A new nematode of the family Capillariidae identified in Cairina moschata (Linnaeus) on Marajó Island in the Brazilian Amazon

Abstract Capillaria Zeder, 1800, parasitizes the organs and tissues of several hosts, including the domestic duck Cairina moschata (Linnaeus). This article describes a new species of Capillaria in domestic ducks identified based on morphological studies and molecular analyses of the ribosomal RNA gene. Thirty-eight specimens of C. moschata from the municipality of Soure, Marajó Island, Pará, Brazil. The organs of the birds' digestive tract were analyzed under a stereomicroscope to confirm the parasitic infection, after which the collected nematodes were identified by light microscopy, scanning electron microscopy, and molecular analysis. Capillariids parasitized the intestine and cecum of the examined birds. These parasites had three bacillary bands and a pair of elongated precloacal papillae on the tail. Phylogenetic analysis indicated that the new species formed a sister clade with Capillaria spinulosa (Linstow, 1890), as described in Indonesia and Japan. Based on morphological distinctions and molecular data, Capillaria cairina n. sp. can be considered a new parasite species of C. moschata in the Brazilian Amazon.


Introduction
Understanding the diversity and biology of nematodes of the family Capillariidae Railliet, 1915 remains scarce. Approximately 390 species have been identified in various organs and tissues in many hosts, such as fish, amphibians, reptiles, birds, and mammals, including humans (Vicente et al., 1995;Moravec, 2001a;Hodda, 2011). However, capillariids are often not identified in faunal surveys and have been reported only as Capillaria (sensu latu) sp. or Capillariidae gen. sp. (Moravec & Beveridge, 2017;Moravec & Justine, 2020).
The taxonomic classification and identification of capillariid nematodes are difficult as they are small, fragile parasites that require intense manipulation for observation (Moravec, 1982;Anderson, 2000;Tamaru et al., 2015). In addition, they exhibit a wide range of ecological adaptation mechanisms, parasitizing vertebrates belonging to the main classes in both terrestrial and aquatic conditions (fresh and marine water). Although many forms have adapted to life in the tissues of various organs (Moravec, 2001b), most capillariids are digestive tract parasites. Deng et al. (2021) demonstrated that using molecular markers to study phylogenetic and systematic relationships within the family Capillariidae is helpful in investigating controversial taxonomic problems in the identification of genera or species.
Muscovy ducks, Cairina moschata (Linnaeus), are well adapted to different climatic conditions and have adjusted to breeding in captivity (Béjcek & Stastný, 2002). However, they are also found in the wild, in areas with adequate water and space (Geromel, 2012). Subsistence farming is common in Brazil, and the trade of live birds, eggs, and meat occurs mainly among small producers, commercial houses, and open markets (Almeida et al., 2016). On Marajó Island, birds are raised extensively, making these animals generalists in their diet; thus, they can act as paratenic hosts by ingesting infected fish viscera or becoming infected after filtering water containing microcrustaceans parasitized by nematodes or eggs (Carvalho et al., 2020). On Marajó Island, Muscovy ducks are commonly used by the local population for food and commercial purposes (Carvalho et al., 2019(Carvalho et al., , 2021. In Brazil, four capillariid species have been identified among the parasitic fauna of C. moschata as follows: Capillaria phasianina Kotlán, 1914;Capillaria sp. Pinto and Almeida, 1935, and Eucoleus cairinae (Freitas & Almeida, 1935) Lopez & Neyra, 1947and Eucoleus contortus Creplin, 1989, in Pará (Vicente et al., 1995;Mattos et al., 2008;Carvalho et al., 2019). This study describes a new nematode species of the genus Capillaria Zeder, 1800, that was found to infect Muscovy ducks in the Brazilian Amazon based on morphological data and phylogenetic analyses.

