Phylogenetic Analysis of Two Species of Procamallanus (Spirocamallanus) (Nematoda: Camallanidae) Parasitizing Indian Freshwater Fishes

Introduction

Parasitic nematodes belong to the family Camallanidae, mostly infect the digestive tract of marine and freshwater fish (and sometimes amphibians and aquatic reptiles), and are distributed worldwide [1, 2, 3, 4, 5, 6, 7, 8]. Camallanid nematodes can be easily distinguished on the basis of morphological features such as the presence of a well-developed buccal capsule that is further supported by different structures like a basal ring, spiral or longitudinal ridges, tridents, etc. [5]. Until now, the camallanid fauna of Indian fish has been poorly studied, and due to a lack of proper studies, numerous species were erected, most of which lacked proper morphological characteristics and molecular data. Recently, in a study by Moravec, et al. [9], it was pointed out that in spite of the large number of taxonomic and faunistic papers for freshwater fish nematodes in South Asian countries, a gap regarding the knowledge of real fauna still persists. According to Moravec, et al. [9], this situation arises because most of the species from this region were not appropriately identified or were poorly described, as well as because many nematode species identifications or their taxonomic problems in the respective groups were just left unsolved.

During parasitological surveys for fish nematodes in the district Muzaffarnagar of Uttar Pradesh and in the Poonch River of the district Poonch of Jammu and Kashmir, India, in 2021, two species of camallanid nematodes were found: Procamallanus (Spirocamallanus) mysti Karve, 1952, and Procamallanus (Spirocamallanus) bilaspurensis Gupta, et al. [10]. In the present study, we provide molecular characterization based on the 18S gene of the found species.

Results

Procamallanus species collected during the present study revealed the presence of Procamallanus (Spirocamallanus) mysti Karve, 1952 and Procamallanus (Spirocamallanus) bilaspurensis Gupta, et al. [10] from Mystus  cavasius, Ompok  bimaculatus, and Mastacembelus armatus, respectively.

Procamallanus (Spirocamallanus) bilaspurensis comprises the following important characteristics: nematodes thick and small, with a transversely striated cuticle, oval mouth aperture present, covered by six flat crescentic elevations with two circles of eight submedian cephalic papillae, each formed by four papillae, including the external circle, which is distinctively larger than others. Buccal capsule looks yellowish, long, with s well-developed basal ring, lateral amphids small and present in a pair, capsule basal part thick, with inner surface of whole capsule shows many spiral thickenings, oesophagus muscular. Deirids small located in the anterior portion at the level of nerve ring. Excretory pore situated between nerve ring and posterior end of muscular oesophagus, both sexes have conical tails.

Procamallanus (Spirocamallanus) mysti encompasses worms having a thick transverse striated cuticle, a pinkish body colour, mouth large and opening of the mouth covered by a membrane ring, submedian cephalic papillae four in number, and two lateral amphids present. Buccal capsule barrel-shaped comprises two sets of spiral thickenings: with male have 17-19 fragmented ridges while females have 19- 21 fragmented ridges; a basal ring present at distal end of buccal capsule, oesophagus anterior end muscular, while the posterior end glandular. Nerve ring present at muscular oesophagus, deirids also present below the nerve ring with a small spike-like protuberance. Excretory pore, situated at the junction of the muscular and glandular oesophagus.

Sequences obtained in the present study for the 18S gene of Procamallanus species were aligned with other sequences downloaded from the GenBank database. The 18S rDNA phylogenetic reconstruction from ML and Bayesian inference (BI) analyses represented a well-supported Procamallanus lineage supported by high bootstrap and posterior probability values (97/1) for both the presently collected specimens (Figure 1). The phylogenetic tree inferred shows that both, Procamallanus (Spirocamallanus) mysti and Procamallanus (Spirocamallanus) bilaspurensis are sister species (Figure 1). They also share a main clade with P. laeviconchus from Sudan and P. pseudolaeviconchus from South Africa, and the relationships among these four species of Procamallanus received strong bootstrap support (97%) and Bayesian posterior probabilities (0.99) (Figure 1). P. (S.) mysti and P. (S.) bilaspurensis exhibit genetic divergence that averages 1.37%. Additionally, the genetic divergence between our isolates with Procamallanus laeviconchus (JF803934) and Procamallanus pseudolaeviconchus (OP602992- OP602994), ranged from 1.97 to 2.23%. The topology of the tree with another Procamallanus species from India, i.e., Procamallanus spiculogubernaculus (KU292357), represents independently in the tree and shows a genetic divergence 2.92% with our isolates (Figure 1).

Click to enlarge

Figure 1: Consensus maximum likelihood and Bayesian inference phylogenetic tree inferred with partial 18S rDNA sequences for species of Procamallanus. Numbers near internal nodes show ML bootstrap and probabilities (BI) values. GenBank accession numbers precede species names. Scale-bar indicates the expected number of substitution per site. Species sequenced in the present study are shown in bold.

