A New Species of the Acanthocephalan Genus Filisoma (Cavisomidae) from Perciform Fishes in Rio de Janeiro, Brasil

Background Twelve species of Filisoma Van Cleave, 1928 are recognized parasitizing tropical and subtropical fish. Four of these species were described from kyphosid fish and it has been suggested that a co-speciation may have occurred among species of Kyphosus Lacepède, 1801 and Filisoma, which could provide valuable information about the evolution history of this host–parasite system. Purpose During a survey of the helminth fauna of Kyphosus sectatrix (Linnaeus, 1758) and Kyphosus incisor (Cuvier, 1831) (Kyphosidae Jordan, 1887) off Rio de Janeiro coast, a new species of Filisoma was found and is described herein based on morphological, genetic, and ultrastructural data. Methods Fish were obtained off Rio de Janeiro coast, Brazil. The parasites found in the intestine were measured and drawings were made with a drawing tube. Type specimens were deposited at the Helminthological Collection of Oswaldo Cruz Institute (CHIOC). The ultrastructure was studied using scanning electron microscope. The genetic analysis included the study of the partial sequences of 18S, ITS1, 5.8S and 28S rDNA, and the mitochondrial cytochrome c oxidase 1 gene (cox 1), with phylogenetic reconstructions based on the maximum likelihood analysis. Results Filisoma caudata n. sp. is characterized by a proboscis with 16‒18 longitudinal rows of 38‒45 hooks each. Hooks are uniform in shape dorsoventrally, gradually decreasing in size towards the base of the proboscis. Anterior hooks are 30‒45 μ long, middle hooks 30‒35 μ long and 5 basal transversal hooks 20‒30 μ long. The new species is differentiated from the closest species Filisoma filiformis Weaver and Smales, 2013 by the size and distribution of hooks, apart from having a subterminal vulva and a curved posterior trunk end (tail) measuring 500‒1,000 long. Phylogenetic analysis based on 18S, 28S rDNA and mtDNA-cox1 markers grouped the new species with Filisoma bucerium Van Cleave, 1940 and Filisoma rizalinum Tubangui and Masiluñgan, 1946 showing a close relationship between these species of Cavisomidae Meyer, 1932 and Echinorhynchidae Cobbold, 1879; the latter represented by species of Acanthocephalus Koelreuther, 1771. The new species can be differentiated from others on morphological and molecular basis. A key to the 13 species of Filisoma Van Cleave, 1928 is provided. Conclusion Filisoma caudata n. sp. is described herein based on morphological, genetic, and ultrastructural data. The topologies of obtained phylogenies suggest that species of Echinorhynchidae should be reevaluated since the family is considered paraphyletic in all analyses conducted.

Four of these species were described from kyphosid fish and it has been suggested that a co-speciation may have occurred among species of Kyphosus Lacepède, 1801 and Filisoma which could provide valuable information about the evolution history of this host-parasite system [1,6,7]. Although studies that use the integrative taxonomy (using different tools that includes morphological, ultrastructural, biochemical, molecular, and behavioral studies to delimit and characterize species) have been increasing in the last few years [8], the number of studies using this approach is still scarce [9,10]).
During a survey of the helminth fauna of Kyphosus sectatrix (Linnaeus, 1758) and Kyphosus incisor (Cuvier, 1831) off Rio de Janeiro coast, a new species of Filisoma was found and is described herein based on morphological, genetic, and ultrastructural data.

Ethical Statement
Collections in this study were authorized by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA, license no. 15898-1).

Fish Collection
A total of 22 specimens of K. incisor were examined from October 2013 to March 2015; nine were acquired from fishermen of Copacabana beach (22°59′08″S, 43°11′18″W) and 13 were obtained from local fish markets. The mean total length of fish was 46 ± 5 (35-53) cm and mean weight was 1672 ± 549 (730-2370) g. Acanthocephalans from a single specimen of K. sectatrix measuring 30 cm long and weighing 525 g, collected in 2005 at Ilha Grande Bay (23°04′04.62″S, 44º13′31.95″W) were also examined for comparison. Fish were identified according to Froese and Pauly [11].

Light Microscopy
The parasites found in the intestine were washed in physiological saline (0.7%) and fixed in AFA, 4% formalin or 70% alcohol. Some acanthocephalans were stained with alcohol chloride carmine, cleared in clove oil and mounted in Canada balsam. Observations were based on the specimens collected from two hosts: K. incisor and K. sectatrix and measurements are given in micrometres, with range in parentheses, unless otherwise stated; holotype measurements are in brackets. Drawings were made with a drawing tube and redrawn using Adobe Illustrator CS6 [12]. The prevalence, intensity, mean intensity and mean abundance follow Bush et al. [13]. Specimens were deposited at the Helminthological Collection of Oswaldo Cruz Institute (CHIOC).

