Introduction

Pinworm nematodes of the genus Trypanoxyuris Vevers, 1923 (Oxyuridae Cobbold, 1864) are common intestinal parasites of primates (Solórzano-García & Pérez-Ponce de León, 2018; Rondón et al., 2021). They have a direct life cycle with no free-living stage and are transmitted by direct contact with food, water or surfaces contaminated with parasite eggs (Adamson, 1989); however, autoinfection and retroinfection are also routes of infection (Anderson, 2000; Felt & White, 2005; Prince, 1950). They appear to be highly host-specific with at least one species of Trypanoxyuris described for almost each primate genus (Hugot, 1999). Nonetheless, in those intensively studied primate species, more than one species of pinworm has been reported. Such is the case of howler monkeys, where Alouatta palliata Gray, A. pigra Lawrence and A. seniculus L. are each parasitized by one or two specific species of pinworms, while sharing Trypanoxyuris minutus Schneider, 1866 with several other species of the genus Alouatta Lacépède (Solórzano-García et al., 2016, 2020). Likewise, spider monkeys (Ateles spp. E.Geoffroy) and uakari monkeys (Cacajao calvus I.Geoffroy) are parasitized by two species of pinworms, T. atelis Cameron 1929, and T. atelophora Kreis, 1932 (Solórzano-García et al., 2015), and T. cacajao and T. ucayalii Conga Giese, Serra-Freira, Bowler and Mayo, 2016, respectively.

Night monkeys (Aotus spp.) are the only nocturnal primates in the Tropical Americas and are characterized by having a relatively small size (approximately 1 kg.), being arboreal, relying on fruits and insects in their diets. Also, night monkeys are known for living in pairs, with one adult male, one adult female and one to three offspring (Fernandez-Duque, 2007). The genus Aotus contains 12 recognized species distributed between Panama and Northern Argentina (Martins-Junior et al., 2022), and many of them are considered to be threatened (IUCN, 2022). Two species of Trypanoxyuris have been described to infect night monkeys (Travassos, 1925; Sandosham, 1950), namely T. microon and T. interlabiata, although the latter was posteriorly synonymized with T. microon (Inglis & Díaz-Ungría, 1960). In this study, we explore the diversity of pinworm species found in the grey-legged night monkeys (A. griseimembra Elliot) and the Andean night monkey (A. lemurinus I.Geoffroy) from two locations in Colombia. We conducted detailed morphological examinations and obtained molecular data from ribosomal and mitochondrial DNA to assess pinworm species identity and validate the existence of two monophyletic clades of Trypanoxiuris in the large intestine of night monkeys.

Materials and Methods

Specimen collection and morphological examination

Pinworm specimens were sampled from six free-ranging grey-legged night monkey individual from San Juan del Cararé, Santander Department; and one individual of Andean night monkey in Pijao, Quindio, Colombia. Pinworms were recovered from fresh monkey feces in situ and fixed in 100% ethanol. Pinworm-positive fecal samples were collected in 15 ml tubes with 70% ethanol for further examination in the laboratory for the recovery of minute male specimens (Hasegawa, 2009). To establish a linkage between morphological features and DNA sequences of parasite individuals, some specimens were cut in half with the anterior portion used for morphological exams, and the remainder posterior portion used for DNA extraction. Full body photomicrographs and measurements were taken using a Leica DM 1000 led microscope (Leica, Germany) for all the specimens before performing the cuts.

For morphological examinations pinworms were cleared with alcohol-glycerol solution and observed using an Olympus BX51 light microscope equipped with differential interference contrast (DIC). En face view observations were made using a modification of the technique proposed by Hasegawa et al. (2004), consisting of mounting the nematode inside a cut micropipette tip filled with commercial hair styling gel, attached to a microscope slide, and placing the cover slide on top (Fig. S1). The gel has the appropriate consistency for holding the nematode in vertical position allowing enface observations; gel cleanup is simple by rinsing the nematode in water, without damaging the tissue for either morphological examinations or even molecular procedures.