Parasitological examination
Each organ was isolated in a petri dish containing 0.9% NaCl saline in the laboratory and analyzed under a stereomicroscope (Leica ES2). Collected nematodes were washed in 0.9% saline, fixed in an A.F.A. solution (93 parts 70% ethyl alcohol, five parts formaldehyde, and two parts glacial acetic acid), and stored in 70% alcohol. In total, 197 adult nematodes were collected (114 females and 83 males), of which 118 were from the ceca, and 79 were from the large intestine. For light microscopy, nematodes were cleared in 0.5% lactophenol amine solution, observed under a Leica DM2500 microscope with a drawing tube, imaged under a Leica DM2500 microscope with a Leica DFC310 FX camera system using Leica Application Suite Software V4.4, and stored in glycerin alcohol (50% of 70% ethyl alcohol and 50% glycerin). For the morphometric analysis, 20 males, 20 females, and 50 eggs were used. Measurements are given in micrometers unless otherwise indicated and are represented as mean values, followed by the minimum and maximum values in parentheses.

Molecular analysis
For molecular and phylogenetic analyses, 20 nematodes (10 females and 10 males) were used. Helminths were isolated from the ceca and fixed in absolute alcohol. Total DNA was extracted using the Purelink ® Genomic DNA Mini Kit (Invitrogen ® ; ThermoFisher, CA, USA), following the manufacturer's instructions. The small subunit ribosomal RNA gene (SSU rDNA) sequence was amplified using primers 18S-E/18S-A27 and 18S-8/Cestode-6 (Olson & Caira, 1999). The final volume for the polymerase chain reaction (PCR) was 25 μL, containing 1 ng of DNA template, 20 mM Tris pH 8.4, 50 mM KCl, 2 mM dNTP (Invitrogen ® ), 1 mM Mg 2 Cl, 0.5 pmol of each primer, and 0.2 U of Taq DNA polymerase (Invitrogen ® ). The amplification profile for the polymerization of any molecules that might have dissociated from the polymerase prior to complete fragment synthesis consisted of 5 min of initial denaturation at 95 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C, with a final extension of 7 min at 72 °C.
The nucleotide sequences obtained from the samples were edited and aligned using BioEdit software (Hall et al., 2011) after a comparison with other sequences available in GenBank (BLAST search). The SSU rDNA sequence was aligned with sequences of 21 capillariids available in GenBank. In addition, the database includes sequences from Haemonchus placei (Place, 1893) and Haemonchus contortus (Rudolphi, 1803), which formed the outgroup for phylogenetic analyses. The consensus nucleotide sequences reported in this study are available in GenBank under accession number OP720889.
(Based on light microscopy and SEM, Figures 1-4) The nematodes were small and filiform, with delicate cuticles that were transversely striated. They exhibited a simple, dorsoventrally oriented oral opening. Their mouth was surrounded by 12 cephalic papillae arranged in two circles, each consisting of six papillae and a pair of small lateral amphids, with a stylet absent. They contained a short, muscular esophagus with a stichosome. The nerve ring surrounded the muscular esophagus in its initial portion. The stichosome consisted of a single row of approximately 40 elongated stichocytes with transverse rings that were difficult to visualize and large stichocytes nuclei with several nucleoli.   (B) Posterior extremity, ventral view, cloacal opening (arrowhead), membrane (*) and the caudal lobes (L). Scale bar = 20 µm; (C) Posterior extremity, lateral and ventral views of the tail with an extruded spicular sheath; lateral (yellow arrowhead) and ventral (white arrowhead) bacillary bands can be observed. Scale bar = 20 µm. In the insert, note the digitiform pre-cloacal papillae (green arrowhead) and the base of the spineless spicular sheath (white arrowhead). Scale bar = 20 µm; (D) Note the digitiform pre-cloacal papillae (arrowhead). Scale bar = 10 µm; (E) Posterior end with partially extruded spinous spicular sheath (ss), rough spicule (s), and membrane of the distal end of the spicule (blue arrow). Scale bar = 50 µm. Insert showing the distal end of the spicular sheath with spines (green arrowhead) and rough spicule (arrowhead). Scale bar = 10 µm; (F) Posterior extremity with spicule (s) and spicular sheath (ss) with fully extruded spines; the spicular canal is observed (arrowhead). Scale bar = 100 µm. Two glandular cells were observed at the esophageal-intestinal junction. Lateral and ventral bacillary bands extended for almost the entire body length in both sexes (Figures 1A-B, G).
Male measurements (based on 20 specimens with an extruded spicular sheath, holotype measurements in brackets) were as follows: body length, 14.7 (11.8-15.8 The sheath was not spiny at the base. A well-developed spicular canal was observed. The proximal end of the spicule was expanded, and the distal end was rounded, with rough transverse grooves observed on the spicule surface. The caudal end was rounded and bifurcated in ventral and dorsal views, without a pseudobursa, supporting two large, round ventrolateral lobes containing a pair of sessile ventrolateral pre-cloacal papillae and a terminal cloacal Female measurements (based on 20 gravid specimens, allotype measurements in brackets) were as follows: body length, 23.