Scanning Electron Microscopy (SEM)
Specimens fixed in AFA or 4% formalin were washed in 0.1 M Na-cacodylate buffer, post-fixed for 40 min in a solution of 1% osmium tetroxide in 0.8% potassium ferrocyanide in 0.1 M Na-cacodylate buffer, dehydrated in an ascending alcohol series, dried by the critical point method with CO 2 , and sputter-coated with gold 60 nm. Samples were examined using a JEOL JSM 6390 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) at the Electron Microscopy Platform, Fundação Oswaldo Cruz.

Genetic Analysis
DNA was extracted using the phenol-chloroform method as described by Billings et al. [14] and a set of primers were used to amplify different regions of the DNA. The partial 28S rDNA gene was amplified by PCR using the primers C1 (5′-ACC CGC TGA ATT TAA GCA T-3′) and D2 (5′-TGG TCC GTG TTT CAA GAC-3′) (Hassouna et al. [15], after Chisholm et al. [16]). For partial 18S, ITS1 and 5.8S, the primers S1 (5′-TTC CGA TAA CGA ACG AGA CT-3′) and H7 (5′-GCT GCG TTC TTC ATC GAT ACT CG-3′) [17] were used. For partial fragment of the mitochondrial cytochrome c oxidase 1 gene (cox 1) primers LCO (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) [18] were used. PCR was carried out using cycling parameters as previously described by those authors. The PCR products were analyzed by electrophoresis in 1.5% agarose gels, stained with SyberGreen (Invitrogen, Eugene, Oregon, USA) and photographed under UV transillumination. Amplified PCR products were purified with ExoSap-IT PCR Product Cleanup (USB ® Products Affymetrix Inc., Cleveland, Ohio, USA). DNA cycle sequencing reactions were performed using BigDye Terminator v.3.1 (Applied Biosystems, Foster City, CA, USA) and automated sequencing was done using the Sequencing Platform at the Fundacão Oswaldo Cruz-PDTIS/FIOCRUZ in Brasil. Sequences of both strands were edited and aligned using the MEGA version 7.0 software [19]. Sequences were compared to others available in the GenBank database using the BLASTN program from the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST ) [20]. The nucleotide sequences were aligned using the CLUSTAL W algorithm [21] of the MEGA 7.0 package. Maximum likelihood (ML) phylogenetic trees were inferred using the best-fit model of MEGA 7.0: the Kimura two parameters  Table 1.

Acanthocephala
Palaeacanthocephala Etymology: The new species is named as an adjective referring to the specific name of the host.
Nine of 22 examined specimens of K. incisor (41%) were infected with 212 acanthocephalans in Copacabana Beach, Rio de Janeiro. Intensity was 4-87 with a mean intensity of 25 ± 28 and mean abundance: 10 ± 21. One specimen of K. sectatrix was also found heavily infected. The acanthocephalans were identified in the genus Filisoma Van Cleave, 1928 because of their long and slender unarmed trunk and proboscis with many hooks decreasing in size anteriorly and posteriorly, long lemnisci, double-walled proboscis receptacle with cephalic ganglion at its base, and four long tubular cement glands. A comparison with known species of Filisoma determined that it is new. This makes F. caudata n. sp. the 13th valid species of the genus and the first to be described in South America. Species of Filisoma appears to be present in fishes inhabiting the tropical and semitropical waters of the Indo-pacific region. The hosts, K. incisor and K. sectatrix, are nektonic, forming schools in shallow waters associated with coral reefs, sand or rocky bottom. In Western Atlantic K. incisor occurs from the United States to Argentina while K. sectatrix occurs from Canada to Santa Catarina (Brasil). These species may also occur in Eastern Atlantic coasts of Spain and Africa. They are diurnal, feeding mainly on plankton, benthonic algae, detritus, small mollusks and crustaceans (Froese and Pauly [11]).

