morphological and molecular evidence …fac.ksu.edu.sa/sites/default/files/42_lsh-lsh.pdf© by psp...

© by PSP Volume 29 – No. 01/2020 pages 633-639 Fresenius Environmental Bulletin

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MORPHOLOGICAL AND MOLECULAR EVIDENCE FOR

MYXOBOLUS TOYAMI AS AN EVOLUTIONARY LINK BETWEEN THELOHANELLUS AND MYXOBOLUS

GENERA OF MYXOZOAN PARASITES

Naireen Fariya1, Daoud Ali2,*, Rehana Abidi1, Fawaz A Falodah2, Saud Alarifi2, Saad Alkahtani2

1Parasitology Lab, Fish Health Management Division, National Bureau of Fish Genetic Resources, Lucknow, India 2Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia

ABSTRACT

Myxobolus toyamai is a gills parasite infecting

Cyprinus carpio in China, Japan and USA. It has been already known for the controversy for its posi-tion between Thelohanellus & Myxobolus. Recent-ly, it was confirmed as Myxobolus spp. after extru-sion of the polar capsule, which is an essential at-tribute for Myxozoan identification. The current article reports the presence of the Myxobolus to-yamai in India, through trans-boundary movement of fishes. The present article deals with the host specific tendency and evidence of the dispersion with the help of molecular analysis of the 18S gene, resemblance to other isolates from North America & Japan. In addition, the study also confirms the presence of the second polar capsule through mor-phological details and extrusion of two polar fila-ments which supports the species as Myxobolus spp.

KEYWORDS: Cyprinus carpio, Geographical, Myxobolus toyamai, India

INTRODUCTION Cyprinus carpio is a well-known freshwater

fish for its taste and extensive availability world-wide. Myxobolus toyamai is a myxozoan parasite infecting the gills of Cyprinus carpio [1]. The para-site has been recorded from Japan, Europe, Asia and North America but has not been reported from the common carp in India. Generally, there no spe-cific approach for combating and restricting the invasive species of myxozoans are available, as they become problematic for the fishes. Additional-ly, the parasite was in argument with its ambiguous classification as Thelohanellus toyamai [2, 3]. Nu-merous species of myxozoans has been poorly de-scribed which created ambiguity to host and geo-graphic distributions. Several workers have sug-gested that the additional molecular data can re-

solve the multiple taxa with the same scientific name [4, 5, 6, 7]. Griffin and Goodwin [8], reported the parasites as T. toyamai through phylogenetic study of the 18S rDNA with the explanation of monophyletic origin of Thelohanellus and Myxobo-lus. Nevertheless, Yokoyama and Ogawa [1], iden-tified and validated the M. toyamai as a Myxobolus species with the presences of the second small polar capsule. The present study discusses the evidences of host specificity, geographical distribution, mo-lecular and morphological study in support of the presence of the small stunted polar capsule of M. toyamai.

MATERIALS AND METHODS Morphology and Morphometry. Collection

of common carp, Cyprinus carpio was done from local cultured farms of Lucknow (Uttar Pradesh, India). Screening of total 104 outwardly healthy fish was done for detection of Myxozoans. Squash preparations of all the internal organs and gills were made and examined through a Nikon E600 micro-scope with 100X objective (plus immersion oil). Numerous myxozoan cysts were observed in the gills (Figure 1). Spores in fresh wet mount were treated with 10-12 % KOH solution for extrusion of polar filaments. Morphometric measurements of fresh spores (n=80) was done with the help of soft-ware NIS-E-Br.

FIGURE 1

Cyts of Myxobolus toyamai in the infected gills of Cyprinus carpio


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Molecular Analysis. DNA was isolated from spore’s samples (i.e. cysts collected from the gills of infected fish) through DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's proto-col. DNA concentration was measured through Nano-drop 2000 spectrophotometer (Thermo Scien-tific, USA). The 18SSU rDNA gene was amplified using polymerase chain reaction (PCR) in Eppen-dorf epgradientS Master Cycler (Eppendorf Inc., USA). Specific primer set MYXF-MYXR, Iwanowicz et al. [9] was synthesized by Sigma-Aldrich. Amplification of the PCR product was commenced with following condition, denaturation at 95oC for 5 min, followed by 35 cycles of 95oC for 20 sec; 58oC for 40 seconds; 72oC for 40 sec and final extension at 72oC for 10 min and rested at 4oC prior to sequencing. Amplicons were excised from the agarose gel electrophoresis. Purified PCR product was sequenced through the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1, using the ABI3730xl Genetic Analyzer (Ap-plied Biosystems, Inc.). Homology search was per-formed using BLAST NCBI database on Genebank [10] for determination of the sequence.