The saved anterior portions of the cut pinworms, as well as some complete specimens, were preserved and processed for scanning electron microscopy (SEM) by dehydration through a graded series of ethanol and then critical point dried with carbon dioxide. The specimens were mounted on metal stubs with carbon adhesive, and then gold coated and examined in a 15 kV Hitachi Stereoscan Model SU1510 scanning electron microscope. Complete specimens were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).

DNA extraction and phylogenetic analyses

Individual pinworms were digested overnight at 56 °C in a solution containing 10 mM Tris-HCl (pH 7.6), 20 mM NaCl, 100 mM EDTA (pH 8.0), 1% Sarkosyl, and 0.1 mg/ml proteinase K. DNA was extracted using the DNAzol® reagent (Molecular Research Center, Cincinnati, OH) following manufacturer's instructions. A fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) and a region of the large subunit of the nuclear ribosomal gene (28S) were amplified by PCR using the primers and conditions specified in Solórzano-García et al., 2020. PCR products were treated with Exo-SAP-IT (Thermo Scientific), according to the manufacturer's instructions, and sequenced at the sequencing facility of the Instituto de Biología, UNAM. All sequences obtained in this study were deposited in GenBank.

Phylogenetic relationships were assessed using Bayesian inference (BI) for each gene independently, using MrBayes v.3.2.6 (Ronquist & Huelsenbeck, 2003) and the CIPRES Science Gateway (Miller et al., 2010). Analyses were complemented with DNA sequences of nine species of Trypanoxyuris and three species of Enterobius Leach, 1853 downloaded from GenBank; Oxyuris equi Schrank, 1788 was included as outgroup (Table S1). Alignments of both genes were performed using MUSCLE (Edgar, 2004) through the EMBL-EBI web interface (Madeira et al., 2019). As an additional check on accuracy, COI sequences were translated into amino acids using MESQUITE v.3.2 (Maddison & Maddison, 2011), and the invertebrate mitochondrial genetic code. Most appropriate evolutionary models were inferred following the Akaike information criterion (AIC) in MrModeltest v. 2.3 (Nylander, 2004). The GTR+I+G substitution model was the best model for both genes. BI analyses included two simultaneous runs of Markov chain Monte Carlo, each for four million generations, sampling trees every 4000 generations, a heating parameter value of 0.2, and a “burn-in” of 25%. A 50% majority-rule consensus tree was constructed from the post burn-in trees. BI outputs were imported to FigTree v. 1.4 (Rambaut, 2014) for graphical visualization and editing. Genetic divergence (p-distance) between Trypanoxyuris from Aotus and the other congeneric species was calculated in Mega v.7 (Kumar et al., 2016) using the pairwise-deletion option; standard error of the distances was estimated by bootstrap resampling with 100 replicates.


Systematics

Order Oxyurida

Family Oxyuridae Cobbold, 1864

Subfamily Enterobiinae Hugot et al., 1995

Genus Trypanoxyuris Vevers, 1923

Trypanoxyuris microon (Linstow, 1907)

Host: Aotus griseimembra Elliot, A. lemurinus I. Geoffroy

Localities: San Juan de Cararé, Santander Department; and Pijao, Quindio Departement, in Colombia.

No. Individuals: 36

Material deposited: CNHE 11896

GenBank accession numbers: COI (OR506585 – 91); 28S (OR509779 – 86)

Redescription

General (Figs. 1, 2A, C, D); based on 27 specimens plus six mature worms used for SEM observations, and 3 complete specimens used for DNA sequencing. White minute nematodes, females larger than males. Cuticle with transverse striations. Cephalic vesicle present. Cephalic tray circular; buccal aperture triangular, delimited by three lips, one dorsal and two subventral. Labial structures surrounded by a circular furrow (Figs. 1B, C). Cephalic papillae readily visible, located in ventral and dorsal extremes of the cephalic tray with ventral papillae closest to the amphids. Two amphids, one on each side of the cephalic tray. Lateral alae present in both sexes. Oesophagus long with posterior spherical oesophageal bulb (Figs. 1A, D).

Fig 1.
figure 1

Trypanoxyuris microon. (A) Female full body, lateral view. (B) Female cephalic end, apical view. (C) Male cephalic end, apical view, (D) Male full body, lateral view; (E) Male posterior end, lateral view; (F) Egg; (G) Apical view of egg.

