A new species of Aplectana Railliet & Henry, 1916 (Nematoda: Cosmocercidae) in the Brazilian Amazon and the taxonomic status of Aplectana longa

Abstract Nematodes of the genus Aplectana Railliet & Henry, 1916 are common parasites of the digestive tract of amphibians and reptiles in the Neotropical region. During a parasite survey on Boana boans (Linnaeus, 1758), we found specimens of nematodes with Aplectana characteristics. We observed a set of characteristics that differs the species of our study from its congeners, and the present study describes a new species of Aplectana parasite of B. boans using light microscopy and scanning electron microscopy. Aplectana pella n. sp. has lateral alae and somatic papillae in males and females; males have equal short spicules, and the gubernaculum is absent. The arrangement of pairs of caudal papillae also differs from other species (2 precloacal,1 adcloacal, and 5 postcloacal+1 unpaired). In females, the vulva is simple, with non-prominent lips, and equatorial. This is the first record of the genus Aplectana parasitizing B. boans and the 58th species described for this genus. Additionally, we added the precloacal papillae pattern of A. delirae, and based on morphological and morphometric characteristics, we propose the reallocation of Aplectana longa to the genus Oxyascaris.


Introduction
Nematodes of the family Cosmocercidae Railliet, 1916 are frequently found parasitizing the digestive tract of amphibians and reptiles worldwide.Among the members of Cosmocercidae, the most common genera found in hosts from the Neotropics are species of Aplectana Railliet &Henry, 1916 andCosmocerca Diesing, 1861, with Aplectana being the most abundant among neotropical amphibians and richest in species (Bursey et al., 2001;Campião et al., 2014;Bursey et al., 2011;González et al., 2021).
The genus Boana Gray, 1825, comprises anuran species distributed throughout Central and South America, from Nicaragua to Argentina, and are also present in Trinidad and Tobago (Frost, 2023).To date, no species of the genus Aplectana have been found in Boana boans (Linnaeus, 1758).Therefore, this study describe a new species of the genus Aplectana parasitizing B. boans in the Brazilian Amazon.Additionally, after analyzing Aplectana spp.from the Neotropics for morphological comparisons, we noted some disparities in the identification of Aplectana longa Alcantara & Silva, 2021.Thus, we proposed a new combination and relocation to the genus Oxyascaris Travassos, 1920.

Materials and Methods
Amphibian samples were collected within the "Beija-Flor Brilho de Fogo" Extractive Reserve, located in the southern part of the municipality of Pedra Branca do Amapari, state Amapá, Brazil (0°47 '30.6'N, 51°58 '42.1'W).The region comprises dense forests with large trees, with the Amapari River as the main watercourse, and the climate is equatorial (Pedroso-Santos et al., 2019).The collections were performed through an active search (visual and auditory) during June and September 2021, totalizing 16 specimens of B. boans (SISBIO: No 48102-2).
The frogs were euthanized with lidocaine (2%) and necropsied to remove all internal organs, which were later transferred to Petri dishes containing NaCl solution (0.9%).The nematodes were killed in heated 70% alcohol and preserved in tubes containing 70% alcohol.For morphological and morphometric analyses, the helminth specimens were hydrated in distilled water, clarified with Amman's Lactophenol, mounted on temporary slides, and analyzed under an Olympus BX41 microscope equipped with a drawing tube.The classification of helminths was performed using catalogs and identification keys, based mainly on Anderson et al. (2009), Gibbons (2010), and other relevant scientific articles.
Four specimens (two males and two females) were selected for analysis by scanning electron microscopy (SEM) and subjected to post-fixation in 1% Osmium tetroxide (OsO 4 ) and then washed in distilled water.Subsequently, they were dehydrated in an increasing series of ethanol (30% to 100%), dried in a CO 2 Critical Point, then mounted on aluminum metal supports, and sputtered with gold-palladium.The specimens were analyzed under a Tescan Vega3 microscope in the Laboratory of Structural and Functional Biology (LABEF -ICB), Federal University of Pará (UFPA), Brazil.
