First report of flesh-fly (Diptera: Sarcophagidae) myiasis in little-devil poison frog (Anura: Dendrobatidae) from Ecuador

Abstract

We report a case of myiasis in the poison frog Oophaga sylvatica from the Canandé Reserve located in the Chocó region of northwestern Ecuador. We identified the causal agents as larvae of flesh flies, Sarcophagidae, by means of DNA barcoding and morphological features. This represents the first record of myiasis in an anuran in Ecuador and the second record for Dendrobatidae in the Neotropics. This observation may constitute a case of facultative parasitism where larvae are deposited in the frog's wounds, but further research is needed to understand the biological mechanisms underlying this interaction.

Graphical abstract

Highlights

• •First report of myiasis in an anuran from Ecuador and the second documented case in Dendrobatidae within the Neotropics.
• •DNA barcoding and morphological identification confirmed Sarcophagidae larvae as the causal agents.
• •Potential case of facultative parasitism, where fly larvae seem to infest wounds in the poison frog, Oophaga sylvatica.
• •Questions arise about the role of chemical defenses of poison frogs in host susceptibility.

1. Introduction

Myiasis is a parasitic disease caused by the infestation of dipterous larvae in living animal tissues (Zumpt, 1965). This disease affects a variety of vertebrates worldwide, including humans, domestic animals, and wildlife (Hall and Wall, 1995; Stevens et al., 2006). In amphibians from temperate regions of Europe, North America and Australia, larvae from the fly families Calliphoridae, Chloropidae, Muscidae, and Sarcophagidae have been identified as obligate or facultative parasites (Bolek and Coggins, 2002; Bolek and Janovy, 2004; Kraus, 2007; Glaw et al., 2014; Mebs et al., 2014). Although myiasis may not be uncommon, cases are rarely reported from Neotropical countries (Crump and Pounds, 1985; Kraus, 2007; D'Bastiani et al., 2020). Nonetheless, case reports are essential for understanding parasitic interactions and the diseases they cause. Here, we present the first documented case of myiasis in an Ecuadorian frog from the family Dendrobatidae.

2. Materials and methods

We encountered a male of Oophaga sylvatica (Funkhouser, 1956), also known as ‘Little-Devil Poison Frog’, on the morning of April 6, 2024 at 0837h, while conducting fieldwork, in the Canandé Reserve located in the Chocó region, Esmeraldas Province, northwestern Ecuador (0.527365 N, 79.211887 W; at 334 m elevation). The frog was active on the leaf litter within a secondary forest undergoing regeneration from previous use as cacao plantation. The temperature on the leaf litter at the time of observation was 24.4 °C. We first observed the presence of a lump and a hole on the right dorsal side of the frog (Fig. 1). We captured the individual and housed it in a terrarium in the laboratory of the scientific station, where it was kept and monitored for health and behaviour for the entire day. During our observations, we discovered three other holes in its body− one on the right forelimb and two on the left dorsal side (Fig. 1). Within these holes we saw white larvae moving. At night, after approximately 12 h in the lab, we noticed a dramatic decline in the frog's health, evident by its reduced activity. As it became obvious that the frog was not going to survive, we decided to euthanize it, using a 10 % lidocaine solution. Just after it was euthanatized, we were able to extract four larvae from its upper right arm and preserved them in 96 % ethanol. After euthanasia, we fixed the frog in 10 % formalin for 48 hours, and thereafter transferred it to 70 % ethanol. Two more larvae were found in the frog's body on its left dorsal side after fixation. These larvae, which have also been fixed in formalin along with the frog, were also preserved in 70 % ethanol. In total we were able to extract six larvae from the frog's body. We deposited the frog (MEPNH-16416) and the associated larvae (MEPN-INV-49152) in the Herpetology and Entomology Collections of the Museum of Natural History Gustavo Orcés V. at the Escuela Politécnica Nacional in Quito, Ecuador.

Fig. 1.

Oophaga sylvatica (MEPNH-16416) with four lesions indicated by black lines: one on the right dorsal side, one on the right forelimb, and two on the left dorsal side of the frog, caused by Sarcophagidae larvae.