Discussion

Of the family Camallanidae Railliet et al. [16] the genus Procamallanus Baylis, 1923, comprises a diverse group of parasites. However, Moravec, et al. [6, 17, 18, 19, 20] cogitated five taxa as subgenera of Procamallanus of the Procamallaninae: Procamallanus Baylis, 1923, Spirocamallanus Olsen (1952), Platicamallanus, Punctocamallanus Moravec et Scholz, 1991; and  Denticamallanus Moravec et al. [20].  Furthermore, Batrachocamallanus  is also considered a junior synonym of  Procamallanus by Moravec, et al. [20], as supported by Rigby, et al. [5]. Procamallanus Baylis, 1923 encompasses a large number of species infected freshwater and marine fishes [21]. From freshwater fishes of southern and south-eastern Asia, especially from India, many nominal species were described [22]. Although, many species of Procamallanus were frequently described from India, often with inadequate morphology, and without molecular data on them, so a thorough comparison of them is impossible [22, 23, 24]. This condition is more complicated when these nematodes are described from similar hosts, creates taxonomic confusions and rarely the type or voucher specimens for most species are available except few by Sood [22].

Although camallanid nematodes have different and sufficient morphological characters to identify them, the differentiation between species is complicated [5]. Though, the use of these characteristics between species is rather confused due to the presence of sexual dimorphism, taxonomy based on morphological structures like the buccal capsule is not stable and therefore requires revision [25, 26]. Consequently, these authors contemplated the subgenera Procamallanus “for practical reasons” [25], to which Rigby and Rigby [5] also diagnosed the Procamallanus subgenera “for the sake of tradition and simplicity”. Besides the buccal capsule, the other trustworthy character for identification was considered the male caudal region. Initially, studies described that camallanid nematodes possess only a right spicule, whereas later descriptions also mentioned that the left spicule is also present in species that were previously described with a right one [7, 20, 27]. The tail tip of adult females of this group of parasites comprises mucrons, and their presence and absence are also treated as reliable characters for identification [28].

According to Sood [22], from the host Mastacembelus armatus, the following species of Procamallanus are listed from India: P. bilaspurensis Gupta, et al. [10], P. fulvidraconis Li, 1935, P. gubernaculus Khera, 1955, and P. meszarosi Arya, 1984. Of these, P. fulvidraconis, P. gubernaculus, and P. kalriai species are in the category of species inquirenda by Sood [22], who predicted that these species were originally described based on misidentifications. The present nematodes collected from M. armatus belong to P. bilaspurensis, which comprises spiral ridges in the capsule, although these ridges are intervallic and not developed properly. The present specimens of P. bilaspurensis are placed in the subgenus Spirocamallanus Olsen, 1952, because of the presence of spiral ridges in the buccal capsule of the males and females, and mentioned as Procamallanus (Spirocamallanus) bilaspurensis [9, 25]. In case of  Procamallanus (Spirocamallanus) mysti Karve, 1952, comprises two unequal spicules, the left one is weakly sclerotized while the right spicule is well sclerotized, and distinctly visible. P.(S.) mysti differentiated from other congeners by having the number of spiral thickenings of buccal capsule and the number of caudal papillae in male while the number of mucrons in the female tail tips [21, 29].

Numerous molecular studies were published recently based on 18S rDNA gene data that included camallanid nematodes, but the phylogenetic relationships between the species and the status of different taxa within the family are still in question and not resolved [7, 30, 31, 32]. To date, in India, only a few molecular studies have been conducted that included very few species, therefore, in the present study, we follow  the genetic identification of the species, collected herein and their molecular characterization based on the 18S gene. Our phylogenetic analysis supported the presence of two different Procamallanus species: Procamallanus (Spirocamallanus) mysti and Procamallanus (Spirocamallanus) bilaspurensis, respectively. Phylogenetic analysis demonstrates that the two species studied here (P. mysti and P. bilaspurensis) are closely related, while another Indian freshwater species, P. spiculogubernaculus, occupies separate positions in the 18S based tree. However, a lack of molecular data from Indian camallanid species can influence the present phylogenetic position of the species in future studies. Thus, according to our opinion, all the species, whether they are previously well-known or new ones, should be supported by proper descriptions/illustrations, and molecular data.

Conclusion

Two species of the genus Procamallanus are clearly recognized in the Indian region using molecular data- Procamallanus (Spirocamallanus) mysti and Procamallanus (Spirocamallanus) bilaspurensis, and the phylogenetic analysis demonstrates that both species are closely related. Using an amalgamation of morphology and molecular data together is the only way to correct species identification with regard to Camallanid nematodes.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgements

We are grateful to the Head, Department of Zoology, D.A.V. College Muzaffarnagar, India and Chaudhary Charan Singh University, Meerut, Uttar Pradesh India, for providing laboratory facilities.

Funding

This research was funded by Research and Development Scheme of the Higher Education Department, Uttar Pradesh Government, India, grant number 93/2021/1401/ sattar-4-2021-4(28)/2021.