Phylogeny Criteria. Evolutionary analyses

were conducted in MEGA6 [11]. The evolutionary history was inferred by using three tree building methods, Maximum Likelihood method based on the Tamura-Nei model [12], Minimum Evolution method [13] and Maximum Parsimony method. The bootstrap consensus tree inferred from 2000 repli-cates is taken to represent the evolutionary history of the taxa analysed and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches [14] for all three methods. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. All posi-tions containing gaps and missing data were elimi-nated. The analysis involved 12 nucleotide se-quences and there were a total of 653 positions in the final dataset. Initial tree for the heuristic search for Maximum Likelihood method were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach (Figure 5). Whereas, the Maximum Parsimony (MP) tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Nei and Kumar, 2000) with search level 1 in which the initial trees were ob-tained by the random addition of sequences (10 replicates) (Figure 6). While the evolutionary dis-tances were computed using the Kimura 2-parameter method [15] and are in the units of the number of base substitutions per site (Figure 7). Furthermore, the Minimum Evolutionary (ME) tree was searched using the Close-Neighbor-Interchange (CNI) algorithm [16] at a search level of 1 with the neighbor-joining algorithm [17] to generate the

initial tree. Codon positions included were 1st+2nd+3rd+noncoding for both the method (MP and ME), respectively.

RESULTS Myxobolus toyamai. Total 104 numbers of

Cyprinus carpio were screened. Out of which 2 fishes were infected with the spore. Thus, preva-lence of the infection was 1.92%. Minute white cysts were observed in the gills (Figure 1). The cysts measured 0.11-0.18 mm. Number of cysts in each fish gill filament was about 5-10. Initially, microscopic study of cysts revealed that parasite belongs to genus Thelohanellus having single polar capsuleat the anterior of the spore body and a tiny mass [8].

Subsequently, spores on higher magnification were showed, the parasite was Myxobolus species as the extrusion of the polar filament from the smaller capsules was observed (Figure 2 a, b, c). Spores were drop-shaped with rounded posterior baseand tapering towards anterior side, ending into a beak like pointed anterior end, slightly curved at one side of the shell valve (Figure 3), measuring 14.01-16.41(15.21±0.52) µm long and 5.02-7.84 (6.06±0.52) µm wide in frontal view. Ratio of the spore (Length of the Spore/Width of the Spore) was 2.51 (Figure 4). Additionally, the spore was 7.08 µm thick in sutural view. Polar capsules were une-qual as pyriform shape measuring the larger polar capsule 4.98-7.60 (5.94±0.57)µm long and 2.28-3.79 (2.90±0.36) µm wide. While smaller polar capsule seems like a tear shaped black structure attached with the anterior tip of the spore measuring 2.43-3.59 (2.83±0.27) µm long and0.53-1.04 (0.88±0.13)µm wide. Length of extruded larger polar filament was 26.05-106.27 (65.03±20.26) µm. Both polar capsules were connected with a fine sutural line at the tip of the spore (Figure 3). Sporo-plasm consists of homogenous granules with 2-3 small sporoplasmic nuclei (Figure 2).

Molecular analysis and phylogeny. To au-

thenticate the morphological results phylogenetic studies were performed. For the confirmation of the identity, resultant 702 bp partial sequence was de-posited to GenBank and accession no. MG800830 was obtained. The consensus sequence of 702 bp was BLAST and showed 99% nucleotide identity with Myxobolus toyamai (LC010116, LC010115), Thelohanellus toyamai (HQ338729), Myxobolus toyamai (FJ710802) and 98% nucleotide identity to M. longisporus (AY364637), Myxobolus pseudoa-cinosus (KX58668), Myxobolus acinosus (KX810022). The resultant tree showed that present Myxobolus toyamai from Cyprinus carpio makes the sister clade with other M. toyamai. Evolutionary relationships displayed that all toyamai species


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were clustered within the clade while other were nested as sister clade. The topology of all 3 phylo-genetic trees inferred were M. toyami is closely related and clusters in sister taxa to other Myxobo-

lus species (Figure 5-7). The out group Henneguya cutanea was attached with the sister branch of ge-nus Thelohanellus namely T.hovorkai, T. kitauei, T. rohitae.

FIGURE 2

(A)Mature Spores of Myxobolus toyamai with coiled stunted polar capsule. (B) Mature same spore with extruded filament from stunted polar capsule of Myxobolus toyamai.

(C) Spores of Myxobolus toyamai with extruded polar filaments.

(A) (B)

FIGURE 3 (A) Mature Spores of Myxobolus toyamai with coiled polar capsule connected with the fine sutural line.

(B) Line diagram of mature spore of Myxobolus toyamai.

FIGURE 4

Histogram showing Spore Length & Width of Myxobolus toyamai

Len

gth

& w

idth

of

Spor

e.

Number of spore.

Length & Width of Spore. Len…Width


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FIGURE 5

Maximum Likelihood tree of the 18S ribosomal DNA sequence of Myxobolus toyamai from Cyprinus carpio (Highlighted in box) with selected myxosporeans.

Numbers at the nodes indicated bootstrap confidence values (2000 replication). GenBank accession numbers are mentioned for each representative species.

FIGURE 6

Maximum Parsimony tree of the 18S ribosomal DNA sequence of Myxobolus toyamai from Cyprinus carpio (Highlighted in box) with selected myxosporeans.