To identify the larvae, we used a morphological and a genetic approach. We used two larvae to take photographs of the whole specimen with a Nikon D7500 digital camera, equipped with an infinite Olympus 4X lens and a Raynox 250 tube lens. We process the images with Zerene Stacker v1.04 software. Another two larvae were cleared with 10 % NaOH, rinsed with 70 % ethanol, disassembled and mounted on slides with Entellan. The photographs of these slides were taken with a Better Scientific BS750 trinocular microscope and a 5 Mpx HD camera. The morphological characters analysed were: body shape, presence of spiny bands in the intersegments, shape of the cephaloskeleton, arrangement of the anterior part of the spiracles, shape and distribution of the spines in the spiny bands, presence/absence of the anterior spiracles, and presence/absence of the ecdysial scar and the peritreme in the posterior spiracles.

Due to the limited knowledge surrounding the morphological identification of dipteran larvae, particularly those in the Sarcophagidae family (Buenaventura, 2013), we also employed DNA barcoding. The cytochrome oxidase I (COI) gene was amplified from the last two individual larvae using the primers LCO1490 and HCO2198 in the EETROP Laboratory at the Universidad de las Américas (UDLA). The sequencing was subsequently performed at MACROGEN. The sequences obtained were edited and analysed with the program Geneious Prime 2023.2 (https://www.geneious.com). We conducted a BLAST search using the GenBank database (National Center for Biotechnology Information, U.S.) and the identification engine on the Barcode of Life Data System (BOLD) to compare our unknown sequences with reference data. To assess the phylogenetic placement of our sequences, we initially constructed Neighbor-Joining (NJ) trees using 41 sequences of closely related taxa, retrieved from both GenBank and BOLD (see Supplementary data). Based on this preliminary analysis, we constructed a reduced taxon NJ tree that includes eight representative sequences along with our newly obtained sequences. Both analyses were conducted in Geneious Prime (2023).2 using the HKY substitution model and 1000 bootstrap replicates. The resulting trees were subsequently edited and visualised using FigTree v1.4.4 (Rambaut, 2018).

3. Results

Of the two entire larvae examined, one preserved in alcohol was greyish-white, while the other, preserved in formalin, displayed a yellowish-white coloration. Both larvae exhibited spiny bands in the intersegments and an elongated shape. The larva preserved in alcohol, measured, 3.61 mm long x 0.71 mm wide, whereas the formalin-preserved specimen was notably shorter, 2.73 mm long and 0.49 mm wide. The difference in size may be due to shrinkage caused by the formalin preservation process. Neither larva showed anterior spiracles, and their posterior spiracles featured two simple openings, characteristic of larvae in the first to second instar stages (Fig. 2A).

Fig. 2.

(A) Lateral view of entire larvae. (B) Lateral view of the cephalopharyngeal skeleton of the larvae; ca: clypeal arch, dc: dorsal cornu, ds: dental sclerite, mh: mouth hook, pb: parastomal bar, ps: pharyngeal sclerite, sh: subhypostomal sclerite. (C) Transversal cut of posterior spiracles of the larvae extracted from the frog.

Of the two larvae mounted on slides, the cephalopharyngeal skeleton shows strongly recurved mouth hooks (Fig. 2B). The dental sclerite and subhypostomal sclerite are of similar size and are completely incorporated into the base of the mouth hook. The pharyngeal sclerite is very dark pigmented and has a slightly upwardly recurved anterior parastomal bar. The ventral edge of the pharyngeal sclerite is nearly straight with rounded ends. The dorsal horn is more pigmented ventrally and is pointed and elongated distally. The clypeal arch is elongated and widely spaced, parallel to the parastomal bar. The posterior spiracles do not show the ecdysial scar and the peritreme is incomplete, with a pair of oval spiracular openings (Fig. 2C).

Overall, the morphology of the four larvae used for morphological analyses showed that the larvae are between a first and second instar. The distinct structure of the cephalopharyngeal skeleton and posterior spiracles are typical for members of the Sarcophagidae family and strongly suggests the larvae belong to the genus Sarcophaga. Notably, the unique shape of the mouth hook differentiates these larvae from related genera, including Oxysarcodexia, Paraphrissopoda, and Sarcodexia. The external morphology of Sarcophagidae larvae is highly similar across different species in all three larval stages, limiting the number of distinguishing features available for use in larval identification keys (Szpila et al., 2015).

