Aquaculture International

https://doi.org/10.1007/s10499-022-00992-7

Reovirus occurrence in mud crab farming systems

and wild‑caught brooders located in eastern coastal area
of India

Sathiyaraj Ganesan1,2 · Babu Baskaran1,2 · Mithun Raj2 · Saravanan Marimuthu3 ·

Velmurugan Krishnasamy3 · Ruban Lamech4 · Anup Mandal2,3,4 ·
Kandan Shanmuganathan2,3,4 · Prabhu Narayanasamy Marimuthu1

Received: 2 August 2022 / Accepted: 3 October 2022

© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022

Abstract
Herein, moribund mud crab (Scylla serrata) samples were collected from various farm-
ing systems, brooders from wild and hatchery quarantine section, located in Tamil Nadu,
Andhra Pradesh, and Odisha. The infected crabs showed clinical signs of lethargic move-
ment with the sleeping position in the culture ponds, tanks, and wild-caught. In the wet
mount study, massive numbers of viral giant cells were observed in the infected crab
hepatopancreas and connective tissue. Mud crab reovirus (MCRV) inclusion bodies were
noticed in the connective tissue of hepatopancreas, gill lamellae, muscle and gonads in
histopathology analysis. Also, viral particles proliferated into the cytoplasm of the cells,
but they did not affect the host nuclei. The infected crab samples were confirmed in the
PCR using specific primers (ReoF and ReoR) and rDNA sequence analysis showed 99%
similarity with mud crab reovirus and Scylla serrata reovirus (OL466868 and OL466869).
The ultrastructural study (TEM) revealed that MCRV viral particles ranged between 70 and
75 nm in diameter, icosahedral shape, and non-enveloped with two capsid layers located
in the cytoplasm of the cells. The prevalence of mud crab reovirus infection was recorded
between 80 and 100% in cultured farms within 25 days and 19–33% in the wild from the
collected samples. In the pathogenicity study, intramuscular injection bioassay reached
100% mortality and a co-habitation assay was 70% mortality on 14 days of MCRV post-
infection. Thus, our results suggest that MCRV is highly pathogenic to mud crab culture.

Handling editor: Brian Austin

* Prabhu Narayanasamy Marimuthu

71gnmprabhu@gmail.com; prabhunm@alagappauniversity.ac.in
1
Disease Control and Prevention Lab, Department of Animal Health and Management, Science
campus, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
2
Central Aquaculture Pathology Laboratory, Rajiv Gandhi Centre for Aquaculture (RGCA),
TTTAC, MPEDA, Mayiladuthurai, Sirkazhi 609 109, Tamil Nadu, India
3
Mangrove Crab Hatchery, Rajiv Gandhi Centre for Aquaculture (RGCA), TTTAC, MPEDA,
Mayiladuthurai, Sirkazhi 609 109, Tamil Nadu, India
4
Central Aquaculture Genetic Laboratory, Rajiv Gandhi Centre for Aquaculture (RGCA), TTTAC,
MPEDA, Mayiladuthurai, Sirkazhi 609 109, Tamil Nadu, India

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 Aquaculture International

Keywords Scylla serrata · Sleeping disease · Reovirus · Mud crab · Disease prevalence

Introduction

Mud crabs (Scylla sp.) are considered one of the commercially important crustacean spe-
cies found in the estuaries, coastal lagoons, and mangrove regions of India with great
demand in the domestic as well as the export market (Sakthivel and Francis 2006). Due
to their huge demand, commercial-scale mud crab farming is expanding very fast in the
coastal areas of India, especially in Tamil Nadu (TN), Andhra Pradesh (AP), Kerala (KL),
West Bengal (WB), Karnataka (KA), Maharashtra (MH), Goa (GA), Gujarat (GU), Odi-
sha (OD), and the Union Territories. In India, the farmed mud crab production was 2000
MT in 2019–2020. Andhra Pradesh (977MT) is the leading producer followed by Odisha
(462MT), West Bengal (325MT), and Kerala (132MT) (MPEDA 2020). Two major spe-
cies of mud crabs, Scylla olivacaea and S. serrata, are reported in the coastal belt of India
(Mandal et al. 2014). However, S. serrata is a potential candidate species for commercial
production due to its behavior, as well as because it grows faster and bigger, has a high
demand in the market, and fetches high profit to the farmer compared to the other species
(Jithendran et al. 2010). In recent years, mud crab farming is becoming an alternative to
shrimp or fish culture systems due to its high-profit margin. Different types of crab culture
practices, such as crab grow-out system (crablet to marketable size), crab fattening (water
crab to hardening), soft-shell crab, and female crab production (for ganoid development),
have been followed in India. However, fattening is the predominant practice followed by
grow-out culture in earthen ponds and tanks, as well as in pen systems, of which, recently,
soft-shell crab production has been flourishing (Rahman et al. 2017). All these farming
systems are entirely dependent on wild seeds due to the lack of sufficient commercial mud
crab hatcheries and nurseries to supply crablets or seedlings on time for stocking (Salam
and Ross 2000; Rahman et al. 2018). The traditional and extensive way of culture practices
has been followed in the crab production system. Technically, however, crab culture has
not taken off to the level of other crustacean species like shrimps and prawns as well as
finfish. To compete with other aquaculture species, scientific crab hatchery and farm man-
agement like feed, water quality, and health are required. In particular, crab diseases and
their management with potential economic impact studies are needed (Lavilla-Pitogo and
Peña 2004).
It has been reported that across the world, many new pathogens and diseases are
adversely affecting the production of mud crabs. Pathogenic bacteria such as Vibrio sp.,
Pseudomonas sp., Aeromonas sp., and Acinetobacter sp. have been reported in the infected
crabs (Senderovichet al. 2010; Lalitha and Thampuran 2012). The parasitic dinoflagellates
(Hematodinium sp.) affect the host digestive organs, gills, and heart because of milky dis-
eases reported in S. serrata (Li et al. 2008) and pink crab disease (PCD) in edible crab
Cancer pagurus caused by hematodinium-like parasitic dinoflagellate (Stentiford et al.
2002; Stentiford 2008). Likewise, parasitic barnacles Sacculina sp. cause major health
issues in edible mud crabs (Knuckey et al. 1995; Waiho et al. 2017). It has been reported
that the fungal species of Lagenidium sp., Fusarium sp., and Atkinsiella sp. affect the eggs
of mud crabs and larval mycosis (Quinitio et al. 2001, 2002; Ghaware and Jadhao 2015).
Several viral diseases have been reported in Scylla sp. that include WSSV (White Spot
Syndrome Virus) (Otta et al. 1999; SahulHameed et al. 2003), MNV (Muscle Necrosis
Virus) (Song et al. 2003), reovirus, baculovirus, and intranuclear bacilliform virus. In

