Introduction
Species of the genus Kalicephalus (Molin, 1861) are gastrointestinal parasites of snakes and lizards (Anderson Reference Anderson2000; Schad Reference Schad1956). To date, 33 species for the genus are known worldwide (Kuzmin et al. Reference Kuzmin, Kinsella, Tkach and Bush2013), with seven reported in the Neotropical region: Kalicephalus inermis inermis Molin, 1861; Kalicephalus inermis macrovulvus (Caballero, 1954); Kalicephalus subulatus Molin, 1861; Kalicephalus inermis coronellae (Ortlepp, 1923); Kalicephalus costatus costatus (Rudolph, 1819); Kalicephalus appendiculatus Molin, 1861; and Kalicephalus rectiphilus neoctiphilus Schad, Reference Schad1962.
Despite the diversity among Kalicephalus species, most taxa have been identified based only on morphological features (Larki et al. Reference Larki, Alborzi, Bahramnejad and Asadi2023; Pereira et al. Reference Pereira, Ferreira, Tomas, Elisei, Paiva and Tavares2018). The current genetic database for this genus remains limited, with sequences for only five species. These include Kalicephalus branchycephalus (Maplestone, 1931); Kalicephalus indicus (Ortlepp, 1923); and Kalicephalus bungari (MacCallum, 1918), all collected in snakes from Hunan Province, China. Additionally, Chilton et al. (Reference Chilton, Huby-Chilton, Gasser and Beveridge2006), also conducted a molecular characterisation of Kalicephalus cristatus from Australia.
The common lancehead, Bothrops atrox (Linnaeus, 1758), is a venomous snake of the family Viperidae widely distributed throughout northern South America (Nogueira et al. Reference Nogueira, Argôlo, Arzamendia, Azevedo, Barbo and Bérnils2019). This species is primarily terrestrial and nocturnal, mainly inhabiting rainforests, although it can also be found near urban areas (Cunha and Nascimento Reference Cunha and Nascimento1975; Fraga et al. Reference Fraga, Lima, Prudente and Magnusson2013; Martins and Oliveira Reference Martins and Oliveira1998; Oliveira and Martins Reference Oliveira and Martins2001). In Brazil, B. atrox is responsible for most snakebite accidents in the Amazon region, highlighting its significant medical importance (Pardal et al. Reference Pardal, Souza, Monteiro, Fan, Cardoso, França and Warrell2004; Siva et al. Reference Siva, Monteiro and Bernarde2019).
The present study describes a new species of Kalicephalus parasitising B. atrox from Amapá state, Brazil. Also, we added the sequences of K. costatus costatus, parasites of the snake Chironius fuscus (Linnaeus, 1758) from the same locality. For the first time, we provide data on internal transcribed spacer 1 (ITS1) for Kalicephalus species in the Neotropical region.
Materials and methods
Host collection and morphological analyses
During a helminthological survey between September 2020 and September 2021, we collected six specimens of Bothrops atrox and one Chironius fuscus in the ‘Beija-flor Brilho de Fogo’ Extractive Reserve, Pedra Branca do Amapari municipality, state of Amapá, Brazil.
The snakes were anaesthetised and euthanised by injection of sodium thiopental; then measured, weighed, and necropsied. The internal organs were removed and dissected under a stereomicroscope. The nematodes found were collected, washed in saline solution (0.9% NaCl), fixed in heated 70% ethanol, and preserved in the same solution at room temperature.
For morphological analyses, the helminths were cleared in Amann’s lactophenol, mounted on temporary slides, and examined in an Olympus BX41 light microscope equipped with a drawing tube. The illustrations were prepared using the CorelDraw 2021 software and processed using Adobe Photoshop Version 21.0.2 software.
Four nematodes (two males and two females), not part of the type series, were post-fixed in 1% OsO4, dehydrated through a graded ethanol series, critical-point dried in carbon dioxide (CO2), mounted on stubs, coated with a thin layer of gold, and analysed using a Vega3 Scanning Electron Microscope (TESCAN) at the Structural Biology Laboratory of the Federal University of Pará (LBE-UFPA).
All morphometric data are provided in micrometres (unless otherwise indicated) as holotype/allotype values, followed by the mean of the paratypes and the minimum and maximum values in parentheses. The vulvar ratio and dorsal rays were evaluated following the criteria established by Schad (Reference Schad1962). The holotype, allotype, and type series were deposited in the Invertebrate Collection of the Museu Paraense Emílio Goeldi (MPEG) (Belém, state of Pará, Brazil).