The COI gene sequencing yielded two sequences, each comprising 674 base pairs (GenBank accession numbers: PQ801123 and PQ801124), and the NJ tree revealed a close genetic relationship to specimens identified as belonging to the genus Sarcophaga within the Sarcophagidae family (Fig. 3, Supplementary data).

Fig. 3.

Neighbor-Joining (NJ) tree based on COI gene sequences from ten Sarcophagidae individuals, including the larvae collected in this study Lucilia sericata was used as the outgroup. Bootstrap values are shown at the nodes.

4. Discussion

In Central and South America, Sarcophagidae are the primary agents responsible for myiasis in anurans (Kraus, 2007; Vázquez-Corzas et al., 2018; da Silva et al., 2019; Junes et al., 2019; Kelehear et al., 2020; D'Bastiani et al., 2020). Within the Dendrobatidae, cases of myiasis have been documented only in three species from Peru: Ameerega bassleri, A. cainarachi and A. trivittata. In the first two species, the larvae causing the myiasis were not conclusively identified, but the authors suspected that they belong to the Sarcophagidae family. In Ameerega trivittata, the larvae were identified to the species level as Sarcodexia lambens (Hagman et al., 2005).

Several dendrobatid species have been recorded in the Canandé Reserve, including Epipedobates aff. espinosai, Hyloxalus awa, and H. toachi (Neira-Salamea et al., unpubl. data) Among these, only Epipedobates aff. espinosai is sympatric with Oophaga sylvatica, as both species occur across various habitat types, from mature forest to disturbed habitats, with a higher prevalence of individuals in the latter (Neira-Salamea et al., unpubl. data). To date, no other cases of myiasis have been reported in any of these co-occurring dendrobatids. However, anecdotal observations from field herpetologists suggest that myiasis-like infections may occur in other anuran families in Ecuador, such as Centrolenidae, Strabomantidae, and Leptodactylidae. These reports are consistent with confirmed cases of myiasis documented elsewhere in the Neotropics. For instance, Medina et al. (2009) described a probable sarcophagid larva in the left thigh of Hyalinobatrachium fleischmanni in Panama. Similarly, Gómez-Hoyos et al. (2012) reported a case of myiasis in Pristimantis thectopternus from Colombia, in which nine third-instar Sarcophagidae larvae were found within a lesion on the frog's right flank. Within the family Leptodactylidae, multiple cases have been reported: Adenomera diptyx, Leptodactylus elenae, and Physalaemus albonotatus in Argentina (Mulieri et al., 2018); Leptodactylus fuscus (Alcántara et al., 2015); Adenomera marmorata, Leptodactylus latrans, and Physalaemus cuvieri in Brazil (D'Bastiani et al., 2020; da Cunha-Martins et al., 2022). In most of these cases, Sarcophagidae larvae were identified as the causal agents. However, in Leptodactylus fuscus, Alcántara et al. (2015) documented a different dipteran family, Phoridae, as the cause of infestation.

Phorid fly larvae have been implicated in other myiasis cases involving Rhinella ornata and Boana faber in the Atlantic Forest of Argentina (López, 2019). Collectively, all these findings support the hypothesis of a broader host range for dipteran larvae among Neotropical anurans, highlighting the diversity of both hosts and parasitic dipterans involved in these interactions.

The results from our phylogenetic analysis indicate genetic similarity of our collected larvae to Sarcophaga species (Figure 3, Supplementary data) which belong to the subfamily Sarcophaginae, that has its highest richness in the Neotropical Region (Buenaventura et al., 2018). The Sarcophagidae catalogues for Ecuador (Pape, 1996; Pape et al., 2004; Salazar and Donoso, 2015) do include species of the genus Sarcophaga. Thus, taking all together, the evidence points to the larvae found in the individual of little-devil poison frog Oophaga sylvatica could belong to an undescribed or misidentified species within the family Sarcophagidae, most likely related with genus Sarcophaga.

Sarcophaga larvae display remarkable variability in their feeding habits, which include saprophagy, and parasitism (Pape, 1996). Parasitism can be obligated, when larvae must develop in living hosts; or facultative, when larvae may develop in decomposing organic matter, as well as occasionally in tissues of living organisms (Hall and Smith, 1993; Arias-Robledo et al., 2019). This type of parasitism is particularly common in weak animals, or those with exposed wounds (Stevens and Wall, 1997; González et al., 2009).