Molecular and phylogenetic analyses
The partial rDNA sequence obtained for Capillaria cairina n. sp. was 1750 bp in length and is available in GenBank (OP720889). Thirty-one taxa were used for the comparison, and the outgroups were H. contortus and H. placei. Among these, four large, well-supported clades were formed as follows: A (Capillaria), B (Eucoleus Dujardin, 1845), C (predominantly Aonchotheca López-Neyra, 1947), and D (Baruscapillaria Moravec, 1982). A BLAST search revealed that the nucleotide sequences with the highest similarity were those of Capillaria pudendotecta Lubimova, 1947 (accession numbers LC052338 and LC052339), observed in swans in Japan, with 95.45% and 95.53% similarity, respectively, and of Capillaria spinulosa (Linstow, 1890) (accession numbers LC424999 and LC425000), in domestic birds from Indonesia and Japan, with 95.32% similarity. Clade A formed two subclades, A1 and A2, in which parasites belonging to the genus Capillaria were observed in the following hosts: Anseriformes (Anatidae), Galliformes (Phasianidae), Passeriformes (Corvidae), and Accipitriformes (Accipitridae), with infection sites in the ceca and small intestine ( Figure 5).
Remarks: The analysis of clade A and subclades A1 and A2 was performed, as the parasites were of the same genus as those in our study and differed due to the morphological characteristics observed in Table 1, with SEM and molecular biology being fundamental for the identification of Capillaria cairina n. sp.
Capillaria cairina n. sp., observed in subclade A1 in this study, formed a sister clade with C. spinulosa with a genetic distance of 6%, which can be justified as these parasites infect hosts of the same Anseriformes order and Anatidae family. Capillaria cairina n. sp. has morphological characteristics that distinguish it from C. spinulosa (Table 1), and it is the only family in this clade to occur in the ceca and large intestine of Muscovy ducks in Brazil. In this subclade, C. pudendotecta had a genetic distance of 6.4% to the results of our study. However, the only similarity was observed in Anseriformes and Anatidae birds.

Etymology:
The specific name cairina (genitive) is related to the generic name of the host bird (Cairina). Baruš et al. (1978) Moravec (1982) and Vicente et al. (1995), the genus Capillaria has the following diagnostic characteristics: the caudal lateral wings are absent in males; the posterior end of the male is rounded and equipped with two lateral lobes, located ventrolaterally or dorsolaterally. This genus lacks a membranous bursa, and sessile pre-cloacal papillae are often observed. These nematodes exhibit a sclerotized spicule and spiny spicular sheath, and they may or may not exhibit a vulvar appendix. They are intestinal parasites of fish, amphibians, reptiles, birds, and mammals, with thetype species being C. anatis. The parasite observed in C. moschata in this study exhibited characteristics of Capillaria. Baruš et al. (1981) identified C. anatis in Anas acuta (Linnaeus) and observed that the posterior end of the male forms a pseudobursa with a smooth cuticle and that the base is formed by two well-developed and rounded lateral processes, with a rounded cloacal opening located ventrally. The species identified in our study had a pair of sessile, longitudinally elongated pre-cloacal papillae, transverse cloacal openings, and lobes without pseudobursa; however, each had a well-marked membrane ( Figure 2G).

