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. 2021 Jul 28;46(1):72–79. doi: 10.1007/s12639-021-01419-5

Hepatic microsporidiosis of mudskipper, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), due to Microgemma sp.

V R Vandana 1, Nalini Poojary 1, Gayatri Tripathi 1, Pavan Kumar 1, N K Sanil 2, K V Rajendran 1,✉
PMCID: PMC8901909  PMID: 35299917

Abstract

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp. in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from the west coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.7% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Small whitish cysts containing microspores of size 0.3–0.5 mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9–3.77 × 1.85–2.67 µm. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

Keywords: Microsporidia, Microgemma, Parasite, Fish, Mudskipper, Boleophthalmus

Introduction

Microsporidia are a diverse group of obligate intracellular, spore-forming parasites that infect a wide range of hosts, including insects, fishes and humans (Dean et al. 2016; Mansour et al. 2020). Among these, fish is the most common vertebrate host for microsporidia and, the infection could cause significant losses to fisheries (Dyková 2006; Abdel-Ghaffar et al. 2011). Some of the microsporidians induce hypertrophic growth of host cells, a well-organized xenoparasitic complex (XC) referred as xenoma (Lom and Dyková 2005). Currently, seven species of Microgemma, namely M. carolinus, M. hepaticus, M. ovoidea, M. tincae, M. vivaresi, M. caulleryi, and M. tilanpasiri have been reported from various fish hosts (Freeman et al. 2015). However, the pathogenic potential of many microsporidians has not been studied as the hosts of these species have relatively low economic value and hence received little attention (Gómez et al. 2014). One such group of fish is the mudskippers (Gobiidae); these diverse species of amphibious teleosts inhabit swamps, estuaries, mudflats, intertidal habitats and mangrove ecosystems. Boleophthalmus dussumieri Valenciénnes, 1837 is one of the most abundant species of mudskippers distributed along the north-west coast of India (Murdy 1989). The mudskippers play an important role in benthic ecology and have been recognized as potential bio-indicators for environmental monitoring (Ansari et al. 2014). The species of mudskippers (eg. Pseudapocryptes elongatus) have been cultured in South-Asian countries and have good demand in the domestic market (Minh et al. 2010). Further, as more species diversification is expected in aquaculture and new potential species such as mudskippers can be brought into culture, diseases caused by parasites such as microsporidians can emerge as a potential threat.

In this background, an investigation was carried out to study the prevalence of parasitic infection in B. dussumieri and we observed a microsporidian infection in the liver of the fish. The present study provides information on spore morphology, morphometrics as well as gross evidence of Microgemma sp. infection in B. dussumieri. The scanning electron microscopic features of spores along with molecular sequence information and phylogenetic relation of the species are also provided. As far as is known, this forms the first report of a microsporidian infecting mudskippers.

Materials and methods

Sampling

A total of 60 live B. dussumieri were collected in September (n = 20), November 2017 (n = 20) and in April 2018 (n = 20) from a brackishwater area located around Pancham Aquaculture Farms, (19°31′32.92’’N and 72°47′57.83’’E), Saphale, Palghar district, Maharashtra, India. The fish (mean length = 11.8 ± 3.17 cm; range = 7.3–17.2 cm) were transported live to the Aquatic Animal Health Laboratory, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, Maharashtra, India for parasitic examination.

Parasitic examination and identification of microsporidian

Fish were killed by pithing without any tissue damage, after immobilizing them on ice for adequate time. This method was as per the accepted guidelines (https://fisheries.org/docs/policy_useoffishes.pdf.). Initially, gross observations were carried out under a stereomicroscope to find out the presence of any ectoparasites, external lesions, discoloration, haemorrhage or cysts. Subsequently, all the external and internal organs of the fish were examined for the presence of parasites. Whitish cysts of the microsporidian parasite were noticed in liver. Besides, a new myxozoan parasite, Ellipsomyxa boleophthalmi was observed in the gall bladder (Vandana et al. 2021). The microsporidian cysts found in the liver were carefully removed, placed on a clean glass slide in physiological saline, ruptured with fine needles, mounted with a clean cover glass, and observed under a phase-contrast microscope. The spores were treated with 1–2% KOH to observe the polar tube extrusion. Smears of the infected tissues were air-dried, fixed in methanol, and stained with Giemsa stain. Photomicrographs of fresh and stained materials were taken using a research microscope (Nikon eclipse 80i, Japan) with image capture software (NIS elements BR, Nikon, Japan).