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Fig 2.
figure 2

SEM pictures of pinworms found in night monkeys. T. microon (A, C, E); T. interlabiata (B, D, F). Buccal structure in females (A, B); dl: dorsal lip; rvl: right ventral lip, lvl: left ventral lip, int: interlobe. Lateral alae (C. Female, D. Male). Buccal structures in males (E, F); dcl: dorsal concave lip; vcl: ventral convex lip.

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Females (N=17): Dorsal lip symmetrically bilobulated; right subventral lip asymmetrically bilobulated with upper lobe significantly smaller than lower lobe; left subventral lip reduced with a truncated inner edge (Fig. 2A). Lateral alae doble crested (Fig. 2C). Excretory pore located anterior to oesophageal bulb. Vulva located in the anterior 3rd of the body; muscular vagina longitudinally oriented, with distal vagina approximately perpendicular to longitudinal body axis. Tail long, conical. Eggs ellipsoidal, symmetric, with 3 longitudinal ridges forming a triangular contour in cross section (Fig. 1F).

Males (N=10): Three unlobulated slightly squared lips, one dorsal and two subventral (Fig. 2E). Lateral alae single crested. Excretory pore located posterior to oesophageal bulb. Four pairs of caudal papillae directed ventrally, all surrounded by ring shaped thickenings; a fifth pair sessile, minute is also present directed postero-laterally (Fig. 1E). Spicule long, slightly wider in the middle. Short tail appendage.


Trypanoxyuris interlabiata (Sandosham, 1950)

Host: Aotus griseimembra Elliot

Locality: San Juan de Cararé, Santander Department, in Colombia.

No. Individuals: 46

Material deposited: CNHE 11897

GenBank accession numbers: COI (OR506592 - 99); 28S (OR509772 – 77)

Redescription

Based on 39 specimens plus six mature worms used for SEM observations, and one complete specimen used for DNA sequencing. General description follows that of T. microon.

Females (N=33): Buccal aperture triangular; dorsal lip symmetrically bilobulated with one interlobe; right subventral lip asymmetrically bilobulated with upper lobe significantly smaller than lower lobe; left subventral lip reduced with a rounded triangular edge (Fig. 2B). Lateral alae doble crested. Excretory pore located anterior to oesophageal bulb. Vulva located in the anterior 3rd of the body; muscular vagina longitudinally oriented, with distal vagina approximately perpendicular to longitudinal body axis. Tail long, conical. Eggs ellipsoidal, symmetric, with 3 longitudinal ridges forming a triangular contour in cross section (Fig. 3C).

Fig 3.
figure 3

Trypanoxyuris interlabiata. (A) Female full body, lateral view. (B) Female cephalic end, apical view. (C) Egg; (D) Apical view of egg; (E) Male cephalic end, apical view; (F) Male posterior end, ventral view; (G) Male full body, lateral view.

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Males (N=6): Three unlobulated lips, rounded shaped at the inner edge, one dorsal concave and two subventral convex (Fig. 2F). Lateral alae single crested (Fig. 2D). Excretory pore located posterior to oesophageal bulb. Four pairs of caudal papillae, directed ventrally, all surrounded by ring shaped thickenings; a fifth pair sessile, minute is also present directed postero-laterally (Fig. 3F). Spicule long, slightly wider in the middle. Short tail appendage.

Remarks

Even though the morphology of these two species is very similar to each other, there are key features in the buccal structure of females that serve as diagnostic traits. Specifically, the interlobe at the dorsal lip is only present in T. interlabiata. Also, lips tend to have a squared shape in T. microon, while in T. interlabiata these are round shaped. Likewise, in males the inner edge of the lips tends to be square shaped in T. microon, and rounded in T. interlabiata (Fig 2E, F); however, these morphological differences between species are less conspicuous than in females making species discrimination in males a challenging task. Female buccal morphology in species of Trypanoxyuris from Aotus is peculiar in comparison with other congeners. For instance, the prominent lobes in the dorsal and right ventral lips and the reduction of the left ventral lip makes it difficult to recognize the three lips. Nonetheless, precisely these features, along with the noticeable sexual dimorphism, differentiate these Trypanoxyuris species from the rest. General morphology of the body, the male reproductive anatomy including the number, shape, and arrangement of caudal papillae and the presence of a spike, as well as the shape and size of the eggs are highly similar among Trypanoxyuris species, hence these traits are not reliable for diagnosing species. Additionally, mixed infections of T. microon and T. interlabiata were recorded in every sample of grey-legged night monkey examined, contrary to the Andean night monkey in which only T. microon were recorded; thus, careful enface observations are needed when characterizing pinworm diversity and infection parameters (Table 1).

