In the present study, we report the macroscopic and microscopic pathological findings of P. elegans infections in squirrel monkeys from Costa Rica. Furthermore, a deep identification of the collected worms was conducted by performing morphometric observations, scanning electron microscopy and DNA genotyping. Herein, we have found the severe nodular lesions caused by P. elegans in monkeys, as well as the cellular infiltrates occurring in intestinal tissues. In addition, the presence of a cryptic clade is suggested given the high nucleotide differences observed in Costa Rican and Colombian specimens as confirmed by phylogenetic, haplotype and PCA analyses.
P. elegans has been detected in a variety of non-human primates including golden-headed lion tamarins (Leontopithecus chrysomelas) and Wied´s marmosets (Callithrix kuhlii) in Brazil 18, in squirrel monkeys (Saimiri sciureus) from Mexico 12, white-footed tamarins (Saguinus leucopus) and white-fronted capuchin (Cebus albifrons) in Colombia 16. Pathology caused by this acanthocephalan is variable and often depends in the underlying immune state of the monkey host due to the stress induced during captivity 13. For instance, two free-living non-human primate species from Brazil were found with good health condition 18. However, different studies have reported that captive animals suffer weight loss, show increased glucocorticoid and neutrophil levels when compared to their wild state 19. Even though the squirrel monkeys studied herein were free-living, they manifested signs of severe intestinal disease, as also observed in a previous report in free-ranging marmosets from Brazil that manifested severe chronic transmural ulcerative enteritis 20. This suggests that other factors such as number of parasites per animal 21, host species 19, age, sex or feeding behaviors may influence the course of the infection.
Prosthenorchis elegans caused a severe intestinal pathology in wild S. o. citrinellus from Costa Rica. An eosinophilic and pyogranulomatous reaction was observed surrounding the worms and extending to the outer muscle layers and serosa. These observations correlate with previous reports that found the worms surrounded by necrotic debris, eosinophils, neutrophils, macrophages, lymphocytes and fibrous connective tissue 8,20,22. However, free-living tamarins and marmosets from Brazil have not manifested severe intestinal manifestations beyond nodular formations where the P. elegans adults are attached 18. As noted before, several primate species are susceptible to infection with this acanthocephalan. Nevertheless, humans have not been reported with P. elegans before. However, their susceptibility to P. elegans infection has been hypothesized and should not be disregarded 10.
Morphological observations confirmed the identity of the collected acanthocephalan as P. elegans. Total body length, number and size of hooks correlated with the descriptions provided by Machado Filho 15, being total body length in males and females in the lower range than original descriptions, but larger than the observations provided by Catenacci, et al. 18. Even though lemnisci were smaller in size in the specimens collected herein when compared to original P. elegans 15, these were cylindrically-shaped rather than claviform, which are typical of Prosthenorchis spirula 15. Several studies have identified the worms without reporting morphometric analyses 16. However, these observations should be performed whenever possible, due to the slight morphological similarities between closely related species. For instance in hook number and shape, total body length, size and shape of lemnisci and presence of ribbon in the anterior end 15. In this sense, molecular assays have facilitated the identification of unknown specimens when their morphological integrity has been damaged or certain structures are difficult to observe to technological limitations 16.
Molecular analyses of the cox1 of P. elegans from Costa Rica showed high nucleotide differences when compared to specimens from Colombia, indicating the presence of cryptic divergence in this taxon. Cryptic divergence occurs when specimens are morphologically similar to the type species but are genetically distant, as described for the Phyla Nematoda, Platyhelminthes 23 and several acanthocephalan species 17,24. This evolutionary process may emerge because of reproductive, ecological, or geographical isolation which eventually leads to heterozygosity and lack of genetic exchangeability within populations 23. Furthermore, these processes may be affected by climate and environmental alterations that may result to faunal mosaics 25. Ribosomal and mitochondrial DNA analyses of worms from the class Archiacanthocephala have found a clear clustering across families 26. The clustering of Prosthenorchis sp. from Russia apart from P. elegans from Colombia 26 was confirmed in our study, as well as the six P. elegans haplotypes previously found in Colombia 16. Genetic variability in P. elegans may have emerged because of allopatric speciation, since host exploitation does not seem to play a role. Falla, et al. 16 found a low genetic divergence among P. elegans collected from two different New World monkey species from Colombia 16. Therefore, P. elegans may be considered a broad generalist that can exploit a wide variety of host species. In this case, geographical separation may have led to genetic drift in these populations, as also hypothesized for the fish acanthocephalan Leptorhynchoides thecatus 17.
Cryptic and genotypic divergence may lead to differences in pathogenicity, as observed in the canid nematode Onchocerca lupi 27. In the present study a severe tissue inflammation was induced by infection with P. elegans in squirrel monkeys. However, pathological observations were not reported for P. elegans from Colombia 16 or Prosthenorchis sp. from Russia. Therefore, an association between cryptic clades and disease severity cannot be drawn with the current data.
Humans and wild primates have been increasingly sharing habitats due to forest fragmentation as a consequence to their activities. Tourism growth and urbanization in Quepos, Puntarenas has come with poor waste management, with the subsequent increase of P. elegans-intermediate hosts such as cockroaches. In addition, change in the environment has directed to modifications in fauna dynamics, including loss of native species and increase in the geographical distribution of others. Altogether, this has favored the concentration of pathogens in a reduced number of host species, leading to a more severe pathology. S.o. citrinellus is an endangered subspecies that has suffered from anthropogenic activities such as car hits, electrocutions, and habitat fragmentation (Solano-Rojas, 2021). As seen here two of these animals from the wild were killed by cars. When bodies were inspected granulomas and parasites were found in the abdomen as described in captivity. This has added another threat to this in situ species' conservation. Economic resources for conservation are limited by the Government 28 even when it has the obligation to anticipate, prevent and attack the causes of biodiversity loss 4. Public policy changes supported by scientific studies should mitigate hosts extinction and are highly relevant for the conservation of this and other endangered species.