©2017 Institute of Parasitology, SAS, Košice DOI 10.1515/helm-2017-0010

HELMINTHOLOGIA, 54, 1: 68 – 76, 2017

Research Note

Procamallanus spiculogubernaculus Agarwal, 1958 (Nematoda: Camallanidae) from Stinging catfi sh, in India: morphological characterization and molecular data

A. CHAUDHARY1*, C. VERMA1, V. TOMAR2, H. S. SINGH1

1Molecular Laboratory, Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), India – 250004, *E-mail: [email protected]; 2Department of Zoology, Dyal Singh College, University of Delhi, Delhi, India – 110003

Article info Summary

Received September 7, 2016 The , Procamallanus spiculogubernaculus Agarwal, 1958 was found from the Stinging Accepted November 23, 2016 catfi sh, Heteropneustes fossilis (Bloch, 1794) from Ghazipur, Delhi, India. Morphological character- ization, including scanning electron microscope observation supplemented with DNA sequences is provided. Specimens recently found are characterized by the presence of an unlined buccal capsule having a small basal ring, esophagus muscular and glandular, vulva position is slightly post-equato- rial, tail conical, long, and ending in three digit-like processes, phasmids present at about mid-length and cloaca located at the posterior end. In this study the P. spiculogubernaculus is validated on the basis of molecular data after 47 years from its original description. In the scanning electron mi- croscope examination, the topology of mouth and sensory pits in anterior portion, while the phasmids and digit like processes in posterior portion is clearly observed. Molecular data of the 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. Molecular phy- logenetic analyses supported the validity of Procamallanus spiculogubernaculus and confi rmed the paraphyletic status of the members of Procamallanus, Spirocamallanus, and Paraca- mallanus. Taxonomic status of members of the family Camallanidae are briefl y discussed along with the results of the systematic evaluation of P. spiculogubernaculus based on molecular data. Keywords: Nematode; Procamallanus; Scanning electron microscopy; Molecular analyses; 18S; cox1; India

Introduction Procamallanus species is based on characters such as esopha- gus structure, presence of spicules, and gubernaculum in males, The Procamallanus Baylis, 1923 (Nematoda: Camallani- while in females it is based on the conical tail with digit-like tail dae) includes species that parasitize freshwater, brackish-water processes, vulva position and shape of the eggs. The rich diversi- and marine fi shes, less often amphibians. More than 20 species ty of the species of Procamallanus from H. fossilis in India made of this genus have been described from Bangladesh, India and Sri the species identifi cation diffi cult, as many descriptions are inade- Lanka (Sood, 1989) from Heteropneustes fossilis (Bloch, 1794). quate for diagnosis. Due to the lack of proper morphological identi- H. fossilis have high economic importance as food fi sh and for fi cation and descriptions, species of this genus remain unclear (De medicinal value (Talwar & Jhingran, 1991; Sherwani & Parwez, & Moravec 1980). Therefore, morphological identifi cation needs 2000; Acharya & Mohanty, 2014). Morphological identifi cation of the use of molecular analyses to supplement the proper identifi ca-