For additional morphological comparisons, we examined holotypes and paratypes of Aplectana delirae deposited in the Helminthological Collection of the Oswaldo Cruz Institute, Brazil (CHIOC), under deposit numbers CHIOC 30591 a-l.
The measurements are presented as the holotype/allotype values, followed by the minimum and maximum in parentheses (reported in micrometers unless otherwise indicated).The prevalence, mean abundance, and mean intensity were calculated according to Bush et al. (1997) andReiczigel et al. (2019).The specimens were deposited in the Collection of Other Invertebrates of the "Museu Paraense Emílio Goeldi" (MPEG), municipality of Belém, state of Pará, Brazil.Etimology: The specific epithet is after the species of hummingbird Topaza pella (Linnaeus, 1758), known as the "Beija-Flor Brilho de Fogo" which gives rise to the name of the "Beija-Flor Brilho de Fogo" Extractive Reserve.

Description (Figures 1-2)
General.Small-sized nematodes.Cuticle with fine transverse striations.Sexual dimorphism evident, males smaller than females.Lateral alae present in both sexes, beginning anterior to nerve ring and ending near tail tip (Figure 2A, 2F).Oral aperture simple, triangular, surrounded by 3 small lips, dorsal lip with one pair of papillae, subventral lips with one papilla and one amphid each (Figure 2C).Esophagus divided into anterior short pharynx, cylindrical corpus, slightly narrower isthmus, posterior bulb with valves (Figure 1B).Excretory pore (Figures 1B, 2D, 2E) slightly anterior to isthmus.Somatic papillae small, with small and non-prominent sensilla in both sexes distributed along whole-body length, very evident in tail of females (Figures 1A,1D,2B,2G).Tail conical with a filament in both sexes.
Aplectana chilensis is easily differentiated from Aplectana pella n. sp. because the latter has an unpaired papilla on the anterior cloaca lip (absent in A. chilensis).In addition, the species have different number and arrangement of caudal papillae pairs (2:1:5+1 in the new species vs. 12:0:6 in A. chilensis), and the spicules are smaller in the new species (104-111 in Aplectana pella n. sp. vs. 260-410 in A. chilensis), as well as the tail length of females (321-394 in Aplectana pella n. sp. vs. 640-670 in A. chilensis) (Lent & Freitas, 1948).
The new species resembles A. crossodactyli because both have an unpaired papilla on the anterior lip of the cloaca.However, these species differ in the number and arrangement of caudal papillae pairs (2:1:5+1 in the new species vs. 20:0:5+1 in A. crossodactyli), the spicules are smaller in the new species (104-111 in Aplectana pella n. sp. vs. 130-140 in A. crossodactyli), males are smaller in total body length (2.3−2.9 mm vs. 2.8-3.7 mm in A. crossodactyli), as well as Aplectana pella n. sp.have a greater distance from the excretory pore to the anterior end in males than that A. crossodactyli (312−344 vs. 290, respectively) and the total length of the esophagus is shorter in females of the new species than that A. crossodactyli (529-594 vs. 610, respectively).Additionally, females have a shorter distance from the vulva to the anterior end (0.6-2 mm vs. 2.1-2.7 mm, respectively) (Vicente et al., 1990).
Aplectana pella n. sp. shares with A. crucifer the absence of the gubernaculum.However, the new species has an unpaired papilla on the anterior cloacal lip, which is absent in A. crucifer.Additionally, they differ by the number and arrangement of the cloacal papillae pairs (2:1:5 +1 in new species vs. 6:1:4 in A. crucifer); the length of the spicules of Aplectana pella n. sp. is three times smaller than that of A. crucifer (104-111 vs. 330, respectively), and the tail of females of the new species is small than that A. crucifer (321-394 vs. 560, respectively) (Travassos, 1931).