Phylogenetic analysis

We obtained DNA from seven specimens of T. microon (two males and five females) and eight specimens of T. interlabiata (two males and six females). The final alignment of COI gene consisted of 47 terminals and 794 bp; while 28S alignment was 1138 bp and included 37 taxa. Phylogenetic analysis of both genes showed both species of Trypanoxyuris from night monkeys, T.microon and T. interlabiata, as reciprocal monophyletic groups with high posterior probability support values (Fig. 4). In the phylogenetic trees of both genes these two species are yielded as sister taxa; nonetheless, their position with respect to other Trypanoxyuris species is slightly different. In COI, this clade is placed as the sister group of Trypanoxyuris species parasitizing howler and spider monkeys, which in turn form one large clade except for T. atelophora which is placed as the sister species of all species (Fig. 4A). In 28S, the clade formed by T. microon and T. interlabiata formed a clade with T. atelophora; this clade is sister to the clade formed by Trypanoxyuris from howler and spider monkeys (Fig 4B). The genetic divergence values between Trypanoxyuris species occurring in night monkeys and the other species are shown in Table 2, with a genetic p-distance higher than 10% between T. microon and T. interlabiata. Intraspecific genetic divergence between T. microon from different host species range from 6.4 to 7.0% between specimens from A. azarae Humboldt and A. lemurinus, and A. azarae and A. griseimembra, respectively, and 5.1% between specimens from A. griseimembra and A. lemurinus.

Fig 4
figure 4

Bayesian phylogenetic tree of Trypanoxyuris species inferred with COI (A) and 28S (B) genes. Dots at the nodes represent posterior probability values higher than 0.95. Each color indicates a clade of Trypanoxyuris found in each primate genus: green – Alouatta; blue – Ateles; orange – Aotus. *T. microon from Aotus griseimembra; **T. microon from A. lemurinus; ***T. microon from A. azarae downloaded from GenBank. GenBank accessions numbers of sequences are shown in Table S1.

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Table 1. Measurements of adult Trypanoxyuris microon and T. interlabiata from night monkeys (Aotus spp.) from Colombia, and its comparison with previous descriptions. Measurements are presented in micrometres (μm) unless otherwise noted. Range followed by mean values in parentheses
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Table 2 Average genetic divergence (percentage of p-distance) between Trypanoxyuris from Aotus and other oxyurids from primates.
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Discussion

The taxonomic history of Trypanoxyuris from Aotus monkeys is rather complex. The first record of Trypanoxyuris infecting Aotus trivirgatus Humboldt from Brazil was T. microon, which was initially described as Oxyuris microon Linstow, 1907. Travassos (1925) reassigned it into the genus Enterobius as E. microon. In 1950, Sandosham described a new species, Enterobius interlabiata, from the large intestine of Aotus infulatus Kuhl from a Zoological Garden in London. In his description, Sandosham mentioned the presence of three interlabia projections between the lips which are absent in other pinworm species, hence serving as the species diagnostic trait (Sandosham 1950). He also proposed that pinworms from platyrrhine primates should belong to a new subgenus named Trypanoxyuris which was characterized by possessing a spiked tail in males. Later, Inglis and Díaz-Ungría (1960) proposed Trypanoxyuris to be raised to genus level, and in the same paper, they synonymized T. interlabiata and T. microon, arguing that the observed differences in buccal structures corresponded to different developmental stages instead of characterizing different species, and thus, leaving T. microon as the only recognized species living in the gastrointestinal tract of night monkeys.