Molecular analyses and phylogenetic study
For molecular analyses, a single specimen of K. atroxi n. sp. and K. costatus costatus were transferred to microtubes containing 100% ethanol and preserved in a freezer at −20ºC. We cut the anterior and posterior portions and stored them separately to confirm each sample’s identity. The middle portion of the nematode body was then kept in absolute ethanol for later molecular characterisation.
Genomic DNA was extracted using Chelex® Molecular Biology Grade Resin according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was performed to amplify the internal transcribed spacer 1 (ITS-1) region using the protocol of Liu et al. (Reference Liu, Wang, Liu, Tan, Liu and Liu2019). The PCR products were visualised on 1% agarose gel electrophoresis and purified using a Qiagen® QIAquick PCR Purifcation kit. The fragments were sequenced according to the protocols for the Big Dye® terminator v.3.1 Cycle Sequencing kit in an ABI 3730 DNA Analyser at the Human Genome and Stem Cell Research Center, Biosciences Institute, University of São Paulo.
Contiguous sequences were assembled and edited using Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock and Drummond2012) and subsequently deposited in the National Center for Biotechnology Information (NCBI). The ITS-1 datasets were aligned and trimmed using Muscle (Edgar Reference Edgar2004) in Geneious 7.1.3 software (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock and Drummond2012). The most suitable evolutionary nucleotide substitution model was TPM3+G, as determined by the Akaike Information Criterion (AIC) in the jModelTest software (Posada Reference Posada2008).
We conducted phylogenetic reconstructions using maximum likelihood (ML) and Bayesian inference (BI). The ML analysis was performed in RAxML, and we considered only nodes with a bootstrap greater than 70% to be well-supported (Guindon and Gascuel Reference Guindon and Gascuel2003). For BI analysis, we used MrBayes and considered nodes with Bayesian posterior probabilities above 90% as well-supported (Ronquist and Huelsenbeck Reference Ronquist and Huelsenbeck2003). Both analyses were conducted in CIPRES Science Gateway (Miller et al. Reference Miller, Pfeifer and Schwartz2010).
The phylogenetic analyses included three genera: Kalicephalus (K. costatus costatus, K. brachycephalus, and K. indicus); Diaphanocephalus Travassos, 1920 (D. galeatus (Rudolphi, 1819)); and Ancylostoma Dubini, 1843 (A. caninum (Ercolani, 1859) Hall, 1913, A. ceylanicum Looss, 1911, A. duodenale Dubini, 1843 and A. braziliense Faria, 1910). We used Oswaldocruzia filiformis (Goeze, 1782) as an outgroup in the phylogenies. The trees were visualised in the software FigTree v1.3.3 (Rambaut Reference Rambaut2009) and edited in Adobe Photoshop 21.0.2 software.
Results
Taxonomic summary
Kalicephalus atroxi n. sp. Haick, Tavares-Costa & Melo, 2025
Type host: Bothrops atrox (Linnaeus, 1758) (Squamata: Viperidae: Crotalinae)
Type material: Holotype, female (MPEG. NEM 000419); allotype, male (MPEG.NEM 000420); and paratypes, 10 females (MPEG.NEM 000421), 11 males (MPEG.NEM 000422) were deposited in the Invertebrate Collection of MPEG, Pará, Brazil
Type locality: ‘Beija-Flor Brilho de Fogo’ Extractive Reserve, Pedra Branca do Amapari municipality, state of Amapá, Brazil (0º47’30,6’’N, 51º58’42,1’ ’W)
Site of infection: Oesophagus, Stomach, and Small Intestine
Parasitological descriptors: Prevalence 88.9% (8 of 9 analysed); mean intensity 18 (2–39)
Zoobank register: The Life Science Identifer for K. atroxi n. sp. is urn:lsid:zoobank.org:act:12B6B0AF-5EA3-403E-9F23-37730CC4F43E
Genbank Accession number: Kalicephalus atroxi n. sp. (PV788965, PV788967), Kalicephalus costatus costatus (PV788966)
Etymology: The species was named in reference to the specific epithet of the type host.
