 24 h, during which time their health rapidly deteriorated. Of the eight infected toads, three died, one became moribund and was euthanized, and the remaining four (although visibly ill) were released at the site of capture. Numerous healthy R. alata (with none of the above signs) that were captured, handled, and retained in the lab under the same conditions remained healthy for the 24-h duration of holding and were released.
We froze (–18 C) three deceased toads from Río Masambi Grande (Fig. 1 and Table 1); at later dissection we confirmed that they had myiasis (Table 2). Rhinella alata number 1 had no ventral swelling but did have a large hole in the ventral skin where one fly larva had exited; two fly larvae were positioned between the skin and the abdominal musculature with their heads inserted through the musculature into the coelom; four larvae were within the coelom, one of which had its head penetrating the abdominal musculature. Rhinella alata number 2 had a fully extended vocal sac and no visible holes in the skin (Fig. 2A); three larvae were present inside the coelom (Fig. 2B–D). Both R. alata number 1 and R. alata number 2 had very enlarged spleens. Rhinella alata number 3 had a grossly inflated ventral surface (from throat to vent) with no visible holes; nine larvae were within the coelom; the spleen was unremarkable.
We identified the 19 larvae in the three dissected toads as third instar larvae of the family Sarcophagidae (flesh flies) based on the appearance and positioning of their posterior spiracles beneath a dissecting scope (Thyssen 2010). Further morphology-based identification is difficult without permitting the fly larvae to metamorphose. We submitted four larvae (n=3 from R. alata number 1; n=1 from R. alata number 2) to the US Department of Agriculture National Veterinary Services Laboratories (Ames, Iowa, USA) for species verification. The larvae were about 10 mm long and almost black in coloration (Fig. 2B–D), both in life and after freezing. Sarcophagid larvae are usually transparent or white (Eizemberg et al. 2008; De Oliveira et al. 2012; Gómez-Hoyos et al. 2012).
Dipterans with adult anuran hosts belong to the Phoridae, Sarcophagidae, Calliphoridae, and Chloropidae. Within Central and South American anurans, only Phoridae and Sarcophagidae occur. Megaselia scalaris (Phoridae) oviposits in frog eggs (Disney 2012) and adult frogs of Leptodactylus fuscus and Hypsiboas caingua in Argentina and Brazil (De Alcantara et al. 2015; López et al. 2016). The larvae we encountered in R. alata were much larger than the 4-mm length of M. scalaris and the appearance of their posterior spiracles did not conform to that of the Phoridae (Thyssen 2010). Four species of Sarcophagidae infect adult Neotropic anurans: Peckia (Sarcodexia) lambens, Lepidodexia centenaria, Lepidodexia adelina, and Lepidodexia (Notochaeta) bufonivora. The first two species are known from single host species in single localities: P. lambens from the dendrobatid Ameerega trivittatus in Peru (Hagman et al. 2005), and L. centenaria from the hylid Hypsiboas beckeri in Brazil (Mello-Patiu and Luna-Dias 2010). Lepidodexia adelina infests leptodactylids (Adenomera diptyx, Leptodactylus elenae, and Physalaemus albonotatus) in Argentina (Mulieri et al. 2018). Lepidodexia bufonivora has a broader geographic distribution and host range including members of the Bufonidae, Ranidae, Leptodactylidae, Craugastoridae, and Hylidae (Kraus 2007; Eizemberg et al. 2008; Gómez-Hoyos et al. 2012; Vázquez-Corzas et al. 2018). Within Neotropic bufonids, L. bufonivora infests Atelopus varius in Costa Rica (Crump and Pounds 1985; Pounds and Crump 1987) and Rhinella granulosa in Venezuela (Lopes and Vogelsang 1953). Within Panama, sarcophagid myiasis (species unknown) has been reported in Hyalinobatrachium fleischmanni (Medina et al. 2009) and the frog tentatively identified as Diasporus (Eleutherodactylus) diastema (Dodge 1968). Considering the broad host range (encompassing bufonids), the large larval size, and the distribution of L. bufonivora, it is likely the species we encountered in R. alata.