* – corresponding author

68 tion and validation of species. Molecular tools are frequently used electron microscopy (SEM) studies, were fi xed in 4 % in nematode identifi cation and systematics (Gasser, 2001; Nadler formaldehyde, post-fi xed in 1 % osmium tetraoxide, dehydrated et al., 2005; Mattiucci & Nascetti, 2008; Borges et al., 2012; Jones through ascending grades of ethanol series, dried using the critical et al., 2012; Liu et al., 2013; Pekmezci et al., 2014; Di Azevedo et point dryer and sputter-coated with gold. The specimens were ex- al., 2015; Goswami et al., 2015, 2016). amined with a JEOL Neoscope JCM5000 scanning electron micro- In this study, supporting morphological and molecular information scope (Tokyo, Japan) at an accelerating voltage of 10 kV. is provided for P. spiculogubernaculus Agarwal, 1958 from the Samples for molecular analysis were fi xed in 95 % ethanol. freshwater fi sh H. fossilis collected from Ghazipur, Delhi, India. Genomic DNA extraction from female worm was conducted using the Qiagen DNeasyTM tissue kit (Qiagen, Germany) according to Materials and Methods the manufacturer’s instruction. PCR amplifi cation for partial 18S (900bp) and cox1 (574bp) gene regions were performed using A total of 25 specimens of H. fossilis (body length 14 – 19 cm) two different sets of the primers, Nem 18SF (5’-CGCGAATRGCT- were collected during January 2013 to March 2014 from Ghazipur CATTACAACAGC-3’) and Nem 18SR (5’-GGGCGGTATCT- (28.7342° N, 77.2728° E), Delhi, India for examination of parasites, GATCGCC-3’) (Floyd et al. 2005); LCO1490 (5’-GGTCAACAAAT- from which 14 fi sh were found infected. Host specimens were kept CATAAAGATATTGG-3’) and HC02198 (5’-TAAACTTCAGGGT- alive in aerated jars at 24 °C until dissection. Nematodes (7 female GACCAAAAAATCA-3’) (Folmer et al. 1994) respectively. Each specimens) from the small intestine of fi sh were collected in 0.6 % 25 μl PCR amplifi cation reaction contained 3 μl of DNA, 2.5 μl of saline solution and then stored in 70 % ethanol until processing 10× PCR buffer, 0.9 μl of each primer, 3.4 μl of deoxyribonucle- for morphology. For the light microscopy study, nematodes were otide triphosphates and 1.5 U of Taq DNA polymerase (Biotools, cleared and mounted with glycerin. Drawings were made with the Spain). The amplifi cation was carried out in an Eppendorf Mas- aid of a camera lucida. All measurements were taken in mm from tercycler (Eppendorf, Germany) with the following cycling profi le: the camera lucida diagram. Photos were taken with a light micro- 94 °C for 3 min (hot start), followed by 35 cycles of denaturation at scope equipped with differential interference contrast and digital 94 °C for 50 s, annealing for 1 min on 54 °C for 18S and 48 °C for image analysis system (Nikon, Eclipse 50i, Kawasaki, Japan). cox1, followed by 1 min extension at 72 °C and a fi nal extension Specimens were deposited in the Museum d’ Histoire Naturelle, at 72 °C for 5 min. The PCR products were electrophoresed on Geneva, Switzerland (MHNG-INVE-91853) and the Museum, De- 1.5 % agarose in TAE buffer stained with ethidium bromide and partment of Zoology, Chaudhary Charan Singh University, U.P., visualized under UV transilluminator. For sequencing, the PCR India under the voucher number Nem/2015/02. For scanning product was purifi ed using PurelinkTM Quick Gel Extraction and Table 1. Comparative morphometric data of P. spiculogubernaculus female specimens P. spiculogubernaculus P. spiculogubernaculus P. spiculogubernaculus Agarwal, 1958 (De & Moravec, 1980) present study Infecting: Host Heteropneustes fossilis H. fossilis H. fossilis Organ Intestine Stomach Intestine Body: Length 4.84 3.56 – 7.49 (3.44) 4.71 (3.25 – 5.31) Width 0.088 0.090 – 0.189 (0.096) 0.090 – 0.189 (0.096) Buccal capsule: Length 0.08 0.069 – 0.084 (0.063) 0.069 – 0.084 (0.063) Width 0.048 0.048 – 0.069 (0.057) 0.048 – 0.069 (0.057) Length of basal ring: – 0.009 0.009 Muscular oesophagus: Length 0.032 0.264 – 0.333 (0.282) 0.264 – 0.333 (0.282) Width – 0.057 – 0.066 (0.057) 0.057 – 0.066 (0.057) Glandular oesophagus: Length 0.046 0.318 – 0.450 (0.336) 0.318 – 0.450 (0.336) Width – 0.042 – 0.072 (0.048) 0.042 – 0.072 (0.048) Nerve ring distance from anterior end: – 0.129 – 0.156 (0.126) 0.129 – 0.156 (0.126) Vulva distance from posterior end: 2.2 1.58 – 3.48 (1.46) 3.26 (2.98 – 3.57) Tail length: 0.1 0.093 – 0.138 (0.090) 0.06 (0.05 – 0.07) Tail process length: – 0.003 0.002