Aplectana delirae is a species that has been described as parasitizing anurans from the Neotropics and does not have a gubernaculum.The description by Fabio (1971) does not include details about the number and pattern of precloacal papillae but rather characterizes them only as "variable".However, when analyzing the type series of A. delirae, we observed that males have the following pattern of caudal papillae pairs: 3 precloacal, 1 adcloacal and 4 postcloacal, and 1 unpaired papilla in the upper cloacal lip (3:1:4+1).Thus, the new species can be distinguished from A. delirae because it has a different pattern of caudal papillae pairs: 2:1:5+1.In addition, the males of the new species are smaller in total body length (2.3−2.9 mm in Aplectana pella n. sp.vs 3.97-4.04mm in A. delirae), distance from the vulva to anterior is shorter (0.6−2 mm in Aplectana pella n. sp. vs. 2.13 mm in A. delirae) and the length of the spicules are smaller (104-111 in the new species vs. 150-170 in A. delirae) (Fabio, 1971).
The new species resembles A. meridionalis in the absence of the gubernaculum and by having an unpaired papilla on the anterior lip of the cloaca.However, they differ in the length and width of the eggs (54-67 × 33-43 in Aplectana pella n. sp. vs. 74-93 × 37-56 in A. meridionalis), distance from nerve ring to the anterior end in males (176-226 in the new species vs. 150-190 in A. meridionalis) and because the spicule length is shorter in the new species (104-111 vs. 130-136).Additionally, they differ by the pattern of cloacal papillae pairs (2:1:5+1 in Aplectana pella n. sp. vs. 7:2:5+1 in A. meridionalis) (Lent & Freitas, 1948).
Aplectana papillifera resembles the new species by having an unpaired papilla on the anterior lip of the cloaca, and the gubernaculum is absent.However, they can be differentiated by the number and arrangement of the caudal papillae pairs.Aplectana pella n. sp. has a pattern of 2:1:5+1, while in A. papillifera, the pattern is 10:1:8+1.In addition, the new species has shorter spicules (104-111 in the new species vs. 400 in A. papillifera), a smaller body length in both males and females (2.3−2.9 mm in males and 2.3−4.3 mm in females vs. 4.4-5.2mm in males and 5.9-8.5 mm in females, respectively) and a shorter distance from the vulva to the anterior end (0.6-2 mm vs. 3.5-5 mm, respectively) (Araujo, 1976/77).

Discussion
Species distinction and taxonomic status of Oxyascaris longum n. comb.
In the present study, the nematodes described have lateral alae, somatic papillae, male caudal papillae without rosettes or plectanes, females with two ovaries anterior to the vulva, and numerous thin-shelled eggs in the uterus.According to Baker (1980) and Gibbons (2010), these are characteristics commonly exhibited by species of the genus Aplectana.Aplectana pella n. sp.can be included in the group of species that do not have a gubernaculum and in which males have an unpaired papilla on the anterior cloacal lip.Currently, Aplectana includes 57 nematode species parasitic of amphibians and reptiles that are distributed worldwide, of which 16 have been recorded in Brazil (Bursey et al., 2018;Alcantara et al., 2021).In the present study, we did not include Aplectana longa Alcantara and Silva, 2021 in species comparisons because the morphological and morphometric characteristics described by the authors demonstrate that the species may have been mistakenly allocated to the genus.
Aplectana longa is a species described as parasitizing Gastrotheca microdiscus (Andersson, 1910) in São Paulo, Brazil.Among the main characteristics proposed for the species, the authors highlight the absence of lateral alae and the total length of both males and females, at that time, being one of the largest species of the genus.In addition, they add the absence of lateral alae to the diagnosis of the genus Aplectana (Alcantara et al., 2021).However, the lateral alae has been widely used as a diagnostic feature of Aplectana [see Baker (1980), Anderson et al. (2009), Gibbons ( 2010)], and it is noteworthy that this would be the only species of the genus known thus far that does not have lateral alae.In addition, according to Baker & Vaucher (1985), mature females of Aplectana spp.do not exceed 10 mm, it is noteworthy that in A. longa, females measure 10.7-20.8mm and they presented numerous eggs and larval development in the uterus, similar to Oxyascaris spp.