General. Body cylindrical, both extremities gradually attenuated. Cuticle finely transversely striated. Cephalic inflation present. Corona radiata absent. Buccal capsule with two valves, formed by chitinous pieces, each valve with an anterior transverse ridge and three parenchymatous extensions (Figure 1a), oral opening is surrounded by two pairs of lateral papillae and one amphid on each side, positioned at the median portion of the oral margin (Figure 2a). Buccal capsule with rectangular anterior plates. Dorsal gutter well-developed, extending from oesophagus to mid-region of buccal capsule. Oesophagus claviform, muscular, wider in bulbar region. Nerve ring located in mid-region of oesophagus and excretory pore at oesophageal bulb (Figure 1a). Intestine straight, with rectal glands at its posterior extremity (Figure 1c).
Line drawings of Kalicephalus atroxi n. sp. from Bothrops atrox. (a), Anterior end of the body, lateral view; (b), vulvar region, lateral view; (c), posterior end of the female, lateral view; (d), copulatory bursa, ventral view; (e), posterior end of the male body, lateral view; (f), spicules, ventral view; (g), gubernaculum, ventral view.
Scanning Electron Microscopy of Kalicephalus atroxi n. sp. from B. atrox. (a), Details of the anterior end (asterisks = amphid; arrowhead = cephalic papillae), inset: detail of the amphid (scale bar: 15 μm); (b), details of the copulatory bursa, genital cone, and dorsal rays; (c), detail of vulvar region; (d), posterior end of the female (arrowhead = phasmids; an = anus).
Females. (Based on the allotype and 10 paratypes, all gravid specimens): Total length 12.13; 10.26 (7.52–12.53) mm, maximum width 487; 430 (267–553). Buccal capsule 213; 206 (181–229) deep, head diameter 272; 261 (235–304). Oesophagus 477; 432 (392–477) long. Maximum width at bulb region 237; 231 (171–272). Nerve ring and excretory pore located at 381; 370 (323–419) and 536; 468 (344–563) from the anterior end. Amphidelphic, vulva with prominent lips situated at 4.13; 3.55 (2.43–4.25) mm from posterior extremity (Figures 1b, 2c), with a vulvar ratio of 1.93; 1.90 (1.54–2.20). Vagina short, bifurcating into ovejectors and short, muscular sphincters (Figure 1b). Eggs at morula stage in uterus and vagina, with a smooth, thin, and elliptical shell 65; 61 (50.5–68) long and 37; 36 (30–41) wide. Rectal glands at final portion of intestine. Anus with posterior lip more prominent. Tail elongated, 427; 417 (344–517) long (Figure 2c), tapering posteriorly, with a terminal spine (Figure 1c).
Males. (Based on the holotype and 10 paratypes, all adult specimens): Total length 9.7; 8.57 (6.72–11.24) mm, maximum width 333; 291 (200–340). Buccal capsule 168; 166 (159–179) deep, head diameter 239; 217 (189–239). Oesophagus 421; 390 (349–421) long, maximum width at bulb region 235; 212 (152–251). Nerve ring and excretory pore situated at 330; 317 (291–333) and 544; 439 (301–544) from anterior end. Testis single, tubular, flexing posteriorly in anterior region. Caudal bursa wide and oblique, typical of the genus (Figures 1e, 2b). Ray 1 indistinct, rays 2 and 3 originating from a common trunk. Rays 4, 5, and 6 arise from a common trunk and run parallel. Rays 7 and 8 separate; ray 7 reduced, not reaching bursal edge. Dorsal ray type IV (according to Schad Reference Schad1962), bifurcating innermost terminal ray (Figure 1d, e). Spicules 720; 692 (629–733) long and equal in size, covered with sheath with fine transversal striations (Figure 1f). Gubernaculum 162; 163 (143–196) long, navicular in shape (Figure 1g). Telamon with crests in lateral view (Figure 1d), genital cone well-developed (Figures 1e; 2b).
Remarks
Kalicephalus atroxi n. sp. was assigned to the genus Kalicephalus based on the following morphological traits: presence of complex buccal capsule, bivalved (each valve with an internal transversal ridge), corona radiata absent, the males have copulatory bursa divided into lobes, and spicules are alate.
Our specimens belong to the subgenus Inermiformes (Lichtenfels, 1980), based on equal spicules, an amphidelphic reproductive system, and parasitising snakes from the New World. Thus, we will compare and differentiate the new species to those included in this subgenus: Kalicephalus inermis inermis; Kalicephalus inermis macrovulvus; Kalicephalus subulatus, and Kalicephalus inermis coronellae.