69 PCR purifi cation Combo Kit (Invitrogen, Germany) following man- es and Hasegawa-Kishino-Yano model (HKY) for mtDNA cox1 ufacturer’s instructions. Purifi ed PCR products were sequenced were selected. The phylogenetic trees were inferred by Maximum (in both directions) using the Big Dye Terminator vr. 3.1 cycle se- Likelihood (ML) using MEGA vr. 6 (Tamura et al., 2013) with boot- quencing kit in ABI 3130 Genetic Analyzer, Applied Biosystems strap analysis based on 1000 replicates. Bayesian Inference (BI) (Foster City, USA) with the same primers cited above. analyses were also carried out in Topali 2.5 (Milne et al., 2009) Obtained sequence fragments of 18S rRNA and mt cox1 gene using MCMC chains searches on two simultaneous runs of four were assembled using MEGA V6.06 (Tamura et al., 2013). Am- chains over 1,000,000 generations with every 100th tree saved. biguous bases were clarifi ed using their corresponding ABI chro- The fi rst 25 % of the sampled trees were discarded as ‘burn in’. matograms. The 18S rDNA and mt cox1 gene sequences were The members belong to the family Physalopteridae, Dracunculi- compared for similarities with other available sequences from dae, Philometridae and Micropleuridae and Dracunculus insignis GenBank using BLAST (www.ncbi.nlm.nih.gov) and the related (EU646538) were used as out-groups for 18S rDNA and cox1 mt sequences based on nucleotide similarities were aligned with the DNA phylogenetic reconstructions respectively. software CLUSTAL W (Thompson et al., 1994). DNA pairwise dis- tances for cox1 and 18S rDNA were calculated in MEGA V6.06 Results and Discussion using the p-distance model. The dataset was tested prior to phy- logenetic analysis for the nucleotide substitution model of best fi t A total of 7 P. spiculogubernaculus specimens were collected from using ModelTest (Posada & Crandall, 1998) and the best-fi tting 14 infected host fi sh, H. fossilis. All specimens were found in the model was chosen using the Akaike Information Criterion (AIC). small intestine, none was recovered from the stomach, liver or The model GTR + G + I (general time reversible) for 18S sequenc- body cavity. The prevalence was 56 % and mean intensity of in-

Fig. 1. Differential interference contrast images of female Procamallanus spiculogubernaculus (dorsal view). A – anterior end; B – buccal capsule of female; C – eggs; D – female tail with processes; E – Vulva. Scale bars: Fig. A = 0.33 mm; Fig. B = 0.11 mm; Figs. C – E = 0.11 mm.

