Systematic assessment of nymphs of Diesingiinae (Pentastomida: Sebekidae) parasitizing Palloceros harpagos (Cyprinodontiformes: Poeciliidae) from Parque Nacional Iguazú, Argentina: filling gaps in an incomplete phylogenetic framework

Abstract

During a search for parasites in fishes from Iguazu River, Argentina, a nymph of pentastomid was found encysted on mesenteries of Phalloceros harpagos. The 28S rDNA and COI mt-DNA were used to compare with the sequences deposited in the GenBank. Pentastomid nymphs belong to the subfamily Diesingiinae (Sebekidae) for having chloride cell pores distributed in a single row per annulus; also, the hooks and rows of spines of our material differ to other genera. Present specimens are most likely Diesingia sp., having 63-74 annuli, a U shaped oral cadre with fibers closing anteriorly and a peg like extension of the oral cadre. The 28S rDNA analysis places our samples into a sister group of Alofia sp., but the COI mt-DNA situate them on the base of the clade. In conclusion, our pentastomid positively belongs to Diesingia sp., however, indisputable assignation requires a reliable description of the nymph, or the availability of sequences linking nymphs and adults, which even could provide evidence of a new hitherto undescribed genus. Aditionally, the systematic position of Sebekia oxycephala previously described by P. harpagos is discussed on the basis of our results, allowing us to suggest a re-assignation of such specimens to the genus Diesingia.

Key words
Iguazu; COI mtDNA analysis; Misiones; Pentastomid; 28S rDNA analysis

INTRODUCTION

As part of a series of ongoing studies on parasites of freshwater fishes from Iguazu River, a pentastomid in the juvenile stage (nymph) was found encysted in the mesentery of Phalloceros harpagos (Cyprinodontiformes: Poeciliidae). This host is a small fish that prefers shallow waters with a muddy and sandy bottom, near the margin of the stream, where the water flow is low (Mazzoni et al. 2011). This fish species has a great variation in diet, consisting of detritus and aquatic insects (Ephemeroptera, Chironomidae, and Simuliidae), being considered as detritivore/insectivore (Uieda & Pinto 2011, Almeida Monaco et al. 2014, Pereira Neves et al. 2015, Carvalho Leitão et al. 2018).

Freshwater fishes have been reported as intermediate hosts for pentastomids of the families Sebekidae and Subtriquetridae. The adult species of Sebekidae are mainly parasites of crocodilians, chelonians, and monitor lizards (Christoffersen & De Assis 2013). In a monograph of Pentastomida, Christoffersen & De Assis (2013) evaluated the classification of this subclass, retaining into Sebekidae the subfamilies Samboninae (Sambonia sp.), Diesingiinae (Diesingia sp., Alofia sp., Selfia sp.), and Sebekinae (Agema sp., Pelonia sp., Sebekia sp.), and erecting the new subfamily Leiperiinae to include three species of the genus Leiperia. The current arrangement in the classification of this group includes an additional genus, Levisunguis, which was described by Curran et al. (2014) and assigned to this latter subfamily.

There are few records of pentastomids in South America. According to Luque et al. (2013), there are reports for Leiperia sp. parasitizing freshwater fishes of the families Characidae, Erythrinidae, Pimelodidae, and Sebekia sp. found in fish hosts of the families Apteronotidae, Arapaimidae, Ariidae, Characidae, Cichlidae, Cynodontidae Electrophoridae, Osteoglossidae, Pimelodidae, Pistigasteridae, Poeciliidae, and Synbranchidae. Among the poeciliid fishes, the species P. harpagos, examined from the Cambé River, Brazil, was recorded as a host of Sebekia oxycephala (Almeida et al. 2009). Additional records for pentastomids have been done from some Argentinian vertebrates such as snakes (Cavalieri 1967, 1970, Troiano & Repetto 1994, Martinez et al. 1999, 2000), cayman (Fernandez et al. 2016), and armadillo (Martinez 1982, Martinez & Resoagli 1982). However, there are no records of other aquatic vertebrates such as Chelonians or fishes. This work aims to provide the record of Sebekidae nymphs found in the poeciliid fish P. harpagos, collected from the National Park Iguazu, Argentina, to describe their morphological features, and to apply molecular phylogenetic tools in order to explore the location of these specimens in the phylogenetic context of Pentastomida.

MATERIALS AND METHODS

Sampling and morphological study

A total of 30 Phalloceros harpagos were caught with hand nets in the Yacaratia stream, which belongs to the flood plain of Iguazu River up to the waterfalls (25°40´34¨ S 54° 27´ 08¨W) inside the Iguazu National Park, Argentina. The fishes were carried to the field camp in the Centro de Investigaciones Ecológicas Subtropicales (CIES), euthanized and examined for parasites under a magnifier glass. Some of the pentastomids were fixed in 10% formalin for morphological procedures, or in 96% ethanol for DNA extraction and sequencing. Samples were transported to the Laboratory of Fishes, Mollusks and Crustaceans Parasites (in the CEPAVE), where some individuals were cleared using lactophenol, and then observed under the microscope. The specimens were photographed with an AmScope MU 1000 10 MP digital camera (Irvine, Wisconsin) attached to an Olympus Bx51 microscope (Tokyo, Japan). The structures captured on microphotographs were measured using the ImageJ software. All measurements are given in micrometers unless otherwise stated, and expressed as the minimum and maximum values, followed by the mean in parentheses. The hooks measurements were taken according to Barton & Morgan (2016). The drawings were made with the aid of a camera lucida attached to an optical interference microscope Olympus BX53 (Tokyo, Japan). Additional specimens preserved in formalin were used for the observation under scanning electron microscopy (SEM).

DNA extraction, PCR amplification and sequencing

Some of the individuals conserved in 96% ethanol were used for the DNA analysis. The extraction was performed using a Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer’s protocol. The 28S rDNA and COI gene fragments were amplified by Polymerase-Chain Reaction (PCR) on an Eppendorf Mastercycler thermal cycler, using the forward primers LSU-5 (5’ -TAG GTC GAC CCG CTG AAY TTA AGC A-3’) and 1500R (5’ -GCT ATC CTG AGG GAA ACT TCG-3’) for the 28S rDNA (Tkatch et al. 2003), and the LCO1490 forward primer (5’- GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and the HCO2198 reverse primer (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) for the COI gen (Folmer et al. 1994). The reactions were prepared using Green GoTaq 5X Buffer (Promega), 2.5 mM MgCl₂ (Promega), 0.2 mM of NEB Nucleotide Mix, and Flexi GoTaq polymerase enzyme (Promega).

The PCR reactions were performed according to the protocols described by Tkach et al. (2003) for the 28s rDNA and by Folmer et al. (1994) for the COI mt-DNA gene. The PCR products were analyzed by electrophoresis in 1% agarose gel using TAE 1X buffer and observed under ultraviolet light. PCR products were purified and sequenced using an ABI 3730XLs sequencer, Macrogen Inc. (Korea).

Molecular data and phylogenetic reconstruction

The sequences obtained were edited using the platform Geneious Pro v.10 (Drummond et al. 2016) and used in the Genbank blast-n tool, searching for homologue sequences for comparison (Table I). For each gene, the sequences were separately aligned using the online version of MAFFT v.7 (Katoh & Standley 2016). The 28S rDNA dataset was analyzed on the Gblocks Website (Castresana 2000, Talavera & Castresana 2007) to detect ambiguously aligned hypervariable regions, and were excluded from the analysis using a less stringent selection (allowing smaller final blocks, gap position within the final blocks, and less strict flanking positions).

Table I
Sequences utilized in the molecular analysis based on the 28S rDNA and COI mtDNA genes.

Family	Specie	host	Country	Sequence		Author
				COI	28s RDNA
Linguatulidae	Linguatula artica	Rangifer tarandus	Norway	KF029443		a
Linguatulidae	Linguatula serrata	Bos indicus	Bangladesh	LC150783 LC150784		b
Porocephalidae	Armillifer agkistrodontis		China	FJ607340		c
Porocephalidae	Kiricephalus coarctatus	Python bivittatus	USA	MG559656 MG559657 MG559658		d
Porocephalidae	Porocephalus cotali	Python bivittatus	USA Germany	MG559647 MG559652 MG559655	EF417058	d,e
Porocephalidae	Waddycephalus sp.	Stegonotus cucullatus	Australia	KF885748 KF885768 KF885769 KF885780		f
Raillietiidae	Raillietiella sp.				AY744894	g
Sebekidae	Alofia Merki		Australia	KU975383 KU975384	KU975382	h
Sebekidae	Alofia sp.	Pseudoplatystoma fasciatum	Peru	MH484065		i
Sebekidae	“ Diesingia sp.”	Phalloceros harpagos	Iguazú, Argentina	MN518003	MN521701 MN521702	This manuscript
Sebekidae	Sebekia mississippiensis	Lepisosteus oculatus		MK248486 MK248488 MK248489		j
Sebekidae	Sebekia purdieae		Australia	KU975386	KU975381	h
Sebekidae	Sebekia sp. 2		Australia	KU975387	KU975379 KU975380	h

• Authors: a= Gjerde (2013), b= Itagaki & Mohanta (unpublished) c= Chen et al. (unpublished) d= Miller et al. (2017), e= Sonnenberg et al. (2007), f= Kelehear et al. (unpublished), g= Giribet et al. (2005), h= Barton & Morgan (2016), i= Gomez-Puerta et al. (unpublished), j= Woodyard et al. (2019).

The best partitioning scheme and substitution model for the 28S rDNA and COI mt-DNA were chosen under the Bayesian Information Criterion (BIC; Schwarz 1978) using the ‘greedy’ search strategy in Partition Finder v.1.1.1 (Lanfear et al. 2012). The appropriate nucleotide substitution model implemented for the 28S rDNA matrix resulting after the Gblock program was K80, and for COI mt-DNA was TrN+G, for the first, F81+G for the second, and HKY+G for the third position.

Additionally, the proportion (p) of absolute nucleotide sites (p-distance) (Nei & Kumar 2000) was obtained to compare the genetic distance between selected copepod species. The p-value matrix was obtained using MEGA X (Kumar et al. 2018), with variance estimated with the bootstrap method (1000 replicates) and with a nucleotide substitution (transition + transversions) uniform rate.

Phylogenetic reconstruction was carried out using Bayesian Inference (BI) through MrBayes v.3.2.3 (Ronquist et al. 2012). Phylogenetic trees were constructed using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 20 million generations each, to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations and the average standard deviation of split frequencies was observed to be less than 0.01, as suggested by Ronquist et al. (2012).

The robustness of the clades was assessed using Bayesian Posterior Probability (PP), where PP > 95 (in percentage) was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for the two runs after discarding the first 25% of the sampled trees.

RESULTS

Based on the morphological features and the molecular analysis, the examined specimens were tentatively assigned to the genus Diesingia Sambon, 1922.

Description of the specimens

Infective nymphs (measurements based on 12 specimens) were found attached to the body wall cavity of the hosts, encapsulated in a thin layer. Fresh specimens were white in color and had intestines with a red content. The body was 3,576-4,387 (3,982 mm) long and 660-956 (808) wide. Annuli margins were easily discernible in whole mounts (Fig 1a), with a total count of 63-74 (67). The annuli lacked interrupted rows of spines, which were similar in length and distributed all over the body. Between each annulus, the chloride cell pore was distributed in a single row (Fig 1b).

Figure 1
Diesingia sp from Phalloceros harpagos. a.- entire nymph. b.- Chloride cell pore distributed in a single row. c.- Detail of the anterior end with the four hooks and the oral cadre.

Hooks were arranged around mouth opening (Fig. 1c). The U-shaped buccal open anteriorly cadre presented almost parallel sides of thick cadre (Fig.2a-c). The posterior edge of the oral cadre was rounded and thick, situated between the fulcra of the anterior hook pair. The anterior edge or oral cadre was approximately at the level of the posterior edge of the anterior hooks. The buccal cadre was 282-319 (300) long and 151-163 (157) wide. The posterior lateral extensions of the cadre were 102-122 (112).

Figure 2
a.- Drawing of the anterior end of Diesingia sp. showing the position of the oral cadre between the hooks. b.- Photograph of the oral cadre. c.- Drawing of the oral cadre.

Anterior and posterior hooks were sharp tipped, all with dorsal accessory pieces (Fig. 1c, 2a, 3a-e). Hooks and dorsal accessory pieces were long, stout, slightly curved fulcra, visible under the dissection microscope. Anterior hooks (Fig 3b, 3d): dorsal accessory piece (DAP) 137-195 (166), blade length (AC) 136-149 (143), hook length (AD) 144-172 (158), base length (BC) 79-93 (86), plateau length (CD) 36-67 (51), hook gape (AB) 55-61 (58), fulcrum length (FL) 216-314 (265). Posterior hooks (Fig 3c, 3e): DAP 143-166 (155), AC 136-149 (143), AD 136-149 (143), BC 90-98 (94), CD 41-52 (46), AB 53-61 (57), FL 271-296 (284). Total hooks: DAP 145-161 (153), AC 138-146 (142), AD 138-146 (142), BC 89-95 (92; 1.5), CD 41-49 (45), AB 55-61 (58), FL 278-297 (287).

Figure 3
Hooks of Diesingia sp. a.- Microphotograph of the anterior end showing the nymphal doble hooks. b.- Photograph of the anterior hook. c.-Photograph of the posterior hook. d.- Drawing of the anterior hook. e.- Drawing of the posterior hook.

Prevalence: 50 %

Intensity: 2,07.

Mean abundance: 0,99.

Genetic results

One sequence of COI mt-DNA with 582bp was obtained and compared with those downloaded from the GenBank (Table I). Almost all the sequences downloaded from GenBank are well supported by the literature, but some of them as Linguatula serrata, Armilifer agkistrodontis, Waddycephalus sp. and Alofia sp. are unpublished. Most of these sequences were reported from Europe, North America and Oceania, while only one was reported from Peru. The Sebekidae family is represented by two genera (Alofia and Sebekia) in the sequences deposited in GenBank. A matrix with 723 bp was performed and used to construct a phyllogram, using the species Raillietiella hebitihamata as outgroup, (Fig 4). The resultant tree showed well resolution. The analyzed sequence constitutes a well-supported clade, having the clade containing species attributed to the families Sebekidae and Porocephalidae as sister group. The clade formed by species belonging to the genus Kiricepahlus, Porocephalus and Amilifer (all of them assigned to Porocephalidae) was well supported (97% PP), but was related (86% PP) to the genus Alofia sp. (assigned to Sebekidae). These relationships, along with those showed in the sister clade, where taxa of the genus Waddycephalus (Porocephalidae) arising as sister group of the specimens of Sebekia (Sebekidae) (PP value = 100%; being the PP between them also higher than 99%), strongly suggest that these families have a paraphyletic arrangement. The p-distance showed values over 20% to separate different genus, and less than 20% to separate between species. The distance between our specimen and the other genera varied from 21 to 25 % (Table II).

Figure 4
Phylogram resulting from Bayesian Inference (20,000,000 generations) of partial COI mtDNA gene sequences of “Diesingia sp.” Branch support values indicate posterior probabilities.

Table II
Genetic distances matrix (uncorrected p-distance) for pentastomids. The percentage values are derived from the COI mt-DNA.

	1	2	3	4	5	6	7	8	9	10	11	12
1.- Raillietiella habitihamat JF975594 (Outgroup)
2.- Kiricephalus coarctatus MG559656-58	29
3.- Porocephalus crotali MG559647,52,55	31	20
4.- Amilifer agkistrodontis FJ607340	30	21	20
5.- Alofia sp. MH484065	29	23	23	24
6.- Alofia merki KU975383-84	32	25	23	24	18
7.- Waddycephalus sp. KF885748, 68-70	31	21	23	25	25	26
8.- Sebekia sp. 2 KU975387	32	24	23	24	26	25	23
9.- Sebekia purdiae KU975386	31	23	24	23	24	26	22	15
10.- Sebekia mississippiensis MK248486, 88-89	32	23	23	24	24	25	23	18	16
11.- Diesingia sp. MN518003	32	21	22	25	24	26	23	22	23	21
12.- Linguatula ártica KF029443	28	29	28	28	31	31	29	32	32	31	30
13.- Linguatula serrata LC150783-84	28	28	30	27	30	30	29	33	32	30	30	10

Two sequences of 28s rDNA with 1167 and 1280 bp, respectively, were obtained and used to download the homologue sequences deposited in the GenBank (Table I). Contrary to the sequences of COI downloaded from GenBank, all of the sequences are well supported by the literature. These sequences are reported from Europe, North America and Oceania..The matrix constructed after excluding the hypervariable regions had 1090 bp, with Raillietiella sp. used as outgroup. The tree obtained (Fig 5) showed that the sequences obtained were related to those assigned to Alofia merki. However, this relationship showed a low support (55% PP). This tree was also related to those sequences assigned to Sebekia with the available sequence of Porocephalus sp. (65% PP). The p-distance between our specimens and the closest genus, Alofia, was 12%, while between the genus Sebekia and Porocephalus varied between 18% and 19% (Table III).

Figure 5
Phylogram resulting from Bayesian Inference (20,000,000 generations) of partial 28 rDNA gene sequences of “Diesingia sp.” Branch support values indicate posterior probabilities.

Table III
Genetic distances matrix (uncorrected p-distance) for pentastomids. The percentage values are derived from the 28S rDNA.

	1	2	3	4	5	6	7
1.- Raillietiella sp. (outgroup)
2.- Alofia merki KU975382	30
3.- Diesingia sp.MN521701-02	30	12
4.- Sebekia sp. KU975379	32	18	19
5.- Sebekia purdieae KU975381	32	17	18	5
6.- Sebekia sp. KU975380	32	18	19	5	4
7.- Porocephalus sp. EF417058	31	17	18	19	19	18

DISCUSSION

Pentastomida has been recently confirmed by genetic analysis as a subclass inside the Crustacea with a much modified parasitic life (Regier et al. 2010, Poore 2012). However, the phylogenetic relationships inside this group are still uncertain. Therefore, the current taxonomical arrangement among families, and genera inside them, arises as a promissory research topic for future years. Since the molecular approach is still far to provide an accurate taxonomical resolution, more species and genera must be sequenced in order to clarify the phylogeny of this fascinating group.

A factor increasing the issues to understand the relationships among Pentastomida are the poor knowledge of the real diversity of this group. In this sense, the nymphs of some genera, as well as their life cycles, and potential hosts remain unknown. Information about pentastomids in Argentina is limited to some eventual records in Ophidia and Dasypodidae (Cavalieri 1967, 1970, Martinez 1982, Martinez & Resoagli 1982, Troiano & Repetto 1994, Martinez et al. 1999, 2000, Fernandez et al. 2016), and nothing is known concerning nymphs or adults of other groups of aquatic vertebrate hosts, like turtles or fishes. In other South American countries such as Brazil and Peru, some works about the diversity of pentastomids have been published (see Garate et al. 2007, Luque et al. 2013), providing some light about this diverse parasitic group. However, the use of molecular tools in these works is very limited.

Most of the published studies in South America have been made for adult pentastomids, being relatively few those records of nymphs infecting different species and organs of fishes (Almeida et al. 2009, Giesen et al. 2013, Luque et al. 2013). In Australia, Barton & Morgan (2016) recently started the study of this group using DNA tools, bringing some new sequences and morphological information about nymphs, which was used here to elucidate the affiliation of the nymphs found inside the poeciliid host Phalloceros harpagos.

The morphology of the nymphs found here agrees with the description of Sebekidae family (Pentastomidae), having a vermiform body, anterior and posterior hooks with two blades (Luque et al. 2013), sinuous alimentary canal longer than the body, and distinctly convex hooks (Christoffersen & De Assis 2013). Inside this family, the subfamily Diesingiinae is composed of Diesignia (2 species), Alofia (3 species), and Selfia (1 species), which are characterized by having a chloride cell pore distributed in a single row per annulus, (Christoffersen & De Assis 2013). This feature agrees with the material examined here, which allows us to distinguish between Sebekinae and Diesingiinae. The hooks of the nymphs of Alofia and Selfia have rows of reduced spines (Christoffersen & De Assis 2013), which were not present in our specimens. However, nymphs of the remaining genus Diesingia had not been described or reported until now. Diesingia was described from material collected from turtles in Brazil (Heymons 1941, Da Fonseca & Ruiz 1956, Self & Rego 1985, Junker et al. 2003), and because the concordant morphological features between this genus and our material, this was tentatively assigned to Diesignia. Further collections of specimens linking the nymphal stage with adults will allow to corroborate this assignation or to suggest the presence of a new genus. In order to evaluate some morphological traits between previously described adults and our material, the species Diesingia megastoma have between 65-70 annuli (Heymons 1941), and the oral cadre is almost twice larger than wide (Heymons 1941, Da Fonseca & Ruiz 1956, Self & Rego 1985, Junker et al. 2003). In our specimens, the oral cadre was U-shaped, with fibers anteriorly closing, as was mentioned by Self & Rego (1985). Junker et al. (2003) confirmed the U-shape. However, they suggest that the presence of the fibers could be caused by pressure during the sample mounting process. About that, we observed that the specimens mounted under different pressure conditions always showed those fibers. Another feature supporting the nearness of our material to Diesingia is the clear peg-like extension of the oral cadre, present in the analyzed nymphs, and also mentioned by Junker et al. (2003) in the re-description of D. megastoma. According to Junker et al. (2003), the oral cadre of D. megastoma exceeds the size of the hooks. Even though this corresponds to the description of adults, Giesen et al. (2013) considered that this feature could be reflected in the larvae. This is not the case of our specimens, where the relation is quite similar between oral cadre and hooks. However, it is possible that in the last moult previous to the adult stage, the oral cadre grew bigger than the hooks. Even if it was not the case, the morphology of the nymphs matches better with the genus Diesingia than the other members of Sebekidae. Further morphological and molecular analyzes, including samples of nymphs and adults from Brazil and Argentina, are necessary to solve the taxonomical identity of the specimens here described.

The only published report of pentastomid nymphs in the fish P. harpagos corresponds to specimens collected from Brazil, which show morphological features very similar to those presented here but registered as Sebekia oxycephala (Almeida et al. 2009). Clearly, this taxonomical assignation must be re-evaluated, since the morphological information linked to the genetic analysis suggests that specimens of the genus Sebekia are phylogenetically far from our samples, with a p-distance of 18-19% in the 28S-rDNA gen and 21-23% in the COI mt-DNA gen. These values of genetic divergence support the inclusion of the samples in another genus (in this case, conservatively assigned to Diesingia).

The phylogenetic analysis based on the COI mt-DNA presented here situates our material as the sister group of a big clade conformed by specimens assigned to different genera belonging to the families Sebekidae and Porocephalidae. These relationships, where taxa such as Porocephalus, Kiricephalus, Armillifer, and Waddycephalus (Porocephalidae) are inserted among specimens of Diesingia, Sebekia, and Alofia (Sebekidae), strongly suggest that these families have a paraphyletic arrangement. This assumption is reinforced by the phylogenetic analysis of the 28S rDNA, which shows a well-supported clade including specimens of Alofia, Sebekia, and the analyzed Diesingia (Sebekidae), with a sequence of Porocephalus inserted as sister of Sebekia. Both phylogenetic trees corroborating the molecular distinction of our samples from the genera Sebekia sp. and Alofia sp. leading us to choose the more parsimonious possibilities, where the nymphs found parasitizing P. harpagos belongs to Diesingia sp. Christoffersen & De Assis (2013) differenced morphologically the families inside the Pentastomida characterizing Porocephalidae by the hooks aligned in adults and Sebekidae by having alimentary canal sinous (longer than body), hooks distinctly convex (with spines large and proeminent), and nymphal double hooks. These features could be insignificant and both families could be synonymized in the future. These observations are concordant with the suggestions of Barton & Morgan (2016) who, based on the analysis of the 18S rDNA and the COI mt-DNA markers, noticed that Sebekia and Alofia showed affiliations with members of the family Porocephalidae.

As a conclusion, the DNA analysis situates our specimens far to the genus Sebekia, and the morphological analysis shows that they are similar to Diesignia. The present finding represents the first mention for this genus in Argentina. Further analysis of adult and nymph samples will allow to corroborate the specific identity of the specimens. Diesingia is the only genus that exclusively uses chelonian as definitive host, and there are reports of this pentastomid in the species Hydromedusa tectifera and Phrynops geoffroanus (= Hydraspis geoffroyana) from Brazil (Diesing 1836, Sambon 1922, Heymons 1941, Da Fonseca & Ruiz 1956, Overstreet et al. 1985, Self & Rego 1985, Riley 1994). Based on these records, future collections must be done having aquatic fish-eating turtles as a principal target, being also advisable to examine other aquatic reptiles such as fish-eating snakes.

ACKNOWLEDGMENTS

We thank Jorge Casciotta, Adriana Almiron, Liliana Ciotek, and Pablo Giorgis for helping us in the field sampling; to the Administration of Parques Nacionales for the permits to collect fishes, and especially to Andres Bosso, Guillermo Gil, Veronica Bernava, Fabian Gatti, Facundo Luque, and all of the people of the National Park Iguazu. We specially thank Patricia Sarmiento for her indispensable help taking the SEM photographs, to M. Marcia Montes for the drawings, and Paula Prince for the English edition of the manuscript. We also thank the Consejo Nacional de Ciencia y Tecnologia (CCT-CONICET-La Plata) for the financial support by a research grant (PIP-0015), SRM and PUE 3334/16, Centro de Estudios Parasitologicos y Vectores; to Universidad de La Plata for the financial support by the research grant N859 to Julia Diaz and Sergio Martorelli and Fondo para la Investigación Científica y Tecnológica (FONCyT) for the financial support by the research grant Prestamo BID PICT 2016-4153 to Martín Miguel Montes. Rogelio Aguilar-Aguilar thanks the Dirección General de Asuntos del Personal Académico (DGAPA, UNAM), for the financial support during a sabbatical in Argentina.

REFERENCES

• ALMEIDA MONACO I, RONDON SUAREZ Y & LIMA-JUNIOR S. 2014. Influence of environmental integrity on feeding, condition and reproduction of Phalloceros harpagos Lucinda, 2008 in the Tarumã stream micro-basin. Maringá 36: 181-188.
• ALMEIDA WO, SILVA SOUZA AT & SALES DL. 2009. Parasitism of Phalloceros harpagos (Cyprinodontiformes: Poeciliidae) by Sebekia oxycephala (Pentastomida: Sebekidae) in the headwaters of the Cambé River, Paraná State, Brazil. Braz J Biol 70: 457-458.
• BARTON DP & MORGAN JAT. 2016. A morphological and genetic description of pentastomid infective nymphs belonging to the family Sebekidae Sambon, 1922 in fish in Australian waters. Folia Parasitol 63: 0.26.
• CARVALHO LEITÃO ML, LIMA E SILVA Í, NUNES HIROKI KA, PELLI A & DE SOUZA F. 2018. Ecomorphological and functional differences in population of Phalloceros harpagos Lucinda 2008 in upper Paraná River basin. Biota Amazonia 3: 34-38.
• CASTRESANA J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17: 540-552.
• CAVALIERI F. 1967. Sobre un nuevo género y nueva especie de Linguatulida, Bothropsiella bicornuta gen. et sp. nov., hallada en Bothrops alternata Duméril et Bibron, 1854, Cephalobaenida: Cephalobaenidae. Neotropica 13: 67-72.
• CAVALIERI F. 1970. Contribution al conocimiento de Cephalobaena tetrapoda Heymons, 1922 (Linguatulida, Cephalobaenida). Physis 30: 291-299.
• CHRISTOFFERSEN ML & DE ASSIS JE. 2013. A systematic monograph of the recent Pentastomida with a compitalion of their hosts. Zool Med Leiden 87, 217 p.
• CURRAN SS, OVERSTREET RM, COLLINS DE & BENZ GW. 2014. Levisunguis subaequalis n. g., n. sp., a tongue worm (Pentastomida: Porocephalida: Sebekidae) infecting softshell turtles, Apalone spp. (Testudines: Trionychidae) in the southeastern United States. Syst Parasitol 87: 33-45.
• DA FONSECA F & RUIZ JM. 1956. Was ist eigentlich Pentastoma megastomum Diesing, 1836? (Porocephalida, Porocephalidae). Sencken Biol 37: 469-485.
• DIESING KM. 1836 Versuch der Monographie der Gattung Pentastoma. Ann Wiener Mus Naturgeschich 1: 1-32.
• DRUMMOND AJ, ASHTON B, BUXTON S, CHEUNG M, COOPER A, DURAN C & WILSON A. 2016. Geneious v5.0.4. Available at: http://www.geneious.com/, Accessed date: 17 April 2020.
  » http://www.geneious.com/
• FERNÁNDEZ L, MOLEÓN MS, POLETTA G & SIROSKI P. 2016. Presencia de Pentastómidos (Sebekia sp.) en Yacare negro (Caiman yacare) en la provincia de Corrientes, Argentina. VI Jornadas de Difusión de la Investigación y la Extensión. Universidad Nacional del Litoral. online: http://www.fcv.unl.edu.ar/media/investigacion/JornadaFCV2016/fscommand/SA_FERNANDEZ_L_PRESENCIA.pdf Accessed date 17 November 2019.
  » http://www.fcv.unl.edu.ar/media/investigacion/JornadaFCV2016/fscommand/SA_FERNANDEZ_L_PRESENCIA.pdf
• FOLMER O, BLACK M, HOEH W, LUTZ R & VRIJENHOEK R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrate. Mol Mar Biol Biotechnol 3: 294-299.
• GARATE C, NAUPAY I, SUYO L, COLQUICHAGUA A & RODRÍGUEZ Q. 2007. Identificación de Porocephalus stilessi (Pentastomida) en la serpiente peruana Lachesis muta. June Rev Inv Vet Peru 18: 89-93.
• GIRIBET G, RICHTER S, EDGECOMBE GD & WHEELER WC. 2005. The position of crustaceans within the Arthropoda: evidence from nine molecular loci and morphology. Crustacean Issues 16: 307-330.
• GJERDE B. 2013. Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene. Parasitol Res 112: 3517-3525.
• HEYMONS R. 1941. Beiträge zur Systematik der Pentastomiden. VI. Die Typenexemplare von Diesingia megastoma. Z Parasitenkd 12: 330-339.
• JUNKER K, RILEY J & BOOMKER J. 2003. Redescription of Diesingia megastomum (Diesing, 1836) Sambon, 1922, a pentastomid parasite from the South American terrapin Hydromedusa tectifera Cope. Syst Parasitol 56: 211-218.
• KATOH K & STANDLEY DM. 2016. A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics 32: 1933-1942.
• KUMAR S, STECHER G, LI M, KNYAZ C & TAMURA K. 2018. Mega X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35: 1547-1549.
• LANFEAR R, CALCOTT B, HO SYM & GUIDON S. 2012. PartitionFinder: Combined Selection of Partioning Schemes and Substitution Models for Phylogenetic Analysis. Mol Biol Evol 29: 1695-1701.