The genera of the family Cosmocercidae are differentiated mainly through the type of ornamentation associated with papillae in the caudal end of males (Anderson et al., 2009).The genera Aplectana, Oxyascaris, Oxysomatium and Raillietnema share the same type of caudal papillae (simple and unadorned).In Oxysomatium, Raillietnema, and Oxyascaris females have a characteristic reproductive system (amphidelphic) and are easily differentiated from Aplectana.In addition, Baker & Vaucher (1985) proposed that large females and numerous eggs or free larvae in the uterus are characteristics of Oxyascaris.Thus, carefully observing the specimens described by Alcantara et al. (2021), we propose that Aplectana longa should be relocated to the genus Oxyascaris.
Additionally, we calculated the bulb width/body length ratio of females and males of Oxyascaris spp.and Neotropical species of Aplectana.When calculating the proportion for the species of Aplectana, we observed that the bulb ratio varied from 1.74% -6% in males and 0.80%-6% in females.On the other hand, the bulb width/body length ratio for Oxyascaris varied from 0.95%-2.15% in males and 0.48%-0.85% in females.Thus, we concluded that in the species of Aplectana, the bulb is larger than that of Oxyascaris spp.Detailed comparisons among bulb width/total length are presented in Supplementary Table S1.
According to the illustration and morphometry presented by Alcantara et al. (2021), the bulb of A. longa is less dilated than that of other Aplectana species.After calculating the bulb width/total length proportion, we found that this structure corresponds to 1.6% of the body length in males and 0.65% in females, similar to Oxyascaris spp.In addition, the body length of the females and the absence of the lateral alae are additional evidence that cast doubt on the allocation of A. longa to the genus.
We conclude that based on the morphological characteristics of the esophagus, the absence of lateral alae, the body length of female specimens, and the presence of eggs and larvae in the uterus of females of A. longa, the species should be reallocated to the genus Oxyascaris and named Oxyascaris longum n. comb.
To validate Oxyascaris longum n. comb., we compared the species with its congeners.We will use the following set of characteristics to differentiate the species: number and distribution of caudal papillae, spicule length, presence/absence of gubernaculum, presence/absence of lateral alae, presence/absence of unpaired papilla in the cloacal anterior lip and position of the vulva in relation to the anterior end of the female's body.According to Freitas (1958), Baker &Vaucher (1985), andFelix-Nascimento et al. (2020), these are the main characteristics that differentiate Oxyascaris spp.
Additionally, the lateral alae are absent in O. longum and are present in O. oxyascaris, O. caudacutus, O. similis, and O. mcdiarmidi.Oxyascaris longum n. comb. is the only species of the genus with a vulva in the equatorial position.In the other species of the genus, the vulva is in the pre-equatorial region of the body (O.caatingae, O. caudacutus, O. similis, and O. oxyascaris) and post-equatorial (O.mcdiarmidi).Thus, O. longum n. comb.becomes the 6th known species of Oxyascaris.

Final Remarks
The nematodes analyzed in our study have characteristics corresponding to the genus Aplectana and represent a new species, which was described using light microscopy and SEM.In the present study, we reallocated A. longa to the Oxyascaris longum n. comb.proposing 6 th species of the genus Oxyascaris.
Furthermore, this is the first record of the genus Aplectana for the hylid B. boans.Thus, we describe the 58 th species of Aplectana, the 28th species for the Neotropics, and the 17 th species for Brazil, adding new data on the diversity of helminth parasites of frogs in the Brazilian Amazon.