Scanning electron microscopy

For scanning electron microscopy, microsporidian cysts were fixed in 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer. The cysts were fixed in 1% osmium tetroxide after washing in cacodylate buffer. Subsequently, after dehydration through graded acetone series, the cysts were transferred to isoamyl acetate and then critical point dried using a Hitachi HCP-2 Critical Point dryer (Hitachi, Japan). The dried cyst was cut open and then mounted on the SEM stub, using an adhesive carbon tape, so as to expose the inner surface of the cyst. Further, it was sputter-coated with gold using Quorum SC76220 mini sputter coater (Quorum Technologies, UK). The processed cyst with microspores was observed and photomicrographs were taken using a TESCAN VEGA 3 scanning electron microscope (TESCAN, Brno, Czech Republic).

Molecular analysis

The total genomic DNA was extracted from the microsporidian-infected liver tissue of fish using DNAzol (Invitrogen®, USA) kit following the manufacturer’s instructions. The small subunit ribosomal DNA (SSU rDNA) of microsporidian spores was amplified using reported primers MicroSSUF: 5’-GGTTGATTCTGCCTGACGT-3’ and MicroSSUR: 5’-GACGGGCGGTGTGTACAAAG-3’ (Baker et al. 1994; Pomport-Castillon et al. 1997). The PCR reaction was carried out in a 25 µL reaction volume with 100 ng of template DNA, 10 mM of dNTP, 10 pmol of each primer, 5 U of Taq DNA polymerase and 1 × Taq buffer with 1.5 mM MgCl2, (Invitrogen®, USA). The thermal conditions for PCR were as follows: 95 °C for 5 min, 30 cycles of 94 °C for 1 min, 62 °C for 1.5 min, 72 °C for 2 min; followed by 72 °C for 10 min final extension. PCR products were visualized on 2% agarose-TAE gel containing 0.5 μg mL¯1 ethidium bromide. The desired PCR amplicon was extracted from the gel using a gel extraction kit and cloned into pTZ57R/T vector using T4 DNA ligase (Thermo Scientific®, India). The recombinant plasmid was transferred into Escherichia coli (DH5α) and the positive clones were selected by blue-white colony selection. The plasmid was isolated using GeneJET® plasmid extraction kit (Thermo Scientific®, India) and the insert was reconfirmed by PCR amplification with microsporidian-specific primers MicroSSUF and MicroSSUR. The confirmed plasmid DNA was further sequenced in both directions using the same microsporidian-specific primers by a commercial company (Xcelris Labs, Ahmedabad, India). The quality of each sequence was verified by the Phred score (q value) of each nucleotide using Finchtv software. The sequences were subjected to BLAST (Basic Local Alignment Search Tool) analysis with NCBI ‘nr’ database and the sequences with more than 85% similarity were downloaded to estimate the genetic divergence values. Kimura 2 parameter model implemented in MEGAX (Kumar et al. 2018) was used to estimate the genetic distance values. JModeltest was used to assess the best evolutionary model (Posada 2008). Based on the Akaike Information Criterion (AIC), the Transitional model with rate variation among sites (TIM3 + G) was selected as the best model to reconstruct the phylogenetic tree using Maximum likelihood and Bayesian Inference. Maximum likelihood and Parsimony methods were used to reconstruct the phylogenetic trees using PAUP software (Swofford 2003). Bayesian inference was also implied to deduce the phylogenetic tree using MrBayes (Ronquist and Huelsenbeck 2003).

Results

Gross examination of fish

A total of 60 B. dussumieri were collected from brackishwater system located around Pancham Aquaculture Farms, Maharashtra. Gross examination of the fish did not show any abnormalities or lesions. However, on internal examination of the fish, a microsporidian infection was observed in the liver. The overall prevalence of the microsporidian infection observed in the liver of B. dussumieri was 11.7% (7 out of 60 fishes). The microsporidian infection was not seen among 20 fishes sampled in September 2017. However, the prevalence was observed in subsequent samplings made during November 2017 (3 out of 20 fish) and April 2018 (4 out of 20 fish). Gross examination of the liver revealed the presence of numerous whitish, round to oval macroscopic cysts (xenoma) 0.3–0.5 mm in diameter. The cysts were either present on the surface or deeply embedded in the liver tissue (Fig. 1 a). Cysts, when ruptured, released numerous microsporidian spores (Fig. 1 b). Fresh spores were pyriform, in the size range of 2.9–3.77 (3.25) X 1.85–2.67 (2.08) µm (Fig. 1 c). The posterior vacuole was seen occupying the posterior third of the spore (Fig. 1 d). A partially extruded polar tube was also observed (Fig. 1 e). Spores stained with Giemsa’s showed the distinct pyriform shape with posterior vacuole (Fig. 1 f).