The results presented here corroborate the existence of these two different morphotypes in mature pinworm individuals from night monkeys. Moreover, molecular phylogenetic analyses show that these two morphotypes are well-supported reciprocally monophyletic clades. Thus, the results of this study support the existence of two different species of Trypanoxyuris parasitizing the genus Aotus. Morphologically, T. microon is characterized by having square shaped lips, and by lacking a dorsal lip interlobe in females. Instead, T. interlabiata is characterized by having round shaped lips, and a dorsal lip interlobe in females. In both species the left ventral lip in females is notably reduced and almost appears as an interlabia structure. Thus, we propose the resurrection of T. interlabiata as a second species of pinworm parasitizing night monkeys, appealing to the utility of an integrative taxonomic approach combining ecological, morphological and molecular information to pursue a more robust species diversity estimation. The molecular information obtained from this study expands the available genetic library, allowing accurate differentiation of Trypanoxyuris species from immature stages such as eggs found in feces; thus, improving the accuracy of noninvasive diagnostic procedures in primate parasitology.

Furthermore, the data presented here supports the notion of Trypanoxyuris as highly host specific parasites, with each genus of primates been parasitized by at least one species of pinworm (Hugot, 1999). Given previous reports on pinworm diversity and what we have observed, it seems highly plausible to postulate that more than one species of Trypanoxyuris is associated with each species of platyrrhine. For example, it has been proposed that T. minutus is shared among all species of Alouatta, whereas each Alouatta species additionally possess another particular pinworm species (Solórzano-García et al., 2016, 2020). However, for spider monkeys only two species of Trypanoxyuris have been reported for the whole genus (Hasegawa et al., 2004; Solórzano-García et al., 2015). In the case of Aotus monkeys, T. microon has been reported in several night monkey species including A. azarae, A. trivirgatus, A. griseimembra, A. lemurinus and A. vociferans (Hasegawa et al., 2012; Hugot, 1985; Michaud et al., 2003), and T. interlabiata has been reported in A.infulatus, A. trivirgatus, and A. griseimembra (Sandosham, 1950; Thatcher & Porter, 1968). A more intense effort to collect pinworms from different Aotus species, and to morphologically and genetically characterize them is needed to explore pinworm diversity in night monkeys, and to assess their host specificity patterns. Furthermore, the COI genetic divergence of T. microon sampled from different host species was higher than 5%. Reports of intraspecific genetic divergence between Trypanoxyuris specimens from different congeneric host species range from 6 – 10%, while divergence between linages of Trypanoxyruis from different host subspecies has been as low as 1.4% (Solórzano-García et al., 2019, 2020). This suggests that the T. microon clade maybe compose by more than one genetic linage probably related to host species. Nevertheless, more information for other Aotus species from different locations is still required to properly examine this hypothesis.

Currently, there are 22 genera and 171 recognized species of primates occurring in the Neotropical biogeographical region (Estrada et al., 2017), and only 24 species of Trypanoxyuris have been described thus far: two in Aotidae Poche, ten in Atelidae Gray, four in Callitrichidae Gray, two in Cebidae Bonaparte, and six in Pitheciidae Mivart (Hugot, 1985; Hugot et al., 1994; Conga et al., 2015; Solórzano-García et al., 2016, 2020). Given the observed patterns of more than one species of pinworm per primate genus, we could expect at least 22 new species still to be discovered. Furthermore, mixed infections seem to be common where one host individual can harbor more than one species of pinworm, and phylogenetic reconstructions and genetic assessments seem to point out towards a coevolutionary hypothesis in which pinworms and primates have shared an intimate and ancient association, showing not only a host-parasite species correspondence (Hugot, 1999), but these associations can be traced to host linage or subspecies (Solórzano-García et al., 2019) and even to host haplotypes (Solórzano-García et al., 2021), making potential pinworm diversity greater than previously expected. As we add morphological, ecological and molecular information of Trypanoxyuris species from different host species obtained from different locations across their range, we will contribute to a better understanding of the evolutionary and ecological dynamics that shape the associations between pinworm and primates, and the implications that diversification of one side of the interaction could have on the diversification processes on the other.