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Hepatic Microsporidiosis of Mudskipper, Boleophthalmus Dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), Due to Microgemma Sp.


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The present study reports a case of hepatic microsporidiosis caused by Microgemma sp in brackishwater fish, 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-specific infection and did not reveal any gross pathology in infected fish. Large 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 X 1.85–2.67 µm. Histological observations of infected liver revealed large xenoma of the microsporidian filled 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) amplification 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 first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.
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Hepatic Microsporidiosis of Mudskipper,
Boleophthalmus DussumieriValenciennes, 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 ( )
Central Institute of Fisheries Education
Research Article
Keywords: Microsporidia, Microgemma, Parasite, Fish, Mudskipper, Boleophthalmus
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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The present study reports a case of hepatic microsporidiosis caused by
sp in brackishwater
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-specic 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) amplication
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
clustered with
M. tilanpasiri.
Even though, the morphomeristic characters of the present
was marginally different from the reported
M. tilanpsasiri;
the SSU rDNA showed considerably higher
similarity with
M. tilanpasiri.
Thus, we report the species of
from a new host. This is the rst report of a microsporidian from
B. dussumieri
and the rst record of the
from India.
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
signicant 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
, 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, mudats, intertidal habitats and mangrove
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 diversication 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.
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In this background, an investigation was carried out to study the prevalence of parasitic infection in
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
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
B. dussumieri
=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 identication 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://shes.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).
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. Paran inltration 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 amplied using reported primers MicroSSUF: 5’-
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, (Invitroge, 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 Scientic®, 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 Scientic®, India) and the insert was
reconrmed by PCR amplication with microsporidian-specic primers. The conrmed plasmid DNA was
further sequenced in both directions using the same microsporidian-specic primers by a commercial
company (Xcelris Labs, Ahmedabad, India). The quality of each sequence was veried 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).
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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
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-
specicity, 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) amplication 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-amplied products were sequenced and almost complete SSU rDNA (1269 bp) was sequenced
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
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distance value (Kimura 2 parameter model) of 0.2% (nucleotide difference of 2) with
The tree topologies reconstructed by different methods were similar and in the consensus phylogenetic
tree, the species of
clustered with
M. tilanpasiri
as a single clade with signicant bootstrap
value (Fig. 5). Further, this group emerged as a sister clade to
M. carolinus
with moderate bootstrap value.
Few species of
sp. (GenBank accession number AB623034 & JQ820238) clustered within the
Tetramicridae family. Family Spragueidae formed a sister group to
This is the rst report of the microsporidium,
sp. from
Boleophthalmus dussumieri
and the
rst record of the genus
from India. The present
sp. showed close similarity
with the diagnostic features described for the genus
(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
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
species reported along with the
present species is given in Table1.
The morphometric values of the present species were found to be closer to
M. vivaresi
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
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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 sucient 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
(1269 bp) showed high genetic similarity (~ 98.9–99.8%) and less genetic divergence value with
M. carolinus
(0.7%), and
M. vivaresi
(1.1%). Several previous studies have also reported
low divergence values between
M. carolinus
M. tilanpasiri
M. tincae
M. vivaresi
M. caulleryi
Tetramicra brevilum
(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 signicant bootstrap. This
clade corresponds to group IV of the classication reported by Lom and Nilsen (2003). Although the
present species of
displayed unique morphological and morphometric features in the new
host, molecular sequence data showed a high anity to
. Hence, the present species can be
considered as
, a species with close anity 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 identies and describes a new record of
infecting the hepatic
tissue of the brackishwater sh,
Boleophthalmus dussumieri
Valenciénnes, 1837, from India.
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
care and use of animals were followed in the present study.
Conict of interest: The authors declare that there is no conict of interest or competing interests.
Page 8/14
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TABLE 1 Comparison of the present species of
with previously reported species
Page 11/14
Species Host The site
Region Reference
hepaticus Chelon labrosus
Liver 4.2 X 2.4 United
Kingdom Ralphs
M. ovoidea Motella tricirrata,
Cepola rubescens, C.
Merluccius hubbsi,
M. barbatus, M. gayi,
M. hubbsi
Liver 3.8 x 1.97 Mediterranean
Atlantic coast
Peru and
Amigó et
, 1996
M. caulleryi Hyperoplus
Liver 2.6 X 1.2 Atlantic coast
(France, and
Leiro et
al., 1999
M. tincae Symphodus tinca
Liver 3.6 × 1.2 Tunisian
coast Mansour
et al.
M. vivaresi Taurulus bubalis
Liver and
3.6 X 2.1 United
Kingdom Canning
et al.,
M. carolinus Trachinotus
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
tilanpasiri Boleophthalmus
Liver 2.9-3.77
(mean,3.25 X
India Present
Figure 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
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
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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.
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Figure 5
Neighbour-Joining tree of selected microsporidians. Labels on the nodes represent bootstrap values.
Branch length shows the divergence between the species.
... Subsequently, a very similar parasite (likely the same species) was described in mudskippers Boleophthalmus dussumier, also in the family Gobiidae, from the north-west coast of India. The infected fish were part of a histologic survey, and the parasite xenomas were observed in the liver of 11% of the fish (Vandana et al. 2021). ...
There have been several significant new findings regarding Microsporidia of fishes over the last decade. Here we provide an update on new taxa, new hosts and new diseases in captive and wild fishes since 2013. The importance of microsporidiosis continues to increase with the rapid growth of finfish aquaculture and the dramatic increase in the use of zebrafish as a model in biomedical research. In addition to reviewing new taxa and microsporidian diseases, we include discussions on advances with diagnostic methods, impacts of microsporidia on fish beyond morbidity and mortality, novel findings with transmission and invertebrate hosts, and a summary of the phylogenetics of fish microsporidia.
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The molecular evolutionary genetics analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from free of charge.
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Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions.
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The gobiid subfamily Oxudercinae was revised to assess the monophyletic nature of the subfamily; the intergeneric relationships within the subfamily; the relationships of oxudercines to other gobiid genera; the recognisable species and their distinguishing characters; and the distribution patterns of the subfamily and component taxa. The following results were obtained: (1) The Oxudercinae can be defined on the basis of derived states of certain neurocranial bones and muscles, eye position, nasal flap morphology, the palatine-ectopterygoid arrangement, reduction in size of the premaxillae ascending processes, and in having a single anal fin pterygiophore anterior to the first hemal spine. (2) Within the subfamily, one undefined and nine monophyletic terminal assemblages are recognised, with relationships amongst them based on derived states of various morphological features. These assemblages are recognised at the generic level and one new genus (Zappa) is described; a key to the genera is provided. Thirty four species are recognised of which one (Boleophthalmus birdsongi) is described for the first time. Each species is described in detail and a key is provided for each genus. (3) Two monophyletic assemblages (one comprising three genera, the other seven) are recognised at the tribal level. Defining characters for each tribe are provided and illustrated. (4) Biogeographic analysis indicated that nine of the ten genera are distributed in an area bounded by the Arabian Gulf to the west, southern Japan to the north, northern Australia to the south, and Papua New Guinea to the east. The remaining genus, Periophthalmus, overlaps and exceeds the above limits, ranging from west Africa eastward to Samoa. Species-specific correlations linking Periophthalmus with mangrove distributions are discussed.
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SUMMARY A new species of Microsporidia Microgemma carolinus n. sp. found in the marine teleost Trachinotus carolinus collected in Florianópolis, Brazil was described based on light, ultrastructural and phylogenetic studies. This parasite developed in the liver forming whitish xenomas that contained different developmental stages with monokaryotic nuclei. The periphery of the xenoma presented some vacuolization and possessed several small projections in the membrane. The mature spores, measuring 3·8±0·4 μm in length and 2·4±0·4 μm in width, were slightly pyriform to ellipsoidal and had rounded ends. The polaroplast was bipartite and the isofilar polar filament was coiled with 8 - 9 turns in a single or double row at the posterior end of the spore. The nucleus was voluminous and in a central position, measuring ∼0·9 μm in diameter. A large posterior vacuole appeared as a pale area, occupying about a third of the spore length. The SSU rRNA gene was sequenced and analysed using maximum parsimony, maximum likelihood and neighbour-joining methods. This study allowed us to conclude that this was a new species of the genus Microgemma, being the first description of this genus from among South America fauna.
Glugea eda n. sp. is described from the mesenteries of the striated fusilier, Caesio striata, collected from the Red Sea coast off Yanbu' al Bahr, Saudi Arabia. Numerous blackish xenomas, ranged from 3 to 5 mm, were found in the body cavity associated with the mesenteries. Mature spores are monomorphic, ellipsoidal with an average size of 5(4-6) μm in length and 2.2 (2-3) μm in width. Observations of the ultrastructure revealed that the development was asynchronous and that the nuclei were isolated throughout the life cycle with uninucleate meronts. Sporoblasts were uninucleated and existed together with sporonts in a fully formed parasitophorous vacuole. The polar filament of the mature spore was isofilar with 24-28 coils, arranged in three rows. Phylogenetic analysis placed the current microsporidia within the clade grouping Glugea species and close to the species described from the Red Sea and Arabian Gulf. The morphometric and molecular comparison with other members of the genus Glugea evidenced the taxonomic novelty of the present form, suggesting that it should be considered as a new species. To the best of our knowledge, the parasite here described represents the first occurrence of microsporidian infection in the fish of the family Caesionidae.
A study of the ultrastructure and life cycle of Microsporidium ovoideum, a hepatic parasite of Cepola macrophthalma, has been carried out. This parasite caused xenomas up to 1-2 mm, which consisted of a hypertrophic cell, filled with different stages of the parasite and rests of the host cell organelles, which had a pychnotic hypertrophic nucleus. Merogonic stages consisted of round multinucleate plasmodia that divided by plasmotomy or exogenous budding, and were not enclosed by any membrane. Sporogonic plasmodia divided by exogenous budding to produce sporoblast mother cells which after a binary fission become sporoblasts. Sporogony occurs in direct contact with host cytoplasm. The spores, which are ovoid and uninucleate, show a posterior vacuole in the posterior third of the spore and an isofilar polar filament with 6-8 coils. The characteristic features permit a reassignation of this microsporidium into the genus Microgemma and give a new combination: Microgemma ovoidea.
A new species of microsporidia, Microgemma tilanpasiri n. sp., is described infecting the burrowing goby, Trypauchen vagina, from Malaysia. The microsporidian forms macroscopic xenomas in the host liver which are packed with mature spores. Mature spores are slightly pyriform to oval in shape measuring 3.92 ± 0.21 μm in length and 2.87 ±0.16 pm in width. No pre-spore stages were observed during electron microscopy studies and mature spores had a single nucleus and 12-13 turns of an isofilar polar filament, arranged in two rows. Sequencing of the ribosomal DNA indicated a strong phylogenetic relationship within the Tetramicridae and to other members of the genus Microgemma. The most similar species in terms of genetic distance is M. carolinus with a similarity of 99.23% over 1295 bases of the small subunit of ribosomal DNA. However, differences in the number of turns of the polar filament combined with host and geographical differences, support M. tilanpasiri as a novel microsporidian species. This represents the first description of Microgemma from the Western Pacific and the first from the Gobiidae family of fishes. Related blackfin eel gobies from the same sampling site were found to be uninfected with M. tilanpasiri; however one fish was infected with Glugea sp. in the visceral mesentery. We conclude that in spite of the low genetic distances observed in ribosomal DNA sequences between geographically distant xenoma-forming microsporidians from both Microgemma and Glugea, that they probably represent a number of different species of parasite that may actually be quite host specific.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Myxozoans are among the most abundant parasites in nature. Their life cycles involve two hosts: an invertebrate, usually an annelid, and a vertebrate, usually a fish. They affect species in their natural habitats but also constitute a great menace for fish aquaculture. Using different strategies they are able to parasitize and cause damage in multiple organs, including mucosal tissues, which they use also as portals of entry. In fish, the main mucosal sites include the intestine, skin and gills. Recently the finding of a specific mucosal immunoglobulin in teleost (IgT), analogous to mammalian IgA, and the capacity of fish to develop a specific mucosal immune response against different pathogens, has highlighted the importance of studying immune responses at mucosal sites. In this review, we describe the major biological characteristics of myxozoan parasites and present the data available regarding immune responses for species that infect mucosal sites. As models for mucosal immunity we review the responses to Enteromyxum spp. and Ceratomyxa shasta, both of which parasitize the intestine. The immune response at the skin and gills is also described, as these mucosal tissues are used by myxozoans as attaching surfaces and portal of entry, and some species also parasitize these sites. Finally, the development of immunoprophylactic strategies is discussed.
. Microgemma hepaticus gen.nov. sp.nov. is described from the liver of juvenile grey mullet, Chelon labrosus (Risso). Development occurs within xenomas (diameter 500μm) which have microvillar surfaces, encircling bands of mitochondria and a reticulate hypertrophic nucleus. Vegetative developmental stages, meronts, are plasmodial and divided by plasmotomy. These stages are enclosed by host membranes. Sporogonic stages are free in the cytoplasm and divide by multiple exogenous budding. Uninucleate spores (2·4μm × 4·2μm) possess 7–9 coils of the polar filament and a lamellar polaroplast. Xenomas are associated with liver connective tissue, and cause necrosis of adjacent liver cells in certain circumstances. Host response to infection involves leucocyte infiltration and granuloma formation, with spores being destroyed by repeated macrophage phagocytosis and necrosis and resulting in gradual resolution of the lesion. Although juveniles apparently tolerate large parasite burdens there is some evidence of a contribution by the parasite to stress-related mortality. The transmission of the disease and its potentially high pathogenicity to larval fishes is discussed.