Fig. 1.

Fig. 1

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a Whitish microsporidian cysts found in the liver tissue of the mudskipper, B. dussumieri observed under a stereomicroscope; b fresh preparation of spores released from the cysts; c enlarged view of spores; d spore showing posterior vacuole (arrow); e spores showing extruded polar tube (arrowhead); f spores stained with Giemsa’s stain

Ultrastructural observations

Infected liver tissues were subjected to ultrastructural observations. Ruptured microsporidian cyst under a scanning electron microscope revealed numerous spores attached to the cyst wall (Fig. 2 a-d). Mature microsporidian spores showed a distinct groove at the anterior end of the spore for polar tube extrusion (Fig. 2 e). Many spores also revealed a prominent ridge/fold-like structure on one side of the spore wall (Fig. 2 f).

Fig. 2.

Fig. 2

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Scanning electron microscopy of spores. a Spores found in the ruptured cyst (arrowhead); b enlarged view of spores attached to the cyst wall; c free spores; d enlarged view of spores. e enlarged view depicting the polar tube extrusion pore (arrow), f enlarged view showing ridge/fold-like structure on the spore wall

Molecular and phylogenetic analysis

Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon (Fig. 3). The PCR-amplified products were sequenced and almost complete SSU rDNA (1269 bp) was sequenced from Microgemma sp. and submitted to GenBank (accession number of MN733420). The sequence similarity analysis using Basic Local Alignment Search Tool (BLAST) with NCBI GenBank database showed ~ 99.8% sequence similarity with M. tilanpasiri (KJ865404) reported from Trypauchen vagina. A total of 26 sequences with more than 85% sequence similarity with the present species were downloaded to reconstruct the phylogenetic tree. Alignment and subsequent trimming resulted in a uniform length of 1219 bp. The number of conserved and variable nucleotides is 752 and 467, respectively. Among the variable nucleotides, 300 nucleotides were parsimony informative. The present species showed a genetic distance value (Kimura 2 parameter model) of 0.2% (nucleotide difference of 2) with M. tilanpasiri.

Fig. 3.

Fig. 3

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Agarose gel electrophoresis of the PCR product. PCR yielded approximately 1340 bp product. Lane M. 100 bp plus molecular weight marker (Fermentas). Lane 1–6 DNA from infected liver

The tree topologies reconstructed by different methods were similar and in the consensus phylogenetic tree, the species of Microgemma clustered with M. tilanpasiri as a single clade with significant bootstrap value (Fig. 4). Further, this group emerged as a sister clade to M. carolinus with moderate bootstrap value. Few species of Sprageua sp. (GenBank accession number AB623034 & JQ820238) clustered within the Tetramicridae family. Family Spragueidae formed a sister group to Tetramicridae.

Fig. 4.

Fig. 4

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Consensus phylogenetic tree using 18S rDNA sequences of selected Microsporidian species. GenBank accession numbers are given in parenthesis. Nodal supports are indicated for MP, ML with a bootstrap of 100 replicates, and Bayesian inference with posterior probabilities, respectively

Discussion

This is the first report of the microsporidium, Microgemma sp. from Boleophthalmus dussumieri and the first record of the genus Microgemma from India. The present Microgemma sp. showed close similarity with the diagnostic features described for the genus Microgemma (Ralphs and Matthews 1986). The resembling features are: pyriform spore shape; posterior vacuole occupying the posterior third of the spore; spore dimensions (4.2 × 2.4 µm of the genus Microgemma and 2.9–3.77 × 1.85–2.67 µm of the present microsporidium); sporogonial development seen within a whitish spherical xenoma (host-parasite complex) in the liver; parasitic in marine fishes.

To date, this genus contains seven species reported from different parts of the world. All the species are known to infect liver except M. vivaresi which has been reported to infect both liver and skeletal muscles (Canning et al. 2005). A comparative account of all the Microgemma species reported along with the present species is given in Table 1.

Table 1.