FIGURE 7

Minimum Evolutionary tree of the 18S ribosomal DNA sequence of Myxobolus toyamai from Cyprinus carpio (Highlighted in box) with selected myxosporeans.



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Geographical dissemination of population. The present M. toyamai has demonstrated the transmission of infection and host specificity worldwide (Figure 8). The occurrence of M. to-yamai from Asia to Europe with a surprising con-stancy in the gene flow and genetic uniformity shows remarkable ability to sustain with the specif-ic host and adaptable to all environmental condi-tions.

Presumably, the dissemination pathway of the parasite is from China (Chongqing) to Japan (Wa-kayama Prefecture & Saku); USA (North Carolina) and India (Lucknow) (Fig. 4). This confirms the adaptive trait of the population survival in the eco-logical niches. However, the estimation of genetic variation with the number of segregating sites and the average number of nucleotide differences was analysed with Tajima’s neutrality test of nucleotide displaying the positive D-value 0.453496. The posi-tive D value signifies the low levels of both high and low frequency of polymorphisms, indicating balancing selection.

DISCUSSION Current studies reveal the presence of Myxo-

bolus toyamai from Cyprinus carpio in India for the first time M. toyamai is already reported from Ja-pan, China and North America but at present is

found in India. The smaller population having no genetic alteration results contraction in population size but existence of M. toyamai in aquatic ecosys-tem proves balancing selection. However, the pre-sent scenario of M. toyamai have slight difference in spore dimension (sizes) which demonstrates that where a species can and cannot maintain population according to their relation to current geographic distributions and environmental factors. Further-more, the availability of the spores in different geo-graphical locations shows there dispersal capabili-ties possibility of (the invasion), and robustness of invading species but host specificity is still complex process. Conversely, this type of myxozoan can be allogenic (transit between two types of ecosystems during their life system) in nature having a defini-tive host. Dispersal of the myxozoan spore is relat-ed with a number of conditions through movement of single or multiple hosts [18]. Hoffman [19], re-ported that rainbow trout from North America was infected with the spread of infected brown trout from Germany. Additionally, El-Matbouli et al. [20], proposed dispersal of spore through vagile predators as a significant element and ultimate ex-tent of geographical dissemination.It is accepted that the abundance and distribution of the fish through anthropogenic movement offers spread of myxozoan parasites from one locality to other [21, 22, 23].

FIGURE 8

Geographical distribution of Myxobolus toyamai from Cyprinus carpio with first date reported.


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Furthermore, the present article also supports the species belongs to the genus Myxobolus, having a large and short (stunted) polar capsule with two extruded filament, evidently. Various discrepancies can arise from unaccompanied morphological stud-ies [4, 5, 6], but occasionally lack of the observa-tion can lead to misapprehension. However, Myxo-bolus toyamai misidentified as Thelohanellus to-yamai by Griffin & Goodwin [8]. Further the spe-cies was screened and identified as Myxobolus spp with the help of detailed description by Yokoyama and Ogaw, [1]. Although, the current M. toyamai is slightly bigger than previous M. toyamai morpho-metrically but the tissue and host specificity was correlated with the previous descriptions. Apparent-ly, the differences in spore size dimension can be a probability of change in the climate and environ-ment or it can be a morphotype of the same species. Although, shape and size plasticity is widely re-ported in myxozoans [24], while Qingxiang et.al. [25] recommended existence of morphotypes and intraspecific morphometric variation among myxo-zoan population. In addition, host specificity with a definitive host is a allogenic nature of myxozoan while they are autogenic i.e. spending their whole life in one type of ecosystem (typically aquatic) was described by Cone et al. [26] for the infracommuni-ty structure of myxozoan in Fundulus diaphanous. Whereas, the genetic study supported previous iso-lates with the homology to the previous sequences indicating subtle alterations. These genetic differ-ences might be attributable to environmental effects [1]. While, Jones et. al. [27] reported that pathogens from cultured fish spilled-over to wild populations to produced genetic exchanges causing genetic var-iations. However, processes of parasite dispersion in natural habitat are random in pattern and com-plex process. Nevertheless, it’s concluded that pres-ence of M. toyamai in India is emergence of an ex-otic species and it has capability to sustain in dif-ferent condition so it might be trans-boundary movement of fishes.

ACKNOWLEDGEMENTS The work was also supported by Researchers

Supporting Project number (RSP-2019/27), King Saud University, Riyadh, Saudi Arabia. The authors acknowledge the Director, National Bureau of Fish Genetic Resources, Lucknow, for providing the necessary research facility.

All the data (tables and figures) used to sup-port the findings of this study are included within the article and the detail will be provided on request due to the registration of petty patent and the tech-nology transfer agreement.

All contributing authors declare no conflicts of interest.

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Received: 02.10.2019 Accepted: 25.10.2019 CORRESPONDING AUTHOR Daoud Ali Department of Zoology College of Science King Saud University BOX 2455 Riyadh 11451 – Saudi Arabia e-mail: [email protected]

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