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Australia, a baculo viral infection was reported in juvenile and matured mud crabs (S. ser-
rata) without much clinical appearance (Anderson and Prior 1992; Humphrey et al. 2009).
Zhang et al. (2002) reported that reovirus was a highly infectious agent to crabs and cause
various diseases, for example, tremble disease reported in Chinese mitten crab (Eriocheir-
sinensis) and sleeping disease (SD) in mud crab or Scylla serrata reovirus (SsRV), causing
high mortality of up to 70% in cultured farms, and thus resulting in the heavy economic
loss (Weng et al. 2007; Chen et al. 2008). The morphology of mud crab reovirus shows that
they are icosahedral, non-enveloped viruses with a particle size of 70 nm (diam), and their
genome comprises 13 linear dsRNA sections (Weng et al. 2007; Chen et al. 2011). This
virus mostly affects the mud crab hepatopancreas connective tissue, gills, muscle, intestine,
and heart. These viral particles proliferate into the cytoplasm of the host cells (Zhao et al.
2021). In recent days, in India, infections and mortality have been observed in the wild
as well as in cultured mud crabs. However, the causative agents for these outbreaks have
not yet been determined. In this study, infected samples were collected from the culture
farms, wild-caught brooders from natural sources, and the hatchery quarantine section for
disease surveillance to investigate the disease-causing agent. The screening was done for
known bacterial, viral, and parasitic pathogens. Then, moribund samples were screened for
mud crab reovirus (MCRV) through histology, molecular, and electron microscopy (TEM)
analysis. The disease agent was confirmed further in the laboratory through a pathogenicity
study. In India, scientific evidence is not yet available on MCRV infection in mud crab (S.
serrata), so the mud crab reovirus (MCRV) infection has been reported, which has been
causing huge mortality (50–70%) in cultured (sub-adult to adult including brooders) and in
wild-caught S. serrata brooders collected from Tamil Nadu, Andhra Pradesh, and Odisha
coastal regions.

Material and methods

Collection of infected samples and gross signs

Mud crab (S. serrata), moribund samples (size 100–400 g) with external signs of lethargy,
and sleepy condition crabs were collected from infected mud crab farms (grow-out and
fattening system) located in Thirumulaivasal (n = 03) in Tamil Nadu, Nellore (n = 05) in
Andhra Pradesh, and Berhampur (n = 03) in Odisha. Furthermore, moribund samples (crab
size 75–200 g) were collected from a re-circulatory aquaculture system (vertical box crab
farming) located at Chennai (n = 04) in Tamil Nadu. The moribund mud crab brooders from
the wild as well as in hatchery quarantine tanks (after 2 days of stocking and size ranging
from 1000 to 1500 g) were taken to the Rajiv Gandhi Centre for Aquaculture (RGCA), at
Thoduvai, Tamil Nadu, during the period of December 2019 to October 2021. They were
sourced from the Punnakayal (n = 18) Tuticorin, Pichavaram (n = 21), Chidambaram, and
Poompuhar (n = 41). The collected crab samples from various sources were observed for
their clinical signs, symptoms, and post-mortem changes. Then, the organs (hepatopan-
creas, gills, muscle, heart, and gonads) were dissected carefully in the field and samples
were preserved individually in 95% ethanol and RNA later solution (Ambion, AM7020)
Davidson’s fixative for histopathology, for electron microscopy analysis, the sample was
fixed in glutaraldehyde (2.5%) the remaining samples were stored in the 2–4 °C. These
preserved samples were brought to the Central Aquaculture Pathology Laboratory (CAPL)
Facility of RGCA (accredited with NABL ISO/IEC 17,025:2017) located at Sirkali, Tamil

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Nadu, in controlled condition for further analysis. Primarily, the collected moribund crab
gill and hepatopancreas were examined for bacterial, fungal, and parasite infection using
selective culture media such as TCBS agar medium, Zobell marine agar medium, potato
dextrose agar medium, and wet mount analysis, respectively. At the time of sample col-
lection, water samples were also collected from sources, hatcheries, and farms to meas-
ure the temperature, salinity, pH, total ammonia, and nitrite using a thermometer (Thermo
and Hygrometer-TA 298), refractometer (Atago-Master-s/Millm), pH meter (Eutech-
pHtestr10), and commercial API Kits (Himedia) respectively.

Genomic identification of the pathogen

Nucleic acid extraction

Ethanol-preserved crab hepatopancreas, gill, and muscle samples were air-dried and
50–60 mg of each tissue was used for nucleic acid extraction. IQ2000 DTAB CTAB extrac-
tion kit and IQ 2000 RNA extraction kit (GeneReach, Taiwan) were used for the isola-
tion of DNA and RNA, respectively. The cDNA was prepared using a high-capacity cDNA
reverse transcription kit (Applied Biosystems). Extracted nucleic acids from crab samples
were quantified using Bio Photometer (Eppendorf) and stored at –20 °C. Approximately
50 μg/mL is maintained for the PCR amplification.

PCR screening for known crab diseases

All the extracted nucleic acids from the crab samples were initially screened by PCR
for the presence of any OIE- and non-OIE-listed crustacean pathogens such as WSSV,
IHHNV, NHPB, YHV, IMNV, TSV, EHP, and MBV using a commercial kit (IQ Real
Quantitative System, GeneReach Biotechnology Corp., Taiwan) at the Central Aquacul-
ture Pathology Laboratory (CAPL) Facility of RGCA (Accredited with NABL ISO/IEC
17,025:2017) located at Sirkali, Tamil Nadu. PCR amplification was performed with the
following universal program for all the pathogens: 42 °C for 30 min followed by 40 cycles
of 93 °C for 15 s and 60 °C for 1 min. The cDNA was prepared using a high-capacity
cDNA reverse transcription kit (Thermo Fisher Scientific) with the cycling conditions of
25 °C for 10 min, 37 °C for 120 min, and 85 °C for 10 min.

PCR amplification and genomic identification for MCRV

The cDNA was prepared using a high-capacity cDNA reverse transcription kit (Thermo
Fisher Scientific) with the cycling conditions of 25 °C for 10 min, 37 °C for 120 min,
and 85 °C for 10 min. The primers used for the PCR amplification (MasterCyclerProS,
Eppendorf, Germany) of mud crab reovirus are shown in Table 1. PCR amplification was
performed with the following cycling parameters, initial denaturation at 5 min for 94 °C
to denature DNA, followed by 35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C
for 30 s, and final extension at 72 °C for 5 min. The UVP-UV transilluminator was used
to visualize the amplified product executed in an agarose gel electrophoresis (1.2% w/v)
stained with 0.5 μg mL−1 ethidium bromide. The PCR product was purified by using the
QIAquick® Gel Extraction Kit and two amplified PCR (amplified DNA) purified prod-
ucts were sequenced using the same primers on Applied Biosystem Genetic Analyzer
3500 (Applied Biosystems, USA) with BigDye Terminator V3.1 cycle sequencing kit.

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Table 1  The following primer sequence used to detection of causative agent of this disease (SsRV/MCRV)
Primers Primer pairs (5′–3′) Amplicon Reference Initial temperature, cycle conditions, and final extension
size (bp)

MF/MR (1st step) TTC​ATT​GGC​ATC​CTG​ACT​TT 433 Guo et al (2008) 94 °C/3 m

TTC​ATT​TGG​TGA​GCC​TTT​GC 30 cycles of 94 °C/30 s, 55 °C/30 s, 72 °C/60 s, and 72 °C/10 m
MNF/MNR (2nd step) ACC​TGA​TTA​TCG​ACC​CAA​TCCCA 304 94 °C/3 m
AGC​CTT​TGC​CTG​ATG​AGC​CTGAA​ 30 cycles of 94 °C/30 s, 60 °C/30 s, 72 °C/60 s, and 72 °C/10 m
ReoF/ReoR GCA​AAT​TGA​ACT​ACT​ACT​ACTTA​ 433 Zhang et al. (2013) 94 °C/5 m
GAT​TCC​TAT​TGT​CAA​CTA​TCTCA​ 30 cycles of 94 °C/30 s, 65 °C/30 s, 72 °C/30 s, and 72 °C/5 m
ReoNF/ReoNR ATA​TCG​TCA​GAA​TGTC​ 304 94 °C/5 m
GTT​CAT​ATC​GTC​AGA​ATG​TCG​TTC​ 30 cycles of 94 °C/30 s, 65 °C/30 s, 72 °C/30 s, and 72 °C/5 m
DicF GCA​CTG​GGT​ACT​CTT​CCT​G 686 Zhang et al. (2013) 94 °C/3 m
DicR AC ACC​TAC​CAA​AGC​CCTAC​ 30 cycles of 94 °C/30 s, 65 °C/30 s, 72 °C/30 s, and 72 °C/1 m
DicNF GGA​TAC​TAT​GGA​TGA​TGT​TTC​ 404 94 °C/5 m
DicNR ACA​AAA​TAC​CAG​ATA​AAG​CAA​ 30 cycles of 94 °C/30 s, 53 °C/30 s, 72 °C/30 s, and 72 °C/1 m

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The obtained sequences were aligned and edited using sequence analysis software MEGA
version 6.0 (Tamura et al. 2013). The aligned sequences were compared with previously
reported nucleic acid sequences and protein databases using BLASTn analysis and pub-
lished in National Centre for Biotechnology Information (NCBI). A phylogenetic tree
using the neighbor-joining method was drawn using MEGA11.

Histopathology

Davidson’s fixative fixed samples were dissected and processed in ascending graded levels
of alcohol. Paraffin wax embedded tissue samples were segmented at 4–5 µm width using a
rotatory microtome (Leica 2125 RTS). Tissue sections were collected in positively charged
slides. The tissue sections were stained with hematoxylin and eosin and mounted with a
DPX (Bell and Lightner 1988). The mounted tissue sections were examined under the light
microscope (Leica DM 750) inbuilt with a camera (Leica Flexicam C1), and the images
were analyzed and captured (40 × magnification) by means of LAS X software.

Electron microscopy

Glutaraldehyde (2.5%) fixed tissue (gill and hepatopancreas) samples in 0.1 M phosphate
buffer (pH 7.2) for 24–48 h at 4 °C were splashed 3–4 times each for 30–45 min with PBS.
Then, the samples were post-fixed for 2 h in aqueous osmium tetroxide (1%); subsequently,
the samples were washed with deionized water 4–6 times each for 30–45 min and dehy-
drated in a series of graded alcohols. Then, the samples were filtrated and embedded in
Araldite 6005 resin or Spurr resin (Spurr 1969), and then incubated for 72 h at 80 °C for
complete polymerization. Ultrathin (50–70 nm) sections were made on ultra-microtome
(Leica ultra-cut UCT–GA–D/E-1/100), mounted on copper grids, and stained using satu-
rated aqueous uranyl acetate. Stained sections were counter-stained with Reynolds’ lead
citrate (LC) and were examined under TEM (model: FEI Tecnai G2 F20 S/TEM) at the
required magnification (Bozzola and Russell 1999).

Pathogenicity studies

Experimental mud crab

Healthy juvenile S. serrata (approximately 50–75 g) were obtained from the mud crab
demonstration farm of RGCA, Karaikal, Puducherry. The obtained mud crabs were stocked
in a 30-L aquarium (10 tanks) at the Central Aquaculture Pathology Laboratory (CAPL)
facility of RGCA for a week. Then, the juvenile crabs were screened (using tissue sam-
ples from part of 2nd walking legs/hemolymph) for mud crab reovirus (MCRV) and OIE
as well as non-OIE-listed pathogens through molecular studies, as described in the earlier
section “Genomic identification of the pathogen.” Then, the healthy crabs were transferred
to the experimental tanks.

Preparation of mud crab reovirus inoculum for infection

An amount of 1 g of virus-infected frozen crab gill and HP tissue was homogenized

in 5 mL of PBS (pH 7.2) using tissue lyser II (Qiagen) at 30 frequencies per min. The

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homogenates were collected and centrifuged at 4000 rpm for 20 min. The supernatant
was collected in sterile vials and filtered with 0.45 µm (Millex® Millipore) syringe fil-
ters. The obtained filtrate was filtrated again using 0.22 µm (Millex® Millipore) syringe
filters. The filtered supernatant was diluted at 1:10 in sterile NaCl at 1.2%. This inocu-
lum was used for intramuscular injection bioassays.

Experimental setup

The acclimatized crabs (for a week) were collected from the holding tank and stocked in
the experimental tanks. After 3 days, the crabs were used for the co-habitation bioassay
(bioassay I) and intramuscular injection (bioassay II) study and observed for 14 days.
For bioassays, co-habitation (bioassay I) and intramuscular injection study (bioassay
II) was carried out along with the control group, and all these studies were performed
in triplicate. In each group, MCRV-free 10 juvenile crabs of weight 50 to 75 g were
stocked in 25-L water tubs. Throughout the experimental period, each day commercial
pellet feed (Growel) was used to feed the crabs at a rate of 2% of crab body weight
in two installments (at 7 am and at 6 pm) and approximately 10% of the tank water
was changed daily. During this study period, the water salinity was 25ppt, temperature
28 °C, and pH 8.2.

Bioassay I—co‑habitation

In this assay, MCRV-free mud crabs (n = 10) were stocked along with the same size
of infected crabs (n = 2) in a plastic tub (group I). The control (group II) tank was
stocked with 10 virus-free juvenile mud crabs. For each group (I and II), triplicates (a
set of three copies with similar carb sizes and numbers) were maintained to validate the
observing results. For each group (I and II), three tanks with similar carb numbers were
maintained. During this experimental period, the feeding procedure, water exchange,
measuring the water salinity, temperature, and pH were recorded as mentioned in the
section of “Experimental setup.” The crab behavior, disease signs, and mortality were
observed on a daily basis until the end of the experiment.

Bioassay II—intramuscular injection

The MCRV inocula extracted from infected samples were injected into the mud crabs to
induce the disease. Briefly, MCRV-free juvenile mud crabs (n = 10) were intramuscu-
larly injected with 50 µL of MCRV-positive inocula in the coxa regions of the abdomi-
nal leg using a sterile 1-mL insulin syringe fitted with a 0.30 × 8 mm needle (group
I). The MCRV-positive inocula injected mud crabs were distributed in tubs. For nega-
tive control, MCRV-free juvenile mud crabs (n = 10) were injected with 50 µL of sterile
1.5% normal saline (NaCl) (group II). This experiment was conducted in triplicates and
the water parameters and other procedures were recorded as mentioned in bioassay I.
The moribund crab samples collected from the two assays were subjected to histopa-
thology and TEM by following the procedures mentioned in the respective sections.

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Ethical declaration

The present study does not need ethical approval as per the “The Committee for the Pur-
pose of Control and Supervision of Experiments on Animals (CPCSEA),” India.

Statistical analysis

The data were articulated as mean ± SD. One-way ANOVA was applied to conclude the
differences between groups (considered at p < 0.05) using the statistical software pack-
age SPSS 17.0.

Results

Disease signs and post‑mortem changes

Outwardly, the sick crabs show sluggish movement or sleeping conditions, no feed
intake, and incomplete molting. Internally, they displayed atrophy with the gray colora-
tion of the hepatopancreas. In culture systems, 60–80% mortality was recorded within
25 to 30 days, and wild-collected brooder crab exhibited 15–25% mortality in the quar-
antine facility within 3 days after stocking. OIE-listed crustacean viral pathogens and
bacterial (RLO, NHPB, vibriosis), parasitic, or fungal infections were not observed in
the collected sample (data not shown). Furthermore, in wet mount analysis, a massive
number of multi-nucleated giant cells were observed in the connective tissues of crab
hepatopancreas (Fig. 1). The above clinical signs indicated that the infection was due
to sleeping disease caused by reoviral (MCRV). The water temperature, salinity, pH,
total ammonia, and nitrite of water samples collected from the crab farms and coastal
areas varied from 27 to 32 °C, 26 to 33ppt, 8.0 to 8.4, 0 to 1 ppm, and 0 to 0.01 ppm,
respectively.

Genomic identification of the pathogen

The OIE-listed crustacean and other pathogens did not show any amplification and
were found to be negative. The primer set of ReoF and ReoR was only amplified with
a product size of 433 bp (Fig. 2). BLASTn result of the two sequences showed 99%
resemblance to that of a mud crab reovirus (MCRV) and Scylla serrata reovirus (SsRV)
strain (GenBank JQ287709 and HQ414137). The sequences were deposited in the NCBI
GenBank database (accession numbers OL466868 and OL466869). The evolutionary
history was inferred using the neighbor-joining method (Saitou and Nei 1987). The tree
was drawn to scale, with branch lengths in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. The evolutionary distances were computed
using the neighbor joining (Tamura et al. 2004). All ambiguous positions were removed
for each sequence pair (pairwise deletion option). There were a total of 315 positions in
the final data set (Fig. 3).

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Fig. 1  Clinical signs, post mortem changes, and wet mount analysis in the collected mud crab samples. A
Normal crab hepatopancreas (pale yellow color, arrow point) and orange color indicate ovary (arrow head);
B MCRV infected brooder crab’s hepatopancreas (pale grayish color, arrow mark) and orange color indi-
cate ovary (arrow head); C experimentally challenged MCRV infected crab’s hepatopancreas (pale grayish
color, arrow mark); D wet mount analyses of infected crab hepatopancreas intertubular spaces were massive
number of multinucleated giant cells (arrow mark)

Fig. 2  PCR products obtained

from amplification of MCRV
represented in agarose gel (prod-
uct size 433 bp). M, marker gene;
lane A, hepatopancreas; lane B,
gills; lane C, muscle; lane D,
heart; lane E, ovary; NC, nega-
tive control; PC, positive control

Histopathology

Diseased mud crabs collected from the farms (grow-out and fattening system in earthen
ponds, pens, and RAS system) and wild broodstock stocked in the hatchery quarantine

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Fig. 3  Phylogenetic tree analysis of relativeness of Scylla serrata reovirus species isolated (*) from the
infected crab samples using the available sequences in the NCBI GenBank database

section showed MCRV inclusion bodies in the hepatopancreatic connective tissue, gill
lamellae, muscle and gonads. In the infected cells, viral inclusion bodies were observed
in the cytoplasm, but not the nucleus. The infected cells were stained eosinophilic to
basophilic and exhibited vacuolar degeneration, multiple giant cell formation, and
necrosis in the infected connective tissue of the hepatopancreas, gill lamellae, heart, and
muscle organs. A massive number of viral inclusion bodies were observed in hepatopan-
creatic connective tissue and gill lamellae. Infected brooder ovaries, and testis showed
moderate numbers of viral inclusion bodies (Fig. 4).

Transmission electron microscopy (TEM)

TEM results revealed that the MCRV virus particles were found in the hepatopancreatic
connective tissues and gill lamellae. Massive numbers of virions with icosahedral shapes
possessing two capsid layers were recorded in the infected cell cytoplasm of the gill and
hepatopancreas. TEM indicated the capsid was about 70–75 nm sized from side to side,
containing an electron-dense core bounded by an electron-translucent zone as well as free
virions and nuclear structures were observed (Fig. 5).

Occurrence of MCRV

This study shows a widespread mud crab reovirus (MCRV) infection in cultured crab (S.
serrata) as well as in the wild located broodstocks collected from the quarantine section in
Tamil Nadu, Andhra Pradesh, and Odisha states in India. The clinical signs, post-mortem
changes, molecular, TEM, and histopathology analysis confirmed the presence of MCRV
in the collected moribund mud crabs. The prevalence shows that 10 out of 11 moribund
crabs collected from three farms (fattening) were detected with MCRV. Similarly, all the
crab samples collected from the RAS vertical crab farming system (4 moribund crabs with
clinical signs of lethargy and off feeding) showed to be MCRV positive. The crab brooders
collected from the wild and stocked in the quarantine Sect. (22 out of 80 crabs) exhibited
similar clinical signs within 3 days of stocking and post-mortem changes, molecular, TEM,
and histopathology analysis confirmed the presence of reovirus. These crabs were collected

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Fig. 4  Histopathology of MCRV infected mud crab internal organs. A, B Crab hepatopancreas intertubu-
lar space showed multinucleated giant cells (arrow mark) with intracytoplasmic viral inclusion bodies; C
muscle tissue showed mild degeneration with necrosis and viral inclusion bodies (arrow mark); D crab gill
lamellae showed few necrotic cells with plenty of viral inclusion bodies (arrow mark); E crab ovarian inter-
tubular space between few numbers of viral inclusion bodies (arrow mark); F crab testicular tissue showed
few number of viral inclusion bodies (arrow mark)

from Tuticorin, Pichavaram, and Poompuhar coastal regions and the reovirus prevalence
was 33% (6 out of 18), 19% (4 out of 21), and 29% (12 out 41), respectively (Table 2).

Pathogenicity study of MCRV

Experimentally challenged mud crabs showed similar clinical signs of farm collected sam-
ples including lethargy and loss of appetite with sleeping position. Mortality of experi-
mentally infected crabs began 4 days post-infection through intramuscular injection (bioas-
say II in group I) of MCRV filtrates and 100% mortality was recorded by 14 days. In the
co-habitation study (bioassay I in group I), the crab commenced showing similar signs of
sleeping diseases (SD) and mortality on the 6th day after the introduction of infected crab
and demonstrated 70% mortality by 14 days. No mortality and clinical signs were observed
in both the bioassay control (group II) groups (Fig. 6). Histopathological studies revealed
viral inclusion bodies with giant cell formation in the hepatopancreas. Degeneration,
necrosis, viral inclusion bodies, and hemocytic inflammation were observed in the gills and
muscle tissue of mud crab in both the challenged groups, while the normal architecture of

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Fig. 5  Electron microscopy images of MCRV viral particles in crab hepatopancreas (A and B) and gill
lamellae (C and D). A–C showed massive core of MCRV viral particles located in the cytoplasm of the
cells (arrow mark) and marginated nuclei (arrow head); D electron dense (long arrow mark) and electron-
translucent (short arrow mark) MCRV viral particles

the hepatopancreas, gills, and muscle was noticed in the control crab (Fig. 7). TEM studies
revealed that MCRV infected the connective tissues of the hepatopancreas and gill lamellae
of mud crab, resulting in a massive number of MCRV virus particles in the cell cytoplasm
(Fig. 8).

Discussion

Recent studies have reported that the mud crab reovirus (MCRV) is extremely patho-
genic to crabs, causing sleeping disease (Zhang et al. 2002). MCRV (also known as S.
serrata reovirus or SsRV) is reported to have caused huge economic losses to crab farm-
ers in China (Weng et al. 2007; Chen et al. 2008). Soft-shell (SS) blue crabs (Callinectes
sapidus) are infected by C. sapidus reovirus 1 (CsRV1), also causing an economic loss
in various farms starting from Louisiana to Massachusetts; this virus was also reported
in the wild crab population at an average prevalence of 20–50% (Rogers et al. 2015a,b;
Flowers et al. 2016a,b). Our study results revealed that disease occurrence was recorded at
90–100% in cultured farms (including RAS). Chaves and Eggleston (2003) reported that
reo-like virus in soft-shell crab production causes a high infection (> 50%) and recognized
that this infection may be due to poor water quality and handling stress (Le Moullac and
Haffner 2000; Lacoste et al. 2002). Twenty percent and 35% of CsRV1 prevalence were

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Table 2  Sampling sites and occurrence (%) of mud crab reovirus (MCRV) detections in farmed and wild collected mud crab samples
Source of mud crabs Latitude Longitude Weight (g) No. of crab sam- Percentage (%)
pling (N) of occurrence of
MCRV

Culture system
Thirumullaivasal, TN (December,2019) 11°14′51.5″N 79°50′34.5″E 300–400 03 100
Nellore, Kavali, AP (October, 2021) 14°55′N 80°03′E 100–200 05 80
Berhmpur, Odisha (November, 2021) 19°07′06.9″N 84°45′32.9″E 250–300 03 100
Soft-shell crab
Vertical Box Farming, Chennai, TN (October, 2021) 13°20′09.7″N 80°07′41.0″E 75–200 04 100
Wild-caught brooder
Tuticorin, Mangroves. TN (December, 2020) 08º39′491″N 78º07′234″E 1000–1250 18 33
Pitcchavaram Mangroves, TN (January, 2021) 11°25′54.6″N 79°46′56.1″E 1000–1250 21 19
Poompuhar Mangroves, TN (October, 2021) 11°09′11.6″N 79°51′41.8″E 1250–1500 41 29

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Fig. 6  Assessment of patho-

genicity studies in mud crab
juveniles challenged with mud
crab reovirus (MCRV) at dif-
ferent experimental groups.
Graphical represented percent-
age of mortality in mud crab at
different experimental groups.
Control groups were no mortality
was recorded

Fig. 7  Histopathological lesions of experimentally MCRV challenged mud crab internal organs. A Normal
histological structure of hepatopancreas; B normal gill lamellae structure; C normal structure of muscle
tissue; D intracytoplasmic viral inclusion bodies (arrow mark) in haemal sinus space of hepatopancreas;
E necrotic cells with MCRV viral inclusion bodies (arrow mark) in gill lamellae; F muscle tissue showed
necrosis with few number of MCRV viral inclusion bodies (arrow mark)

reported in wild crab populations and soft-shell production systems in the northeast United
States and the Chesapeake Bay, respectively (Flowers et al. 2016a,b; Spitznagel et al.
2019). In the present study, moribund samples collected from the RAS vertical crab farm-
ing system show 100% occurrence, and this may be due to the recirculation of the same
water without awareness of the infection. Also, MCRV disease occurrence was at 19–33%
in the wild brooders stocked in the quarantine section, collected from Tamil Nadu, India.
In India and other countries, very limited disease surveillance reports are available on
mud crab, especially on sleeping diseases caused by reovirus in cultured farms and brood-
stocks. The mud crab reovirus (MCRV) outbreak was noticed in S. serrata wild-caught
brooders and cultured crabs from juvenile to sub-adult in the eastern coastal regions of
Tamil Nadu, Andhra Pradesh, and Odisha in India (December 2019–October 2021). It has
been stated that the clinical signs of reovirus-infected crabs showed sluggish movement or
sleeping conditions, loss of appetite, the gray coloration of hepatopancreas, and massive

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Fig. 8  Electron microscopy images of MCRV viral particles in experimentally challenged crab hepato-
pancreas and gill lamellae. A (hepatopancreas) and B (gill) showed massive core of MCRV viral particles
located in the cytoplasm of the cells (arrow mark) and marginated nuclei (arrow head)

mortality of up to 60–80% on farms (Weng et al. 2007). Hayakijkosol and Owens (2011)
reported that the clinical signs of crayfish C. quadricarinatus infected with reovirus exhib-
its sluggishness, deprived appetite, a debilitated tail-flip response, and red color append-
ages and mouthparts. In the present study, similar symptoms and clinical signs of lethargy,
sleeping position, and loss of appetite were observed externally, and internal discoloration
of the hepatopancreas was recorded in infected mud crabs.
Guo et al. (2008) and Chen et al. (2011) confirmed the MCRV infection by RT-PCR,
one-step RT-PCR, and RT-loop-mediated isothermal amplification (RT-LAMP). Also, it
has been stated that the nested PCR is more reliable to detect MCRV and also useful for
epidemiology study of MCRV (Flowers et al. 2016b) or any virus. Similarly, in the pre-
sent study, nested PCR was used to confirm the MCRV infection in the collected samples
of wild-caught brooders and farmed mud crabs. Mud crab reovirus is noticed in the vari-
ous organs gill, gut, intestine, heart, muscle, gonad, hemolymph, and thoracic ganglion of
disease-affected crabs. Therefore, the study result suggests that similar to shrimp culture
practices, before stocking in the crab hatchery and farms, PCR detection is essential to pre-
vent the spread of the virus (Yang and Rothman 2004; Guha et al. 2015).
In an earlier study, Weng et al. (2007) and Chen et al. (2008) documented that MCRV
infects the hepatopancreas, gills, muscle, and intestine connective tissues of crabs and also
causes degeneration and necrosis in these tissues. Johnson (1977, 1984) reported that the reo-
like virus affected the ectodermal and mesodermal derived tissues in the host. Similarly, in
blue crabs, Callinectes sapidus reovirus (CsRV) infects the hepatopancreas and gill and also,
eosinophilic to basophilic, cytoplasmic inclusions have been reported in the hemocytes and
cells of connective tissues (Tang et al. 2011). The present study results show that mud crab
reovirus (MCRV) mostly affected the hepatopancreas connective tissue, gills, muscle, intes-
tine, heart, and gonads of crab. The viral particles proliferated into the cytoplasm of the cells
but did not affect the host nuclei. Previously, MCRV-infected crab hepatopancreas, gill, mus-
cle, and intestine were the only organs displaying histopathological lesions, but in our study,
viral inclusion was observed in wild-caught brooder gonads (testis and ovary); therefore, there
is a possibility of vertical transmission. Furthermore, in wet mount and histopathological anal-
ysis, numerous viral infected giant cells were observed in the hepatopancreas of infected crab.
Previously, electron microscopy studies revealed that the MCRV particle size is 70–75 nm
in diameter, icosahedral in shape with two capsid layers, and non-enveloped, non-turreted

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reoviruses with a smooth plane surface appearance (Mertens et al. 2005; Ye et al. 2012). The
size of virus particles was similar to the Aquareovirus and grass carp reovirus (Mertens et al.
2005; Weng et al. 2007; Ye et al. 2012). In the present study, similar size and morphological
features were observed in TEM analysis.
Several authors reported that intramuscular injection, bath inoculation, and oral inoculation
of MCRV cause 100% mortality in S. serrata whereas co-habitation study results show 80%
mortality (Bonami et al. 1976; Mari and Bonami 1988; Weng et al. 2007; Bonami and Zhang
2011; Bowers et al. 2010). The pathogenicity of reovirus-challenged juvenile C. quadricarina-
tus exhibited less mortality by injection methods (20%), or by feeding (5%) (Hayakijkosol and
Owens 2011). In the present study, the first mortality was recorded on the 4th day post-infec-
tion by intra-muscular injection, while in co-habitation bioassays, mortality was observed on
the 6th day. At the end of the experiment (after 14 days), 100% and 70% mortality was docu-
mented in intra-muscular injection and co-habitation assay, respectively. However, no mortal-
ity occurred in the control groups. This study result shows that reovirus is transmitted through
horizontal (enteric, water, and body surface routes) in mud crab culture systems.
In mud crab culture practices, many factors aggravate the vulnerability of infection and
mortality such as high stocking density, the stress due to poor water quality, temperature
fluxes, or hypoxia (Mohanty et al. 2018). Additionally, disposal of dead and moribund crabs,
as well as untreated culture water into the nearest coastal resources, promotes the spread of
disease (Lafferty et al. 2015; Hajeck and Shields 2017; Flowers et al. 2018). Crabs entirely
depend on their innate immune system, so preventive measure is always the best, to help the
farmers to escape from the virus including other pathogens. Spitznagel et al. (2019) reported
CSRV1 virus loads and mortality in live and dead mud crabs in recirculating and flow-through
systems due to various environmental factors and poor management. Mortality was recorded
as twofold higher in flow-through aquaculture systems (33%) than in recirculating aquaculture
systems (16%) due to higher fluctuation in temperature and hypoxic measures in flow-through
systems. Similar to the shrimp culture system biosecurity measures and disease, screening
procedures may help crab farmers avoid the disease. Also, broodstock should be screened for
the presence of MCRV and preference should be given to specific pathogen-free crab brood-
stocks and crablets for stocking in hatcheries and farms respectively. In addition, water treat-
ment procedures might be followed before stocking and during the production system. Gener-
ally, in RAS, only suspended matters and organic metabolites can be filtered; this might be
helpful to maintain the good physical and chemical condition of water parameters (Tom et al.
2021). However, to overcome the disease problems in RAS, further studies are required, espe-
cially in water quality management (disinfection procedure). The disease can be managed by
following biosecurity procedures that include the elimination of potential live vectors, patho-
gens at various stages, and by implementing the appropriate disinfection procedures for the
non-living vectors including vehicles, equipment, and nets (Shelley and Lovatelli 2011). In
this line, further study is essential for the survival of crab farmers and the industry.

Conclusion

We found that mud crab reovirus (MCRV) causes sleeping disease (SD) in wild brood-
stocks as well as farmed S. serrata in India. The presence of the virus was associated with
high mortality rates of 70–80% in cultured and 20–30% in wild brooders. This infection
was confirmed systematically by external symptoms, wet mounts, nested PCR, histology,
and TEM analysis. The MCRV virus particles are found in most internal organs such as the

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hepatopancreas connective tissue, gill, heart, muscle, and gonads. The viral particles mul-
tiply in the cytoplasm of host cells and do not appear to damage the nucleus. The MCRV
particle size was 70–75 nm in diameter with aicosahedral shape observed under electron
microscopy study. Furthermore, the infection was reconfirmed by bioassays in the labora-
tory and the clinical signs and histopathology results were similar to the samples collected
from the farms and wild broodstocks. The experimental study indicated that the virus is
highly infectious and can also be transferred horizontally via co-habitation. Also, reovirus
infection was observed in the mud crab gonad, which may indicate the possibility of verti-
cal transmission. However, further research is needed to investigate this possibility. The
outcome shows that the reovirus is a great concern for mud crab aquaculture and needs to
be controlled with stringent biosecurity measures similar to shrimp culture practices such
as stocking of SPF crab broodstocks in the hatchery, disease screening procedure, water
treatment, and health management in culture systems.
Acknowledgements The authors acknowledge the chairman of the Marine Products Export Development
Authority (MPEDA), Government of India, Director and Project Directors of Rajiv Gandhi Centre for
Aquaculture (RGCA), officials, and colleagues for their continuous support and encouragement towards this
research. They express their gratitude to the farmers of Tamil Nadu, Andhra Pradesh, and Odisha for pro-
viding the samples. They also thank the National Surveillance Programme for Aquatic Animal Diseases,
the National Bureau of Fish Genetic Resources, Lucknow, and the National Fisheries Development Board,
Hyderabad. They thank ICAR-National Bureau of Fish Genetic Resources (NBFGR), Lucknow, and ICAR-
Central Institute Brackishwater Aquaculture (CIBA), Chennai, and for revalidation of the research work.
Sincere thanks are also due to the Department of Fisheries, Ministry of Fisheries, Animal Husbandry &
Dairying, and the Government of India.

Author contribution Ganesan Sathiyaraj conducted the experiment and analysis and wrote the manuscript.
Baskaran Babu, Mithun Raj, Marimuthu Saravanan, Krishnasamy Velmurugan, Lamech Ruban, Anup Man-
dal, and Sanmugam Kandan contributed to the data analysis and manuscript revision. Narayanasamy Mari-
muthu Prabhu contributed to the study conception, design, and manuscript revision.

Data availability The data that support the findings of this study are available from the corresponding author
upon reasonable request.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication All the authors of this article agree to the publication.

Competing interests The authors declare no competing interests.

Conflict of interests The authors declare no competing interests.

Human ethics Not applicable.

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