Comparison of the present species of Microgemma with previously reported species

Species Host The site of infection locus Spore dimension (µm) Country/Region References
Microgemma hepaticus Chelon labrosus Liver 4.2 × 2.4 United Kingdom Ralphs and Matthews 1986
M. ovoidea

Motella tricirrata, Cepola rubescens, C. macrophthalma,

Merluccius hubbsi,

M. barbatus, M. gayi, M. hubbsi

 Liver 3.8 × 1.97

Mediterranean Sea,

Atlantic coast

(France),

Peru and Patagonia

(Argentina)

 Canning and Lom 1986; Amigó et al. 1996
M. caulleryi Hyperoplus lanceolatus Liver 2.6 × 1.2

Atlantic coast

(France, and Spain)

 Leiro et al. 1999
M. tincae Symphodus tinca Liver 3.6 × 1.2 Tunisian coast Mansour et al. 2005
M. vivaresi Taurulus bubalis

Liver and

Skeletal Muscle

 3.6 × 2.1 United Kingdom Canning et al. 2005
M. carolinus Trachinotus carolinus Liver 3.8 × 2.4 Brazil Casal et al. 2012
M. tilanpasiri Trypauchen vagina Liver 3.92 × 2.87 Malaysia Freeman et al. 2015
M. aff. tilanpasiri Boleophthalmus dussumieri Liver 2.9–3.77 × 1.85–2.67 (mean,3.25 × 2.08) India Present study
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The morphometric values of the present species were found to be closer to M. vivaresi and M. tilanpasiri. However, the species has been recorded from a different host and different geographical location. There were no gross signs of the microsporidian infection in infected fish in the present study. This is in accordance with the previous observations made in M. tincae (Mansour et al. 2005) and M. tilanpasiri (Freeman et al. 2015).

Under the scanning electron microscope, numerous spores were seen attached to the cyst wall and mature spores were observed to have a distinct groove on the anterior end of the spore for polar tube extrusion and a ridge/fold-like structure on the spore wall. Though M. caulleryi spores were studied using SEM, there was no clear description of the surface morphology of the spores (Leiro et al. 1999). As far as is known, the present study forms the first detailed SEM description of a Microgemma sp.

Molecular data, particularly small subunit ribosomal DNA, have been used to study the microsporidian phylogeny (Baker et al. 1995; Cheney et al. 2000; Kent et al. 1999; Moser et al. 1998; Nilsen 2000; Bell et al. 2001). Lom and Nilsen (2003) stated that the level of genetic variation between closely related species of microsporidians varies as per the host group. Several reports showed a lack of sufficient genetic variation among closely related species of microsporidia that infect fishes (Nilsen et al. 1998; Cheney et al. 2000; Casal et al. 2012; Freeman et al. 2015). The sequence of the present species of Microgemma (1269 bp) showed high genetic similarity (~ 98.9–99.8%) and less genetic divergence value with M. tilanpasiri (0.2%), M. carolinus (0.7%), and M. vivaresi (1.1%). Several previous studies have also reported low divergence values between M. carolinus and M. tilanpasiri (0.7%), M. tincae and M. vivaresi (0.7%), and M. caulleryi and Tetramicra brevifilum (0.3%) (Freeman et al. 2015; Casal et al. 2012). This could be due to the recent evolution of the species and subsequently less divergence time from their most recent common ancestor. However, accurate species delimitation relies on the occurrence of high genetic distance value (minimum 2%) between species.

In the phylogenetic tree, the present species clustered with M. tilanpasiri with significant bootstrap. This clade corresponds to group IV of the classification reported by Lom and Nilsen (2003). Although the present species of Microgemma displayed unique morphological and morphometric features in the new host, molecular sequence data showed a high affinity to M. tilanpasiri. Hence, the present species can be considered as Microgemma aff. tilanpasiri, a species with close affinity to M. tilanpasiri. However, more molecular markers (large subunit ribosomal DNA and ITS) are required for further resolution of these recently evolved microsporidian species. In conclusion, based on the light and scanning electron microscopic studies together with molecular sequencing and phylogenetic analysis, the present study identifies and describes a new record of Microgemma aff. tilanpasiri infecting the hepatic tissue of the brackishwater fish, Boleophthalmus dussumieri Valenciénnes, 1837, from India.

Acknowledgements

The authors are thankful to the Director, ICAR-CIFE, Mumbai, India, and the Director, ICAR-CMFRI, Kochi, India, for providing the facilities. The first author is grateful to a fellowship support by the Indian Council of Agricultural Research.

Author’s contribution

The study was designed and guided by KVR and NKS. VRV carried out the study in detail. The manuscript was written by all the authors.

Declarations

Conflict of interest

The authors declare that there is no conflict of interest or competing interests.

Human and animal rights

All applicable institutional, national and international guidelines for the care and use of animals were followed in the present study.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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