Most species within Sarcophaginae subfamily are facultative or obligate parasitoids of insects, small invertebrates, and vertebrates (Buenaventura et al., 2018), and a smaller subset of sarcophagines includes coprophagous species and known producers of myiasis (Pape, 1996; Bänziger and Pape, 2004; Hall &Wall, 1995; Hagman et al., 2005; Bermúdez et al., 2010). The infested male individual of Oophaga sylvatica was found in a formerly cacao plantation that has undergone approximately 20 years of natural recovery. Despite this regeneration, the location was just 30 m from a road frequently used by local people to move cattle and mules. This proximity to human activity and livestock may imply that organic matter and faeces may attract saprophagous or coprophagous flies with the potential to behave as opportunistic parasites. Additionally, males of O. sylvativa exhibit territorial and aggressive behaviour, often engaging in intense physical combat with other individuals of the same sex, which can generate a variety of skin lesions (Pröhl, 2005; Summers, 2000; Mendez-Narvaez and Amézquita, 2014). Given these ecological factors, we propose that the observed case of myiasis may have been facilitated by such skin lesions, representing a possible instance of facultative parasitism. These intraspecific interactions could increase the susceptibility of individuals to infections and ectoparasitism. However, further research is needed to determine the extent to which territorial behavior and associated injuries influence vulnerability to parasitic infestations in this species.

Myiasis in anurans, particularly in species of the family Dendrobatidae, represents a poorly documented phenomenon compared to other anuran groups (D'Bastiani et al., 2020). It is essential that research on myiasis in anurans from neotropical regions focuses on the specific mechanisms that facilitate this type of parasitism. Although the skin secretions of Oophaga sylvatica possess chemical properties that provide protection against predators, bacteria and fungi (Saporito et al., 2012; Hantak et al., 2016; Murray et al., 2016; Bolton et al., 2017), it is evident that the flesh fly larvae can deal with the skin toxins. According to Mebs et al. (2014), the parasitism observed in the European common toad (Bufo bufo) by blowfly larvae Lucilia bufonivora may be attributed to the larvae's ability to metabolize bufadienolides, toxins present in toad skin, with remarkable efficiency. This capability is likely the result of physiological adaptations that enhance the excretion of these toxins, representing a crucial strategy to counter their harmful effects. These results highlight the need to explore how ectoparasites manage to overcome chemical defenses in tropical anurans, since they could share similar mechanisms with those proposed by Mebs et al. (2014). Research focusing on these interactions will not only contribute to understanding the dynamics of parasitism, but also to assessing its impact on the conservation of vulnerable species in highly biodiverse and fragmented ecosystems.

5. Conclusion

The documentation of myiasis in Oophaga sylvatica represents a significant contribution to the understanding of host-parasite interactions in tropical amphibians. As the first reported case of myiasis in an anuran from Ecuador and the second within Dendrobatidae in the Neotropics, this finding highlights the potential susceptibility of poison frogs to opportunistic parasitism. The identification of Sarcophagidae larvae through DNA barcoding and morphological analysis provides robust evidence of the parasitic agent involved. Future studies should explore the ecological and physiological factors influencing this host-parasite relationship, including those that allow or facilitate the flies to deposit its eggs in the frog, as well as the potential impact of this kind of parasitism on amphibian health and conservation.

CRediT authorship contribution statement

Michelle Vélez: Writing – original draft, Writing – review & editing, Methodology, Investigation, Conceptualization. Mark-Oliver Rödel: Writing – review & editing, Resources, Funding acquisition. Vladimir Carvajal: Writing – original draft, Methodology, Investigation. David A. Donoso: Writing – review & editing, Resources, Funding acquisition. Mónica A. Guerra: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

Funding

This study is part of the Research Unit ‘REASSEMBLY’ (FOR 5207; sub-project SP-2, grant RO 3064/5-1 and gender equality funds of the core project) funded by the ‘Deutsche Forschungsgemeinschaft’ (DFG). Additional financial support was provided by Escuela Politécnica Nacional through Proyecto Semilla PIS 23-22. The Ministerio del Ambiente, Agua y Transición Ecológica issued the research permits: MAATE-ARSFC-2023-0305 and MAATE-DBI-CM-2022-0262.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: