Systematics of Mexiconema cichlasomae (Nematoda: Daniconematidae) Based on Sequences of SSU rDNA

Systematics of Mexiconema cichlasomae (Nematoda: Daniconematidae) Based on Sequences of SSU rDNA Author(s): H. H. Mejia-Madrid and M. L. Aguirre-Macedo Source: The Journal of Parasitology, 97(1):160-162. 2011. Published By: American Society of Parasitologists DOI: 10.1645/GE-2569.1 URL: http://www.bioone.org/doi/full/10.1645/GE-2569.1

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J. Parasitol., 97(1), 2011, pp. 160–162 F American Society of Parasitologists 2011

Systematics of Mexiconema cichlasomae (Nematoda: Daniconematidae) Based on Sequences of SSU rDNA H. H. Mejia-Madrid* and M. L. Aguirre-Macedo, Laboratorio de Patologı´a Acua´tica, Centro de Investigacio´n y de Estudios Avanzados del Instituto Polite´cnico Nacional, Unidad Me´rida, Antigua Carretera a Progreso Km. 6, Colonia Gonzalo Guerrero, C.P. 97310 Me´rida, Yucata´n, Me´xico. *To whom correspondence should be addressed. e-mail: [email protected] TAATGGTGAAACCGCGAAC) and reverse D-1R (CCGGTTCA AGCCACTGCGATTA) primers of Wijova´ et al. (2006). PCR cycling parameters included 36 cycles of denaturation at 94 C for 30 sec, annealing at 54 C for 30 sec, and extension at 72 C for 60 sec. The final product was sequenced with an ABIPrism 3700 Genetic Analyzer (Applied Biosystems, Foster City, California). Segments amplified were compared to positions in the SSU sequence of Caenorhabditis elegans GenBank X03680 (Ellis et al., 1986). CodonCode Aligner (Version 3.5.7, CodonCode Corporation, Dedham, Massachusetts) with Phred base calling was used for assembly of contigs. Sequence alignment was performed with Clustal X version 2.0.11 (Larkin et al., 2007). Homologous SSU fragments determined from GenBank sequences used for generating an unambiguous alignment were those of DQ442665 Procamallanus pacificus, DQ442664 Camallanus sp., EF180076 Spirocamallanus istiblenni, DQ442672 Alinema amazonicum, DQ442671 Nilonema senticosum, DQ442673 Dentiphilometra sp., AB185161 Margolisianum bulbosum, AY852267 Philometra obturans, DQ442674 Philometra sp., DQ442675 Philometra cyprinirutili, DQ442677 Philometra ovata, DQ442676 Philometroides sanguineus, DQ442678 Micropleura australiensis, AY947719 Dracunculus insignis, AY947720 Dracunculus medinensis, AY852269 Dracunculus oesophageus, DQ442668 Molnaria intestinalis, DQ442669 Skrjabillanus scardinii, DQ442670 Philonema oncorhynchi, and U81574 Philonema sp., because they are well within the range for alignment. A total of 21 taxa were employed. Phylogenetic analyses of DNA sequences were performed with the use of PAUP* v4.0 b10 (Swofford, 2000). Sequence alignment was analyzed by maximum parsimony (MP) and maximum likelihood (ML). MP analyses were based on a heuristic search with random addition sequence of 1,000 replicates, on a tree bisection-reconnection (TBR) branch swapping, and on a branch-and-bound search with furthest addition sequence. ML was set with an initial NJ tree and an MP generated by the aforementioned search. The best-fit model of evolution identified was based on the Akaike Information Criterion implemented in jModelTest 0.1.1 (Guindon and Gascuel, 2003; Posada, 2008). In all cases, trees were treated as unrooted and outgroups were rooted during analysis with default outgroup (5 leading taxon of the sequence alignment matrix). Clade support was assessed using 1,000 replicates of bootstrap resampling (Felsenstein, 1985) with heuristic and 100 replicates with branch-andbound searches for MP and ML analyses with confidence level of 50%. A single contig resulted from assembling 12 (6 forward, 6 reverse) sequence segments of SSU recovered from 6 different females. Sequences varied in length from 398 to 858 bp. The complete consensus sequence (1710 bp) of M. cichlasomae has been deposited in GenBank with accession number HM566890. Alignment resulted in a matrix with 2,031 characters. After running analyses for MP (heuristic and branch and bound) and ML, our results indicated that 588 characters are constant, 215 are variable and parsimony uninformative, and 1,228 are parsimony informative. In the case of ML analysis, a general time reversible model plus gamma distribution of variable sites (GTR + G) was selected as the most appropriate for our data (nucleotide frequencies A 5 0.25450, C 5 0.21630, G 5 0.27340, T 5 0.25580). All analyses recovered single trees (Fig. 1, ML tree shown) (MP heuristic 5 1, MP branch and bound 5 1, ML 5 1 with NJ, ML 5 1 with heuristic search-generated tree) with identical topologies and a length of 2,233 steps, CI 5 0.818 (excluding uninformative characters), HI 5 0.161 (excluding uninformative characters), RI 5 0.891, and RC 5 0.747. Procamallanus pacificus Moravec, Justine, Wu¨rtz, Taraschewski, and Sasal, 2006, Camallanus sp., and Skrjabillanus istiblenni Noble, 1966 appeared as outgroups to a monophyletic dracunculoid taxon. A

ABSTRACT: The molecular characterization of the daniconematid dracunculoid Mexiconema cichlasomae Moravec, Vidal, and SalgadoMaldonado, 1992 through the sequencing of SSU rDNA from adult individuals is presented herein. Additionally, preliminary genetic relationships of this nematode are inferred from alignment of sequences generated previously for other dracunculoids. Maximum parsimony and maximum likelihood analyses recovered identical trees. As anticipated by previous taxonomic work, M. cichlasomae is putatively closely related to skrjabillanid dracunculoids represented by Molnaria intestinalis (Dogiel and Bychovsky, 1934) and Skrjabillanus scardinii Molna´r, 1966 SSU rDNA sequences, but the relationships of this newly discovered clade to other dracunculoid clades remain unresolved.

Philometrids are dracunculoids that are parasites of the abdominal cavity and tissues of fishes (Moravec et al., 1998; Anderson, 2000). Recently, there has been a growing interest in philometrid nematodes of fishes because they infect reproductive organs and, therefore, cause serious losses to fisheries (Moravec, 2004, 2006, 2007; Moravec and SalgadoMaldonado, 2007; Moravec et al., 2008). Special concern exists about the classification of dracunculoids, especially those that parasitize wild and economically important fishes (Moravec, 2004). It has been argued that the current classification of this nematode group is not based on phylogenetic relationships and that a taxonomic revision, based on detailed morphological, life history, and molecular studies, is still due (Moravec, 2004). Moreover, despite the economic importance of dracunculoid species, data on their biology are scarce (Moravec, 2004). A small number of dracunculoids have been molecularly characterized (Wijova´ et al., 2005, 2006) or included in recent phylogenies of nematodes (Blaxter et al., 1998, Dorris et al., 1999; Blaxter, 2001; De Ley and Blaxter, 2002; Wijova´ et al., 2006; Nadler et al., 2007; Van Megen et al., 2009). Such phylogenies have been published with a considerable amount of taxonomic sampling, but philometrids have remained undersampled. An example of a group of philometrid species that has not been sampled so far is the Daniconematidae Moravec and Køie, 1987. Species of this family are common parasites in freshwater and marine fishes (Molna´r and Moravec, 1994; Moravec, 2006). Therefore, the molecular characterization based on SSU (18S) rDNA sequences and preliminary genetic relationships of a daniconematid philometrid, Mexiconema cichlasomae Moravec et al., 1992, is presented herein. Mexiconema cichlasomae is a nematode parasite common in cichlid fishes of southeastern Me´xico coastal basins (Vidal-Martı´nez et al., 2001; Garrido-Olvera et al., 2006) and is especially abundant in Cichlasoma urophthalmus Gu¨nther in the Yucata´n Peninsula. These hosts were gill netted in March 2009 from Laguna de Celestu´n in Yucata´n, Me´xico, near the northern end of the lagoon at 20u459000N, 90u159000W. Fishes were transported immediately to the laboratory and kept alive until necropsy. Parasites were collected only from immediately necropsied fishes. Voucher specimens were deposited in the Coleccio´n Helmintolo´gica CINVESTAV Me´rida. Worms were either fixed in 100% ethanol and processed later, or were immediately processed for DNA extraction from fresh tissue with 20 ml of 5% Chelex 100 (Sigma, St. Louis, Missouri) mixed with 1 ml proteinase K and incubated at 60 C for 1 hr. Final extracts had a total DNA concentration of ,100 ng/50 ml. A fragment of the DNA nuclear SSU was amplified with the use of the forward D-1F (GCCTA-

Received 10 June 2010; revised 20 September 2010; accepted 21 September 2010. DOI: 10.1645/GE-2569.1 160

RESEARCH NOTES

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FIGURE 1. Maximum likelihood (ML) tree of 18 species of dracunculoids based on SSU rDNA sequences. Bootstrap values for MP heuristic/MP branch and bound/ML analyses are shown near their respective branches or are indicated by arrows.

diphyletic dracunculoid clade was recovered. One clade includes (M. cichlasomae [M. intestinalis, Skrjabillanus scardinii]); the other comprises the rest of the philometrids + dracunculids. Bootstrap tests (MP and ML methods) yielded similar branch confidence levels, except for the philometrid + dracunculid clade, which had less than 50% support in ML analyses and none in MP. Phylogenetic analyses revealed that M. cichlasomae is genetically related to M. intestinalis and S. scardinii and is grouped in a well-supported monophyletic clade. Trees recovered indicate a preliminary phylogenetic relationship between Daniconematidae and Skrjabillanidae that can only be confirmed when new taxa of both families are added to further phylogenetic analyses. This relationship had been mentioned previously in the discussion of the taxonomy of these nematode families, because the presence of 3 small caudal processes in females and males and a sclerotized copulatory plate in males is common to species of the aforementioned philometrid families (Moravec et al., 1992, 1998, 2009). Additionally, the mode of infection is similar among species of this subclade (Moravec et al., 1999). The rest of the taxa incorporated into this phylogeny exhibit a similar topology as in previous wider-ranging phylogenies, where the ‘‘philometrid’’ group appears always paraphyletic, but remains within a monophyletic dracunculoid subclade (Wijova´ et al., 2006; Nadler et al., 2007; Van Megen et al., 2009). Since publication of the description of M. cichlasomae, 2 other congeneric species have been described from other parts of the world, namely, M. africanum Moravec and Shimazu, 2008 and M. liobagri Moravec and Nagasawa, 1998 (Moravec and Shimazu, 2008; Moravec et al., 2009). Only females are known from the latter (Moravec and Shimazu, 2008). The aforementioned species have not been sequenced up to this time. The inclusion of more sequences from these and other species of

dracunculoids is necessary to recover a phylogenetically based classification that can generate meaningful information on the evolutionary relationships between species and parasite–host relationships of this economically important group of nematode parasites. Thanks are due to the staff of the Laboratorio de Patologı´a Acua´tica, CINVESTAV-IPN Unidad Me´rida, Yucata´n, Me´xico, for field collections and dissection of fish hosts. Special thanks go to Dr. Rossana Rodrı´guezCanul for lending space in her lab at CINVESTAV-IPN Unidad Me´rida. Sequencing was undertaken at Laboratorio de Servicios Geno´micos, LANGEBIO CINVESTAV-Campus Irapuato facility by IBQ Karla Iveth Pe´rez Ma´rquez and Corina Dı´az. The present work was supported by a postdoctoral fellowship from the CONACYT, ‘‘Estancias Posdoctorales y Saba´ticas Vinculadas al Fortalecimiento de la Calidad del Posgrado Nacional 2008-2009’’ to H.H.M.M., and to an operating grant from CINVESTAV-IPN to M.L.A.M.

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