70 Fig. 2. Scanning electron micrographs of Procamallanus spiculogubernaculus (female). A – anterior part of female showing mouth and sensory pits; B – tail of female showing posterior papillae (phasmids) and digit-like processes. Scale bars: Fig. A = 50 μm; Fig. B = 20 μm. fection was 2.7. Morphological identifi cation was based on female culogubernaculus was deposited in GenBank under the accession worms and the specimens were tentatively identifi ed as P. spicu- number KU292358 in this study. Subsequently, we compared our logubernaculus due to the presence of an unlined buccal capsule cox1 sequence by BLAST similarity search with available se- with a small basal ring, muscular and glandular esophagus, the quences in the database. The sequences which show similarity position of vulva slightly post-equatorial, tail conical, long, ending with cox1 sequence of P. spiculogubernaculus were from Spiro- in three digit-like processes (one dorsal and two ventrolateral), camallanus and Camallanus genera (Fig. 4). There is no cox1 phasmids occur at about mid-length, cloaca located at the posteri- sequence available in the GenBank database for any species of or end in female (Fig. 1 and Fig. 2). These specimens are similar to Procamallanus. Pairwise comparison between the cox1 sequence the description of P. spiculogubernaculus and the redescription by of P. spiculogubernaculus and Spirocamallanus and Camallanus De & Moravec (1980). Measurements were taken and compared species showed 0.17 – 0.21 % nucleotide differences. The phy- to other described species in the Table 1. logenetic analyses of the cox1 gene sequence of P. spiculoguber- Moreover, we obtained 18S rDNA sequence of P. spiculoguber- naculus isolated from H. fossilis were conducted with two methods: naculus (900 bp), deposited in GenBank under the accession ML and Bayesian inference. We present only the ML tree as the BI number KU292357 and compared it with the sequences available tree shows a similar topology in the clade formation (Fig. 4). This in the GenBank. The phylogenetic relationship of 18S rDNA was phylogenetic tree was based on the sequences of cox1 mtDNA. analyzed on the basis of ML and Bayesian inference (Fig. 3). The This tree is focused on the representatives of the family Camal- trees obtained by these two methods showed similar topologies, lanidae with Dracunculus insignis (Leidy, 1858) (Dracunculidae) therefore, only the ML tree is presented (Fig. 3) together with pos- as an out-group species. The phylogenetic tree constructed from terior probability values for BI. The newly generated 18S rDNA dataset containing representatives of genera Camallanus and sequence of P. spiculogubernaculus was used along with 67 pub- Spirocamallanus displays in fi gure 4. The phylogenetic analyses lished taxa for phylogenetic reconstruction. The 18S gene analysis gave a tree with two major clades (Fig. 4). P. spiculogubernacu- comprises of total 68 sequences from which the members belong lus incorporates between clades of Camallanus and Spirocamal- to the family Physalopteridae Leiper, 1908, Dracunculidae Leiper, lanus. This is unfortunate to encorporated any sequence of cox1 1912, Philometridae Baylis and Daubney, 1926 and Micropleuridae gene of Procamallanus, due to unavailability of cox1 sequence in Baylis and Daubney, 1926 were taken as out-group from which the GenBank. To bring out more detailed view on the relationships be- family Camallanidae arises. P. spiculogubernaculus is found to tween species of Procamallanus, more sequences of cox1 gene form a clade with representatives of genera Procamallanus Bay- have to be generated. lis, 1923, Spirocamallanus Olsen, 1952, Camallanus Railliet and Both phylogenetic trees fairly illustrated the relationship of P. Henry, 1915 and Paracamallanus Yorke and Maplestone, 1926. P. spiculogubernaculus with the genera Camallanus and Spirocama- spiculogubernaculus represented sister taxa to the Procamallanus llanus. One represents the species of Camallanus and the other laeviconchus Wedl, 1861. This is the fi rst sequence for any Proca- belonged to the species of Spirocamallanus and Procamallanus mallanus species from India available on GenBank. (Fig. 4). The specimens of P. spiculogubernaculus sequenced in Partial sequences of the cox1 (574 bp) gene obtained from P. spi- the present study show close relationships with species of Pro-

71 77/1.00 Camallanus oxycephalus GU170847 24/0.66 Camallanus oxycephalus GU170850 61/0.87 Camallanus oxycephalus GU170849 Spirocamallanus istiblenni GU170860 Spirocamallanus istiblenni GU082491 39/0.72 Spirocamallanus istiblenni GU082495

