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Hepatic Microsporidiosis of Mudskipper,
Boleophthalmus DussumieriValenciennes, 1837
(Perciformes: Gobiidae), Due to Microgemma Sp.
V.R Vandana
ICAR-CIFE: Central Institute of Fisheries Education
Nalini Poojary
ICAR-CIFE: Central Institute of Fisheries Education
Gayatri Tripathi
ICAR-CIFE: Central Institute of Fisheries Education
Pavan-Kumar A
ICAR-CIFE: Central Institute of Fisheries Education
N.K. Sanil
Central Marine Fisheries Research Institute
Rajendran Kooloth Valappil ( rajendrankv@hotmail.com )
Central Institute of Fisheries Education https://orcid.org/0000-0002-2987-5236
Research Article
Keywords: Microsporidia, Microgemma, Parasite, Fish, Mudskipper, Boleophthalmus
DOI: https://doi.org/10.21203/rs.3.rs-344161/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
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Abstract
The present study reports a case of hepatic microsporidiosis caused by
Microgemma
sp in brackishwater
sh,
Boleophthalmus dussumieri
(Valenciennes, 1837) (n = 60), from north-west coast of India. An eight-
month study from September 2017 to April 2018 revealed a prevalence of 11.6% for this parasite. The
microsporidian showed tissue-specic infection and did not reveal any gross pathology in infected sh.
Large whitish cysts containing microspores of size 0.3–0.5 mm were observed in the liver of sh. The
range of pyriform microsporidian spore size varied from 2.9–3.77 X 1.85–2.67 µm. Histological
observations of infected liver revealed large xenoma of the microsporidian lled with spores and
encircled by a cyst wall-like layer. 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) amplication
of the DNA extracted from the microsporidian spores 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 rst report of a microsporidian from
B. dussumieri
and the rst record of the
genus
Microgemma
from India.
Introduction
Microsporidia are a diverse group of obligate intracellular, spore-forming parasites that infect a wide-
range of hosts, including insects, shes and humans (Dean et al. 2016; Mansour et al. 2020). Among
these, sh is the most common vertebrate host for microsporidia and the infection could cause
signicant losses to sheries (Dyková 2006; Abdel-Ghaffar et al. 2011). Some of the microsporidians
induce hypertrophic growth of host cells, a well-organized xenoparasitic complex (XC) referred to 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 sh 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 sh is the Mudskippers (Gobiidae); these diverse
species of amphibious teleosts inhabit swamps, estuaries, mudats, 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. Further, as more species diversication 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.
Page 3/14
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 sh. The present study provides
information on spore morphology, morphometrics as well as gross and histological 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 rst report of a microsporidian infecting mudskippers.
Materials And Methods
Sampling
Live
B. dussumieri
(
n
=60) were collected 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
sh (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 identication 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://sheries.org/docs/policy_useoshes.pdf.).
Initially, gross observations were carried out under a stereomicroscope to nd out the presence of any
ectoparasites, external lesions, discoloration, haemorrhage or cysts. Subsequently, all the external and
internal organs of the sh were examined for the presence of parasites. Microsporidian cysts found in the
liver were carefully removed, placed on a clean glass slide in physiological saline, ruptured with ne
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, xed 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).
Histology
The infected tissues were xed in neutral buffered formalin (NBF) for 24-72 h and washed thoroughly to
remove the xative. The tissues were dehydrated in an ascending series of alcohol followed by acetone
and cleared in xylene. Paran inltration and embedding of processed tissues were carried out using a
histoembedder (LEICA EG 1140C, Germany). Tissue sections of 3-5 µm thickness were made using a
rotary microtome (LEICA RM2125RT, Germany) and stained with Harris haematoxylin and eosin. The
sections were dehydrated through different grades of alcohol and acetone. Xylene was used for clearing
and sections were mounted in DPX (Sigma-Aldrich, USA).
Scanning electron microscopy
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For scanning electron microscopy, microsporidian cysts were xed in 2.5% glutaraldehyde in 0.2 M
sodium cacodylate buffer. The cysts were xed 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 microsporidian-infected liver tissue of sh using DNAzol
(Invitrogen®) kit following the manufacturer’s instructions. The small subunit ribosomal DNA (SSU rDNA)
of microsporidian spores was amplied 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 nal 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 Scientic®, 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 Scientic®, India) and the insert was
reconrmed by PCR amplication with microsporidian-specic primers. The conrmed plasmid DNA was
further sequenced in both directions using the same microsporidian-specic primers by a commercial
company (Xcelris Labs, Ahmedabad, India). The quality of each sequence was veried 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
Page 5/14
Gross examination of sh
A total of 60
B. dussumieri
were collected from brackishwater system located around Pancham
Aquaculture Farms, Maharashtra. Gross examination of the sh did not show any abnormalities or
lesions.
Microgemma
sp.
A microsporidian infection was observed in the liver with a prevalence of 11.6% (7 out of 60 shes).
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 2.9-3.87 (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).
Histopathology and ultrastructural observations
Infected liver tissues were subjected to histological observation. The parasite showed strict tissue-
specicity, as cysts were noticed only in the liver of
B. dussumieri
. Infected liver tissues were subjected to
histological observation. Large xenomas surrounded by distinct layer/wall were observed in the
histological section (Fig. 2 a). Multiple xenomas were frequently observed. Granuloma formation was not
observed in any of the tissue sections examined. A large number of spores were noted inside the xenoma
(Fig. 2 b). Ruptured microsporidian cyst under a scanning electron microscope revealed numerous spores
attached to the cyst wall (Fig. 3 a-d). Mature microsporidian spores showed a distinct groove at the
anterior end of the spore for polar tube extrusion (Fig. 3 e). Many spores also revealed a prominent
ridge/fold-like structure on one side of the spore wall (Fig. 3 f).
Molecular and Phylogenetic analysis
Polymerase chain reaction (PCR) amplication of the DNA extracted from the microsporidian spores
using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~1340 bp amplicon (Fig. 4).
The PCR-amplied 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 33 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
Page 6/14
distance value (Kimura 2 parameter model) of 0.2% (nucleotide difference of 2) with
M
.
tilanpasiri
(Table
1)
.
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 signicant bootstrap
value (Fig. 5). 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
.
Discussion
This is the rst report of the microsporidium,
Microgemma
sp. from
Boleophthalmus dussumieri
and the
rst 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 X 2.4 µm of the genus
Microgemma
and 2.9–3.87 X 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 shes.
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 Table1.
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 sh 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). Histological observations revealed large, multiple xenomas surrounded by a
distinct layer. Previous studies on
M. tilanpasiri
infection in
T. vagina
have also reported similar
observations as in the present study (Freeman et al. 2015). Granuloma formation and extensive necrosis
in host shes have been reported in response to
M. caulleryi
(Leiro et al. 1999) and
M. tilanpasiri
(Freeman et al. 2015) infections. However, such pathological changes were not observed in the present
study. This could be attributed to the fact that a symbiotic co-existence might have developed between
the host cell and the microsporidian parasite leading to the formation of the xenoparasitic complex as
observed by Lom and Dyková (2005).
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 rst detailed SEM description of a
Microgemma
sp.
Page 7/14
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 sucient genetic
variation among closely related species of microsporidia that infect shes (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 brevilum
(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 signicant bootstrap. This
clade corresponds to group IV of the classication 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 anity to
M
.
tilanpasiri
. Hence, the present species can be
considered as
Microgemma
aff.
tilanpasiri
, a species with close anity 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 histopathology, molecular sequencing and phylogenetic analysis, the
present study identies and describes a new record of
Microgemma
aff.
tilanpasiri
infecting the hepatic
tissue of the brackishwater sh,
Boleophthalmus dussumieri
Valenciénnes, 1837, from India.
Declarations
Acknowledgments The authors are thankful to the Director, ICAR-CIFE, Mumbai, India, and the Director,
ICAR-CMFRI, Kochi, India, for providing the facilities. The rst author is grateful to a fellowship support by
the Indian Council of Agricultural Research.
Author’s contribution: The study was designed and guided by Rajendran K.V. and Sanil N.K. Vandana V.R.
carried out the study in detail. The manuscript was written by all the authors.
Compliance with ethical standards: All applicable institutional, national and international guidelines for
the
care and use of animals were followed in the present study.
Conict of interest: The authors declare that there is no conict of interest or competing interests.
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Page 8/14
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Tables
TABLE 1 Comparison of the present species of
Microgemma
with previously reported species
Page 11/14
Species Host The site
of
infection
locus
Spore
dimension
(µm)
Country/
Region Reference
Microgemma
hepaticus Chelon labrosus
Liver 4.2 X 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 x 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 X 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 X 2.1 United
Kingdom Canning
et al.,
2005
M. carolinus Trachinotus
carolinus
Liver 3.8 X 2.4 Brazil Casal et
al., 2012
M. tilanpasiri Trypauchen vagina
Liver 3.92 X 2.87 Malaysia Freeman
et al
.
,
2015
M.
aff.
tilanpasiri Boleophthalmus
dussumieri
Liver 2.9-3.77
X1.85-2.67
(mean,3.25 X
2.08)
India Present
study
Figures
Figure 1
Page 12/14
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
Figure 2
Histological section of infected liver a) xenoma (arrow) observed in the hepatic tissue; b) enlarged view of
the xenoma showing spores (arrowhead) (H&E).
Figure 3
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
Page 13/14
Figure 4
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.
Page 14/14
Figure 5
Neighbour-Joining tree of selected microsporidians. Labels on the nodes represent bootstrap values.
Branch length shows the divergence between the species.