The new species resembles Kalicephalus inermis inermis by having a type IV copulatory bursa; however, in K. atroxi n. sp. the internal branches of the dorsal rays are bifurcated (simple in K. inermis inermis). Kalicephalus atroxi n. sp. is smaller than Kalicephalus inermis inermis in body dimensions (9.56–15.67 mm × 530–820 in K. inermis inermis vs 7.52–12.53 mm x 267-553 in K. atroxi n. sp.); oesophagus total length (580–730 × 260–360 in K. inermis inermis vs 392–477 × 171–272 in K. atroxi n. sp.), and spicules length (680–1000 in K. inermis inermis vs 629–733 in K. atroxi n. sp.). Also, the females of the new species have an elongated and tapered tail, while in K. inermis inermis it is short and conical.
Kalicephalus atroxi n. sp. can be easily distinguished from Kalicephalus inermis macrovulvus by several characteristics. Firstly, the new species features a type IV copulatory bursa, whereas K. inermis macrovulvus has a type V. Additionally, they differ in the length of spicules (629–733 in K. atroxi n. sp. vs 700–900 in K. inermis macrovulvus). Vulvar lips are less protruded in K. atroxi n. sp., while in K. inermis macrovulvus vulvar lips are very protruded. Lastly, the tail of the new species has a tapered end, in contrast to the rounded tip end in Kalicephalus inermis macrovulvus.
Although the new species and Kalicephalus subulatus are found in the Amazon region, the females of K. atroxi n. sp. have a terminal spine at the end of the tail, which is absent in K. subulatus. Additionally, the new species features a type IV copulatory bursa, while K. subulatus has a type III. Furthermore, K. atroxi n. sp. has larger spicules (630–730 in K. atroxi n. sp. vs 510–650 in K. subulatus) and longer tails (344–517 K. atroxi n. sp. vs 240–390 in K. subulatus).
Kalicephalus atroxi n. sp. resembles Kalicephalus inermis coronellae by the presence of slight cephalic cuticular inflation. However, the new species differs in the type IV copulatory bursa (type III in Kalicephalus inermis coronellae), the greater dimensions of buccal capsule (181–229 × 235–304 in K. atroxi n. sp. vs 120–150 × 210–240 in K. inermis coronellae), longer oesophagus (392–477 in K. atroxi n. sp. vs 300–360 in K. inermis coronellae), and larger spicules (630–730 in K. atroxi n. sp. vs 480–560 in K. inermis coronellae). In addition, while Kalicephalus inermis coronellae is distributed from the northeast of the USA to southern Mexico, the new species has been exclusively found in Brazil.
Currently, only three species of the genus are registered from Brazil: Kalicephalus costatus costatus, Kalicephalus appendiculatus (subgenus Kalicephalus), and Kalicephalus rectiphilus neorectiphilus (subgenus Rectiphiloides Lichtenfels, 1980).
We already differentiated K. costatus costatus from the new species. Thus, species in the subgenus Kalicephalus can be easily distinguished from K. atroxi n. sp. by their female reproductive system, which is prodelphic. In these species, the vulva is located closer to the anus than to the middle of the body. In addition, all species found in Brazil have the terminal branches of the dorsal ray classified as type III.
Kalicephalus rectiphilus neorectiphilus, a parasite of Palusophis bifossatus (Raddi, 1820), can be distinguished from the new species by its dorsal ray, which is of type II, exclusively found in species assigned to the subgenus Rectiphiloides. In contrast, Kalicephalus atroxi n. sp. belongs to the subgenus Inermiformes, in which the dorsal ray is of the type IV. Furthermore, the members of the subgenus Rectiphiloides typically infect the rectum of snakes.
Molecular analyses and phylogenetic study
The ITS1 region sequencing of Kalicephalus atroxi n. sp. resulted in 568bp, while a BLASTn search revealed no identical match with any other Diaphanocephalidae available in the NCBI database. The sequencing of the ITS1 region of Kalicephalus costatus costatus resulted in a sequence of 627 bp.
A pairwise genetic comparison between congeners of the new species revealed the lowest genetic distance to Kalicephalus costatus costatus with a nucleotide divergence of 7–8%. This was followed by K. indicus, which showed a difference of 54-55% (see Supplementary 1). The alignment of our sequences with those available in GenBank generated a matrix of 379 bp.