32/0.70 Paracamallanus sp. AM-2008 KJ566936 Spirocamallanus istiblenni GU170859 56/0.87 Spirocamallanus istiblenni GU082492 Camallanus oxycephalus GU082498 18/0.57 Camallanus oxycephalus GU170848 Camallanus oxycephalus GU170852 Spirocamallanus istiblenni GU170858

62/- Paracamallanus sp. GC-2009 GU082487 72/0.99 Camallanus oxycephalus GU082499 Spirocamallanus istiblenni GU082494 Camallanus oxycephalus GU082497 61/0.92 Paracamallanus sp. GC-2009 GU082488 Paracamallanus sp. GC-2009 GU082486 51/- Camallanus oxycephalus GU082496 68/1.00 Spirocamallanus istiblenni GU170861

24/0.70 Paracamallanus sp. GC-2009 GU170855 Camallanus oxycephalus GU170851 91/1.00 54/1.00 Camallanidae 28/0.72 Paracamallanus sp. GC-2009 GU170854 95/1.00 Spirocamallanus istiblenni EF180076 Spirocamallanus istiblenni KC505629 71/0.99 Procamallanus monotaxis JF803931 57/0.90 Spirocamallanus istiblenni KC505630 Procamallanus rebecae DQ442667

42/0.73 Spirocamallanus philippinensis JF934736 40/0.67 Procamallanus annulatus JF803932 51/0.63 93/1.00 Procamallanus sigani HM545908 61/0.87 Procamallanus laeviconchus JF803934 54/0.72 52/0.93 Procamallanus spiculogubernaculus KU292357 99/1.00 Procamallanus pacificus DQ442665 99/1.00 Spirocamallanus rarus DQ494195 97/1.00 Procamallanus fulvidraconis JF803914 99/1.00 53/0.67 Procamallanus fulvidraconis DQ076689 Camallanus sp. MW-2006 DQ442664 51/0.74 Camallanus cotti 18S EF180071 Camallanus cotti DQ442662 99/1.00 73/0.99 Camallanus hypophthalmichthys JF803915 72/0.95 DQ442663 71/- Camallanus oxycephalus DQ503463 Spirocamallanus rarus JF803912 Procamallanus pintoi DQ442666 99/1.00 99/1.00 Philonema sp. U81574 Philometridae Philometra cyprinirutili DQ442675 Micropleura australiensis KP116004 Micropleura australiensis KP116008 99/1.00 Micropleura australiensis KP116007 Micropleuridae Micropleura australiensis DQ442678 67/1.00 Micropleura australiensis KP115997 Philometroides sanguineus DQ442676 78/0.87 Philometroides pseudorasbori DQ076687

91/1.00 Philometroides carassii DQ076683 89/1.00 Philometra ovata DQ442677 91/1.00 Philometroides cyprini DQ076688 31/0.63 Philometra cyprinirutili DQ442675 Philometridae 53/0.85 Philometra lagocephali KP122959 99/1.00 Philometroides seriolae FJ155811

57/0.90 Philometra bagri JF803948 62/1.00 Philometra morii JF803933 60/0.85 Philometra fujimotoi DQ076680 Dracunculus medinensis AY8522668 Dracunculidae turgid DQ503459 Physalopteridae 99/1.00 Physaloptera sp. SAN-2007 EF180065 Toxocara canis JN256976

0.02

Fig. 3. A phylogenetic tree is based on the 18S rDNA sequences and demonstrating the position of Procamallanus spiculogubernaculus with other nematode species. The tree was generated by maximum likelihood (ML) method. Numbers of nodes indicate the bootstrap values ML and posterior probabilities (BI) respectively. GenBank accession numbers are listed along the species names. Species examined in this study are indicated in bold. Unsupported nodes by BI are marked with a hyphen.