Phylogenetic Analysis
The phylogenetic reconstruction based on the partial sequence spanning the 18S rDNA shows that our consensus sequence of F. caudata n. sp. is grouped with F. bucerium (AF064814) forming with F. rizalinum (JX14229) a clade of the family Cavisomidae. The sister clade with 54% support is formed by species of Acanthocephalus. A major clade with 99% of support encloses the two former clades and Pseudacanthocephalus Petrochenko, 1958 species (Fig. 3). The paraphyletic Echinorhynchidae Cobbold, 1879 family is separated between the two major clades, with species of Echinorhynchus Zoega in Müller, 1776 clustering with species of Rhadinorhynchidae Travassos, 1923 and Pomphorhynchidae Yamaguti, 1939 with 79% support, while the other genera grouped with Cavisomidae (Fig. 3).
On the partial 28S rDNA ML tree, our sequences of F. caudata n. sp. are grouped with F. bucerium (AY829110) with a statistical support of 85% in a clade of the family Cavisomidae, separated from two Echinorhynchidae clades, one formed by the species of Acanthocephalus and Pseudoacanthocephalus and the other with species of Echinorhynchus (Fig. 4).
On the ML reconstruction for the partial region of the mtDNA-cox1, all sequences of F. caudata clustered together with 100% of bootstrap support. The species of the genus Acanthocephalus appear as a sister group of our F. caudata. F. bucerium (DQ089722) is placed outside this cluster and shares a common ancestor with the cluster F. caudata + Acanthocephalus spp. Genus Echinorhynchus and Rhadinorhynchus Lühe, 1911 are placed in another cluster as sister groups with 80% of bootstrap support (Fig. 5).      ). The absence of a long female tail can also be an inferred difference, considering that this character was not mentioned in the original description. The above key further differentiates our new species from these four species and others of the genus from other host species. The phylogenetic analysis of the 28S rDNA sequences (Fig. 4) grouped F. caudata n. sp. with F. bucerium (AY829110) forming a clade of the family Cavisomidae between two clades in the family Echinorhynchidae confirming that different genera of Echinorhynchidae are paraphyletic [23][24][25]. Our phylogenetic tree for 28S rDNA is similar to those of Braincovich et al. [26] and Gárcia-Varela and Nadler [23], where the family Cavisomidae grouped close to the Echinorhynchidae genus Acanthocephalus. Although García-Varela and Nadler [23] commented that the 28S rDNA is not the most appropriate for taxonomic studies at the generic level, the 18S rDNA sequences appear to be more suitable to infer phylogenies among Acanthocephalans. The clade with the Cavisomidae had a good statistical support for the genus Filisoma. The phylogenetic analysis of the 18S rDNA sequences (Fig. 3) confirmed a clade of Cavisomidae with F. caudata n. sp. and F. bucerium (AF064814) that are well separated from Echinorhynchidae, Arhythmacanthidae (see Braincovich [26]), Rhadinorhynchidae, and Pomphorhynchidae.
Our phylogenetic analysis of the mtDNA cox-1 gene (Fig. 5) also grouped Filisoma spp. as sister groups of Acanthocephalus spp. However, more sequences of other species of Cavisomidae are necessary to better understand the relationship between species of these two genera, since Filisoma is the only genus with sequences available in the GenBank. Benesh et al. [27] discussed the reliability of the use of mitochondrial DNA amplified with universal primers for taxonomy since amplification could result in fragments of nuclear pseudogenes that have sequences similar to mitochondrial genes. In this work, the results of the phylogeny did not differ much in topology from the analysis made with nuclear genes indicating that the mitochondrial sequences used are reliable to infer phylogenies. The greater impediment to robust analysis was the reduced number of sequences deposited for the family Cavisomidae in the GenBank.
Amin [3] discussed the need of reevaluation of the families of Palaeacantocephala considering that the classification of families based only on morphology, e.g., the number of cement glands, can be doubtful. After Braincovich et al. [26] families with these characteristics may not be related as Cavisomidae and Rhadinorhynchidae, both usually with four cement glands that are grouped into different clades. Gymnorhadinorhynchidae Braicovich et al., 2014, for example, with four cement glands, group with Transvenidae Pichelin & Cribb, 2001, which has only two cement glands [26].
The molecular data analyzed also suggest that families of Palaeacanthocephala must be reevaluated, since the delineation of monophyletic families was not clear in any of the topologies obtained, especially for species of Echinorhynchidae. The lack of sequences of different genera of Acanthocephala demonstrates that the use of molecular tools in defining species of Acanthocephala is still scarce and a large number of studies still describe and redescribe species based only on morphology [2,4,[28][29][30][31][32]. For Cavisomidae, for example, there are genetic sequences of only three species available in the GenBank, all of them from the genus Filisoma (F. bucerium, F. rizalinum and now F. caudata n. sp.). Therefore, new integrative studies with morphological, molecular and geographical distribution data help to determine species with reliability and are necessary to better understand the classification of acanthocephalans [23,25]. Keys to species of Filisoma were previously provided by Van Cleave and Manter [1], Amin and Nahhas [5] and Weaver and Smales [2]. An updated key for species of Filisoma including F. caudata n. sp. is now provided. and ultrastructural analysis. JNB performed molecular analysis. OMA developed and revised the manuscript. All authors wrote the paper and approved the final version of the manuscript. This study is part of the Master dissertation of VCF at the Postgraduate course in Biodiversity and Health at Institute Oswaldo Cruz, Fiocruz, Brasil.
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