Maximum likelihood and Bayesian inference phylogenetic analyses based on 11 taxa resulted in similar topologies, forming two main clades (clades A and B). The sequences of K. atroxi n. sp. formed a well-supported monophyletic group with Kalicephalus costatus costatus parasites of Chironius fuscus (Linnaeus, 1758) (Serpentes: Colubridae), from the same locality as the new species (clade B2).
The clade B is a well-supported monophyletic group, which included the sequences of Kalicephalus spp. (clade B2 and B3) and three sequences of Diaphanocephalus galeatus (MN589649, MH134561, MN589648) from Dracaena paraguayensis (Amaral, 1950) (Sauria: Teiidae) from the State of Mato Grosso do Sul, Brazil (clade B4). The clade B2 consists of K. atroxi n. sp. and Kalicephalus costatus costatus with a high support and forms a sister group with Kalicephalus brachycephalus (MK685982) + Kalicephalus indicus (MK630251), both of which were found parasitising the snake Elaphe carinata (Günther, 1864) (Serpentes: Colubridae) from Hunan Province, China (clade B3).
Finally, the clade formed by nematodes belonging to the family Diaphanocephalidae (Kalicephalus and Diaphanocephalus) constitutes a sister group distinct from the other members of the internal group, which belong to the family Ancylostomatidae (clade A) (Figure 3).
Maximum likelihood phylogenetic of Ancylostomatidae and Diaphanocephalidae based on ITS1 gene sequences using Oswaldocruzia filiformis as outgroup and indicating the position of Kalicephalus atroxi n. sp. (shown in bold). GenBank accession numbers follow each taxon. Support values are presented as posterior probabilities/bootstrap values; when posterior probabilities <0.90 and bootstrap values <70 are represented by a dash.
Discussion
Kalicephalus atroxi n. sp. is the eighth species of the genus described in the Neotropical region, the sixth identified in Brazil, and the first recorded from the state of Amapá. The main morphological characters distinguishing the new taxon from its congeners include cuticular inflation in the cephalic region, the morphology of the female reproductive system, tail shape, type of dorsal ray (which has a single bifurcation in the deepest rays), and the length of spicules.
Observations using Scanning Electron Microscopy (SEM) have been made for the genus Kalicephalus, including Kalicephalus subulatus from Xenodon merremii (Wagler, 1824) in Argentina (González et al. Reference González, Schaefer and Duré2018); Kalicephalus guangdongensis (Zhang et al. Reference Zhang, Zhang and Zhang2011), and Kalicephalus schadius fotedari (Kalia and Nayital, 1989) from Varanus salvator (Laurenti, 1768) in China (Zhang et al. Reference Zhang, Zhang and Zhang2011); and Kalicephalus viperae viperae (Rudolphi, 1819) from Macrovipera lebetina (Linnaeus, 1758), from Iran (Larki et al. Reference Larki, Alborzi, Bahramnejad and Asadi2023). The SEM analyses allowed us to observe the ultrastructural aspects of the anterior end of Kalicephalus atroxi n. sp., the vulva opening, the arrangement of the phasmids on the female tail, and details of the copulatory bursa in males. However, using this technique, we do not observe the excretory pore or the presence of cervical papillae (deirids).
The ITS1 sequence of the ribosomal gene from Kalicephalus atroxi n. sp. forms a well-supported monophyletic clade with Kalicephalus costatus costatus, a species found distributed across the Neotropical region. This group also showed a low genetic difference (only 7% nucleotide divergence), besides these species being very different morphologically, particularly on the copulatory bursa and the female reproductive system.
The dorsal rays of types III and IV are the most common trait shared among Kalicephalus species (Schad, Reference Schad1962). The morphology of the male copulatory bursa and the female reproductive system in Kalicephalus spp. do not necessarily reflect the phylogenetic relationships within the group, as these characters appear to be more related to functional adaptations than to an evolutionary lineage. Thus, based on this and our findings, we suggest that these traits can be useful for species-level differentiation; however, they may have evolved independently and might not carry a true phylogenetic signal.
In contrast, the genetic divergence between the new species and those from the Oriental region is significant, showing 55% of nucleotide divergence between K. atroxi n. sp. and K. indicus and 54% between K. atroxi n. sp. and K. brachycephalus. These differences indicate a significant separation between Kalicephalus species that parasitise snakes in the Neotropical and Oriental regions. However, because of low nodal support, the addition of new sequences to the phylogeny of Kalicephalus spp. may alter the phylogenetic relationships found here for these taxa.
Our phylogenetic analyses and the high nucleotide divergence between K. costatus and K. indicus (52% of divergence) indicate that those should be considered as independent and valid species, rather than subspecies as proposed by Schad (Reference Schad1962). We also should highlight that the division of Kalicephalus into subgenera is an artificial classification, since we clearly observed the paraphyly of the group (separation of clades B2 and B3). Additionally, this reinforces the fact that the morphology of the female reproductive system does not reflect the evolutionary relationships among this species.
The phylogenetic reconstruction from this study shows groupings similar to those found by Liu et al. (Reference Liu, Wang, Liu, Tan, Liu and Liu2019). Notably, we found the formation of a sister group within the clade that encompasses Diaphanocephalidae and Ancylostomatidae. However, it should be noted that the authors did not use sequences from D. galeatus. Overall, this study supports the close relationship between the Diaphanocephalus and Kalicephalus lineages, which form a monophyletic group with high nodal support, thereby reinforcing the monophyly of the Diaphanocephalidae family.
The clades grouping Diaphanocephalidae and Ancylostomatidae are well-supported in ML analysis (87% of bootstrap). According to molecular phylogenies of Chilton et al. (Reference Chilton, Huby-Chilton, Gasser and Beveridge2006), Diaphanocephaloidea and Ancylostomatoidea are more closely related to each other groups within Strongylida.
Finally, Kalicephalus atroxi n. sp. is the first species of Kalicephalus from the Neotropical region to be characterised using molecular data, and the second species of Kalicephalus found parasitising the snake Bothrops atrox. Thus, our study contributes to the knowledge of the parasitic fauna of this important snake species from the Amazon region and adds data on these parasites’ geographical distribution.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25100436.
Acknowledgements
We are grateful to Ph.D. Edilene Oliveira da Silva and Ph.D. Yuri Willkens from the Federal University of Pará, Belém, Brazil, for their assistance with the SEM analysis; the students from the Laboratory of Cellular Biology and Helminthology “Prof. Dra. Reinalda Marisa Lanfredi” (Federal University of Pará, Belém, Brazil); the students from the Herpetology Laboratory of the Federal University of Amapá (Federal University of Amapá, Macapá, Brazil); the professionals from the Chico Mendes Institute for Biodiversity Conservation for granting the necessary collection permit; PROPESP/UFPA; and Professor Dr. Edson A. Adriano and Rayline Figueiredo from the Laboratory of Ecology and Evolution at the Federal University of São Paulo (UNIFESP) for their support in conducting the molecular analyses.
Author contribution
F. Haick wrote the main manuscript, J.K.S. Neves and A.J.S. Félix prepared the figures. L.F.S. Tavares-Costa performed the phylogenetic analyses. M.I. Müller carried out the PCR and sequencing. C.E. Costa-Campos collected the host specimens. G.F. Maschio Santos and F.T.V. Melo contributed to the writing of the manuscript, reviewed the content, and prepared the line drawings. All authors reviewed the final manuscript.
Financial support
This work was supported by Coordination for the Improvement of High Higher Education Personnel, Brazil (CAPES); Postgraduate Program in Zoology (PPGZOOL); University Federal of Pará (UFPA); PROPESP/UFPA; Amazon Foundation for Research and Studies Support (FAPESPA)/CNPq–PRONEM (01/2021); The National Council for Scientific and Technological Development (CNPq) Productivity Scholarship Grant (CNPq) F.T.V. Melo. CNPq (process: 314116/2021-4); Carlos Eduardo Costa-Campos (process: 307697/2022-3). M.I. Müller was supported by a postdoctoral fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (fellowship #2017/16546-3).
Competing interests
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.
Ethical standard
All applicable institutional, national, and international guidelines for the care and use of animals were followed. Host specimens were collected under permits Institute for the Environment and Renewable Resources – IBAMA/ICMBio (SISBIO: Nº 48102-2) and Ethics Committee on the Use of Animals of the Federal University of Pará (CEUA/UFPA: Nº 8341260821).