Prevalence of infection at a site during a single survey varied from 0% to 100% (mean SE=4.32±3.48) and intensity ranged from three to nine larvae (n=3 toads); similar intensities (one to 10 larvae) were encountered in A. varius (Crump and Pounds 1985). Considering that anurans afflicted with myiasis generally do not survive longer than a few days (Crump and Pounds 1985), and that dead frogs are quickly consumed by the larvae (Bolek and Janovy 2004; Lopez et al. 2016), the window of detection is limited in this host-parasite relationship. Further, early asymptomatic stages of infection may go undetected in a visual encounter survey as only active frogs are observed (any sick frogs refuging under cover would not have been detected). As such, the prevalence we report is likely far lower than the actual prevalence.
In our study, all eight infected R. alata were male. There was, however, a sampling bias toward males (302 males vs. 23 females inspected) since we surveyed water-body margins where males congregate throughout the breeding season, whilst females are dispersed, coming to the water only briefly to breed (Bull 2006). During breeding periods male anurans may be at greater risk of predation and parasitism (e.g., by flies) since they are aggregated and often conspicuous (e.g., calling). Indeed, the myiasis cases overlapped the R. alata breeding season (June–January), rather than the wet vs. dry season (n=6 cases and n=2 cases, respectively). Previous studies on A. varius only encountered myiasis in the dry season (Crump and Pounds 1985) and found that females and males were attacked equally when corrected for their relative abundance (Pounds and Crump 1987).
Common anatomic sites of infection with flesh fly larvae in anurans include the dermis, oral cavity, cloaca (Eizemberg et al. 2008; Carvalho-Filho et al. 2010), and body cavity (Gómez-Hoyos et al. 2012). Sarcophagid flies generally lay their larvae on the dorsal surface of the thighs of their hosts and larvae burrow in to feed on the musculature and organs (Pounds and Crump 1987). We did not observe any holes in the dorsal skin or wounds on the thighs of the R. alata in our study, as described in other frogs with sarcophagid myiasis (Crump and Pounds 1985; Mello-Patiu and Luna-Dias 2010). We did observe a large hole in the ventral skin of two of the infected R. alata (one was not dissected); these wounds, coupled with the positioning of two larvae between the skin and the abdominal musculature in one dissected toad, imply that the ventral skin may have been the route of entry in these instances.
Anuran cases of sarcophagid myiasis are usually fatal and we suspect myiasis as the cause of death for the R. alata that died in the current study. Most prior myiasis studies are single-case reports, ours is one of few that report multiple conspecifics infested by the same species of fly larvae (Crump and Pounds 1985; Bolek and Coggins 2002; Bolek and Janovy 2004). One similarity between R. alata and other anurans (A. varius, juvenile Rana sylvatica, juvenile Bufo americanus) regularly afflicted by myiasis is their diurnal lifestyle—implying that diurnal anurans may be at higher risk of myiasis.
We thank Mark Torchin and Kristin Saltonstall for logistic support, and Anette Garrido and Jenifer Benítez for field assistance. C.K. is grateful for funding received through the A. Stanley Rand Fellowship, the George E. Burch postdoctoral fellowship, and a National Geographic Research and Exploration Grant (9945-16). R.I. was supported by the Panama Amphibian Rescue and Conservation Project, Minera Panamá S.A., and the Sistema Nacional de Investigación of Panama. Collection of toads was conducted under the permits SE/AO-2-14 from the Autoridad Nacional del Ambiente and SE/A-32-15 from the Ministerio de Ambiente of Panama. Fly larvae were exported from Panama to the US under the permit SEX/A-93-18 from the Ministerio de Ambiente of Panama. We thank James Mertins at the US Department of Agriculture National Veterinary Services Laboratories (NVSL) for identifying the fly larvae and depositing them in the NVSL Parasitology Reference Collection (accession 18-039330, case TE18-27).