72 Spirocamallanus istiblenni KC517382 Spirocamallanus istiblenni KC517391 Spirocamallanus istiblenni KC517390 Spirocamallanus istiblenni KC51740 Spirocamallanus istiblenni KC517386 Spirocamallanus istiblenni KC517385 Spirocamallanus istiblenni KC517394

98/1.00 Spirocamallanus istiblenni KC517402 Spirocamallanus istiblenni KC517393 Spirocamallanus istiblenni KC517388 70/0.85 Spirocamallanus istiblenni KC517392 91/0.94 78/0.95 Spirocamallanus istiblenni KC517384 Spirocamallanus istiblenni KC517403 Procamallanus spiculogubernaculus KU292358 Camallanus hypophthalmichthys EU598816 Camallanus cotti EU598890 90/0.99 Camallanus cotti EU598879

99/1.00 Camallanus cotti EU598881 Camallanus cotti EU598882 84/0.96 Camallanus cotti EU598836 Camallanus cotti EU598868 56/0.75 Camallanus cotti EU598868

70/0.84 Camallanus cotti EU598838 Camallanus cotti EU598883 Camallanus cotti EU598871 Camallanus cotti EU598873 Dracunculus insignis EU646538

0.01

Fig. 4. Maximum Likelihood (ML) tree inferred from mtDNA cox1 gene sequence data from Procamallanus spiculogubernaculus obtained in this study. Numbers of nodes are bootstrap values higher than 50% and indicate the ML and posterior probabilities (BI) respectively. GenBank accession numbers are also given in the tree. P. spiculogubernaculus examined in this study are indicated in bold. camallanus. It is also fi gured out the paraphyletic nature of Pro- other in morphology. The following species, i.e., Procamallanus camallanus from phylogenetic reconstruction as it grouped in four daccai Gupta, 1959; Neocamallanus heteropneusti Chakravarty different clades included members of Camallanus, Procamallanus et al., 1961; P. chakravartyi Fernando & Furtado, 1963; P. con- and Spirocamallanus. P. spiculogubernaculus in the present study fuses Fernando & Furtado, 1963; P. mathurai Pande et al., 1963; shows the close relationships with species of Spirocamallanus that P. hindenensis Lal, 1965; P. magurii Lal, 1965; P. devendri Sinha depicts them as putative sister species because the genus Spiro- & Sahay, 1966; P. ottuei Varma & Varma, 1971 and P. sprenti camallanus was retain as subgenus of Procamallanus by Moravec Bashirullah & Hafi zuddin, 1974 were synonymized with P. spicu- & Sey (1988) to accommodate the species possessing a buccal logubernaculus by De & Moravec (1980) as they all were mor- capsule with spiral thickenings. phologically similar. P. spiculogubernaculus was inadequately de- A large number of species are placed in Procamallanus Baylis, scribed before the study of De & Moravec (1980). This species is 1923 that are mostly parasites of freshwater and marine fi shes (De distinguished from others on the basis of taxonomic features such & Moravec, 1980; Andrade-Salas et al., 1994; Rigby, 1999; De & as the length of the body, spherical mouth opening, mouth papillae Maity, 2000; Moravec et al., 2006; Moravec & Van As, 2015). In four in number, buccal capsule barrel shaped, esophagus divided the present study, the collected specimens were all of the females; into muscular anterior and glandular posterior parts; in females, no male was found. According to De & Moravec (1980) many de- tail ends conical with three small processes. As these digit-like pro- scribed species of the genus Procamallanus reported from the cesses were also found in female of P. laeviconchus and P. pseu- same host from India and nearby countries are very close to each dolaeviconchus (Moravec & Van As, 2015) confi rmed by the SEM