• LUQUE JL, VIEIRA FM, TAKEMOTO RM, PAVANELLI GC & EIRAS JC. 2013. Checklist of Crustacea parasitizing fishes from Brazil. Check list 9: 1449-1470.
• MARTINEZ F. 1982. Ninfas de Porocephalus crotali Humboldt,1808 (Arachnida; Pentastomida) en edentados de Argentina. Bol Chil Parasit 37: 74-75.
• MARTINEZ F & RESOAGLI EH. 1982. Artropodos y helmintos en Dasipodidos del nordeste argentino. Gaceta Vet 373: 799-803.
• MARTINEZ FA, TROIANO JC, GAUNA AÑASCO L, DUCHENE A, JUEGA SISCARDI A, NÚÑEZ S, STANCATO MR, FESCINA N & JARA D. 1999. Frecuencia de infestación por pentastómidos en ofidios. Com Cient Tecnol IV: 58-60.
• MARTINEZ FA, TROIANO JC, GAUNA AÑASCO L, FESCINA N & JARA DN. 2000. Infestación por ninfas de pentastómidos del género Porocephalus (Humboldt, 1811) en Hydrodinastes gigas (Ophidia-Colubridae. Analecta Vet 20: 39-42.
• MAZZONI R, NOVAES VC & IGLESIAS-RIOS R. 2011. Microhabitat use by Phalloceros harpagos Lucinda (Cyprinodontiformes: Poecilidae) from a coastal stream from Southeast Brazil. Neotrop Ichthyol 9: 665-672.
• MILLER MA, KINSELLA JM, SNOW RW, HAYES MM, FALK BG, REED RN, MAZZOTTI FJ, GUYER C & ROMAGOSA CM. 2017. Parasite spillover: indirect effects of invasive Burmese pythons. Ecol Evol 8: 830-840.
• NEI M & KUMAR S. 2000. Molecular Evolution and Phylogenetics. Oxford University Press. Oxford, 333 p.
• OVERSTREET RM, SELF JT & VLIET KA. 1985. The pentastomid Sebekia mississippiensis sp. n. in the American alligator and other hosts. Proc Helminthol Soc Wash 52: 266-277.
• PEREIRA NEVES M, DELARIVA RL & WOLFF LL. 2015. Diet and ecomorphological relationships of an endemic, species –poor fish assemblage in a stream in the Iguaçu National Park. Neotrop Ichthyol 13: 345-254.
• POORE GCB. 2012. The nomenclature of the recent Pentastomida (Crustacea), with a list of species and available names. Syst Parasitol 82: 211-240.
• REGIER JR, SHULTZ JW, ZWICK A, HUSSEY A, BALL B, WETZER R, MARTIN JW & CUNNINGHAM CW. 2010. Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463: 1079-1083.
• RILEY J. 1994. A revision of the genus Alofia Giglioli, 1922 and a description of a new monotypic genus, Selfia: two genera of pentastomid parasites (Porocephalida: Sebekidae) inhabiting the bronchioles of the marine crocodile Crocodylus porosus and other crocodilians. Syst Parasitol 29: 23-41.
• RONQUIST F, TESLENKOVAN M, VAN DER MARK P, AYRES DL, DARLING A, HÖHNA S, LARGET B, LIU L, SUCHARD MA & HUELSENBECK JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across large model space. Syst Biol 61: 539-542.
• SAMBON LW. 1922. A synopsis of the family Linguatulidae. J Trop Med Hyg 25: 188-206.
• SCHWARZ G. 1978. Estimating the dimension of a model. Ann Stat 6: 461-464.
• SELF JT & REGO AA. 1985. Reassessments and revisions of certain genera and species of the family Sebekidae (Pentastomida) including description of Sebekia microhamus n. sp. Syst Parasitol 7: 33-41.
• SONNENBERG R, NOLTE AW & TAUTZ D. 2007. An evaluation of LSU rDNA D1-D2 sequences for their use in species identification. Front Zool 4: 1-6.
• TALAVERA G & CASTRESANA J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56: 564-577.
• TKACH VV, LITTLEWOOD DTJ, OLSON PD, KINSELLA JM & SWIDERSKI Z. 2003. Molecular phylogenetic analysis of the Microphalloidea Ward, 1901 (Trematoda: Digenea). Syst Parasitol 56: 1-15.
• TROIANO JC & REPETTO RM. 1994. Pentastomidos en ofidios argentinos. Jornadas de Ciencias Naturales del Litoral V. Posadas, Misiones, Argentina.
• UIEDA VS & PINTO TLF. 2011. Feeding selectivity of ichthyofauna in a tropical stream: space-time variations in trophic plasticity. Com Ecol 12: 31-39.
• WOODYARD ET, BAUMGARTNER WA, ROSSER TG, BODIN EN, FERRARA AM, NOTO TW, FORD LM & RUSH SA. 2019. Morphological, molecular, and histopathological data for Sebekia mississippiensis Overstreet, Self, and Vliet, 1985 (Pentastomida: Sebekidae) in the American Alligator, Alligator mississippiensis Daudin, and the Spotted Gar, Lepisosteus oculatus Winchell. J Parasitol 105(2): 283-298.

Publication Dates

• Publication in this collection
  20 Nov 2023
• Date of issue
  2023

History

• Received
  1 May 2020
• Accepted
  13 Sept 2020