Discussion
By light microscopy, the parasite seemed to be similar to C. anatis, C. spinulosa, and C. pudendotecta. However, with SEM, our capillariid showed three bacillary bands (lateral and ventral), a delicate membrane in each lobe, and a pair of elongated pre-cloacal papillae, which differed from the descriptions of C. anatis reported by Baruš et al. (1978Baruš et al. ( , 1981. They observed microscopically the posterior end of males, and they could observe a delicate membrane between ventro-lateral lobes, called "pseudobursa," and a pair of short pre-cloacal papillae in males. The description differed from that of C. spinulosa, provided by Mettrick (1959) and Sakaguchi et al. (2020), who observed males with a caudal end showing two small, non-elongated papillae. Oyarzún-Ruiz et al. (2019) identified the presence of a spiny sheath with a distal expansion without a thorn in males of C. pudendotecta, which differed from the description of males in our study ( Figure 4F) since the absence of spines was at the base of the sheath.
All female specimens in our study were morphologically similar to C. pudendotecta described by Oyarzún-Ruiz et al. (2019), with a small vulvar appendix. However, they could be easily distinguished from C. anatis and C. spinulosa described by Baruš et al. (1981), Tanveer et al. (2015), andYevstafieva et al. (2020). They identified females without a vulvar appendix, and Mettrick (1959) and Sakaguchi et al. (2020) reported that C. spinulosa females had a vulva without a vulvar appendix. These differed from the females in our study but corroborated Moravec's (2001b) observations, who reported that capillariid females may or may not have a vulvar appendix. Thus, the organisms identified in this study differed from those already registered ( Table 2). Tamaru et al. (2015) and Sakaguchi et al. (2020) recorded C. pudendotecta and C. spinulosa, respectively, only in the ceca of Anas platyrhynchos var. domesticus (Linnaeus), Anser cygnoides domesticus (Linnaeus), and Cygnus olor (Gmelin). The morphometric comparisons with other capillariids that parasitize the large intestine and ceca of birds are described in Table 2. Yevstafieva et al. (2020) reported specific morphological characteristics and biometric parameters of male and female С. anatis observed in A. platyrhynchos raised on poultry farms in Ukraine. As reported by Moravec (1982Moravec ( , 2001b and Melnychuk et al. (2020), males have characteristics such as a pseudobursa, spicules, and spicular sheath ornamentation, and in females, the morphology of the vulvar area and eggs in the uterus should be considered. Cram (1936) and Moravec (2001b) considered the shape and arrangement of hypodermic cells (bacillary bands) as a complementary characteristic for the differentiation between genera and species of capillaries. In the capillariids identified in this study, three bacillary bands were observed in both males and females, which differed from those observed in other species. In addition, the male was reported to have a spicular sheath with sclerotized spines oriented toward the cloaca. The spines were sparse in the proximal part of the spicule sheath, whereas they were more densely distributed in the middle and distal parts.
The morphological details of capillariids, such as the anterior extremity, bacillary bands, and tail, are not visible under light microscopy (Moravec, 2001a;Moravec & Barton, 2018;Carvalho et al., 2019). In this study, we observed the conical shape of the cephalic region with large and flat papillae in both males and females using SEM. Females have a well-defined vulvar appendix and a pair of papillae close to the vulva. In addition, a large "rectangular" area without a bacillary band was observed around the vulva. The tail of the male exhibited a pair of thin and elongated papillae, a spicular sheath armed with small spines, and three bacillary bands, with the ventral bacillary band being wider in females. None of these aspects have been previously reported in capillariid species and are described for the first time in the genus Capillaria in our study (Figures 3C-D).
The descriptions of many capillariids and the establishment of unjustified new genera within the family Capillariidae have confused the taxonomy and systematics of this group of parasites (Moravec et al., 1981).
The pairwise genetic distance between our isolate and C. spinulosa was 6%, this distance is considered high because most distances between capillariid species belonging to the same genus are less than 1%, according to Tamaru et al. (2015), Sakaguchi et al. (2020) and Garbin et al. (2021). Sakaguchi et al. (2020) noted that integrative approaches are highly recommended for identifying capillariids. Thus, in the phylogenetic tree constructed in our study, the species were grouped according to the site of infection, order, and host family. These factors determine the degree of proximity between the Capillaria species analyzed in this study.

Conclusion
Through morphological, morphometric, and phylogenetic analyses of the capillariid nematodes in our study, we identified a new species, Capillaria cairina n. sp., which parasitizes the large intestine and ceca of C. moschata.