73 study. In the present study SEM observation was performed for P. the accurate identifi cation, distinction of species and for providing spiculogubernaculus for the fi rst time. It is evident that most Proca- reliable phylogenies. Further studies in future with more sequence mallanus species from India were inadequately described, making data are needed for congruent analysis and comparison which will identifi cation solely on the basis of light microscopy diffi cult, espe- clear phylogenetic position of parasitic nematodes of the family cially when many species of Procamallanus harbor the same host Camallanidae in India. fi sh. Of many described Procamallanus species in India, only few studies are available using the SEM (Moravec et al. 2013). Struc- Acknowledgments tures like topology of mouth, sensory pits at the anterior region, the phasmids and digit like processes in posterior region visible by The authors thank to the Head of the Department of Zoology, SEM can act as an important taxonomic feature through which P. Chaudhary Charan Singh University, Meerut, for providing labora- spiculogubernaculus can easily be distinct from other species of tory facilities. Special thanks to Dr. František Moravec, Institute of Procamallanus. Although, morphological information contributes Parasitology, Academy of Sciences of the Czech Republic, České to species identifi cation, but DNA sequence data are required for Budějovice, Czech Republic for identifi cation of parasite species substantiating the fi ndings. To date, no sequence is available in (P. spiculogubernaculus) and for mailing us some literature on P. the GenBank database for any Procamallanus species from India. spiculogubernaculus. This study was supported by grants from This study provides the phylogenetic status of P. spiculoguber- the UGC (University Grants Commission) India, under the Post naculus with combined use of 18S rDNA and cox1 mt DNA mo- Doctoral Fellowship [(F.15-191/2012 (SA-II)] to AC. The authors lecular data. Further, this is the fi rst molecular data for this taxon declare that they have no confl ict of interest. and it does establish the phylogenetic relationship with some other members of Procamallanus. By comparing the sequence differ- References ences, it is easy to identify closely related nematode species. The molecular markers based on nuclear and mitochondrial genes are ACHARYA, G., MOHANTY, P.K. (2014): Comparative hematological an excellent tool for differentiating nematode species (Floyd et al., and serum biochemical analysis of catfi shes Clarias batrachus 2002; Blaxter et al., 2005; McLean et al., 2012; Singh et al., 2013; (Linnaeus, 1758) and Heteropneustes fossilis (Bloch, 1794) with Nakacwa et al., 2013; Chaudhary et al., 2015; Mikaeili et al., 2015; respect to sex. J. Entomol. Zool. Stud., 2: 191 – 197 Goswami et al., 2015). The present molecular analyses using 18S AGARWAL, S.C. (1958): On a new species of Procamallanus Baylis, rDNA and cox1 mtDNA genes support the placement of P. spic- 1923 (Nematoda). Curr. Sci., 27: 348 – 349 ulogubernaculus in family Camallanidae. Comparison of a large ANDRADE-SALAS, O., PINEDA-LÓPEZ, R.F., GARCÍA-MAGAÑA, L. (1994): number of sequences of representatives of Camallanidae also Spirocamallanus rebecae sp. n. Nematoda: Camallanidae) from supports the paraphyly of genera Procamallanus. The current phy- freshwater fi shes in south-eastern Mexico. Folia Parasitol., 41: logenetic reconstruction also indicated the paraphyletic nature of 259 – 270 Camallanid genera Camallanus, Procamallanus and Spirocamal- BASHIRULLAH, A.K.M., HAFIZUDDIN, A.K.M. (1974): Two new nema- lanus. The genus Spirocamallanus was established as a distinct tode species of Procamallanus Baylis, 1923 from freshwater fi shes genus from Procamallanus with species having presence of spiral of Dacca, Bangladesh. Norw. J. Zool., 22: 53 – 55 thickenings on the buccal capsule. Many authors agreed with the BLAXTER, M., MANN, J., CHAPMAN, T., THOMAS, F., WHITTON, C., FLOYD, distinct status of Spirocamallanus (Ivashkin et al., 1971; Chabaud, R., ABEBE, E. (2005): Defi ning operational taxonomic units using 1975; Soota, 1983; Andrade-Salas et al., 1994). Whereas Proca- DNA barcode data. Philos. Trans. R. Soc. Lond., B, Biol. Sci., 360: mallanus have smooth buccal capsule lack of spiral thickenings. 1935 – 1943. 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