Mucus properties of Chinese carp and Indian carp: Physical barrier to pathogens
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Iranian Journal of Fisheries Sciences 19(3) 1224-1236 2020
DOI: 10.22092/ijfs.2019.119394.0
Mucus properties of Chinese carp and Indian carp: Physical
barrier to pathogens
Abbas F.1; Hafeez-ur-Rehman M.1; Ashraf M.1; Iqbal K.J.2*;
Andleeb S.3; Khan B.A.4
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Received: February 2017 Accepted: February 2018
Abstract
The study was aimed to investigate the probable role of mucus in reception and/or
repulsion of Lernaea in Indian and Chinese major carps. Ctenophryngodon idella,
Hypophthalmichthys moiltrix, Labeo rohita, Cirrhinus mrigala, Catla catla and
Cyprinus carpio averaging 830±316 g each were collected and disinfected with KMnO4
(8.0ppm) for collections of contamination free mucus. Total protein contents and size
categorization were determined by Bradford Micro Assay and SDS-PAGE analysis.
Lectin and alkaline phosphatase activities were also measured by hemagglutination
(HA) titer and alkaline phosphatase test, respectively. Protein concentrations were the
highest in C. idella and C. catla and the lowest in C. carpio. Considering protein
profiles mucus samples from C. catla contained the highest molecular weight proteins
while C. carpio has one unique protein band of 14.13 kDa the weight of which
resembles the weight of lysozyme, a protective element of mucus. Lectin activity was
highest in C. idella indicative of low resistance while it was the lowest in H. molitrix.
Alkaline phosphatase level was the highest in C. catla, and was the lowest in C. carpio.
Overall results indicted C. carpio as the most resistant species as it showed better
values for the immune components. Higher values of protein contents and alkaline
phosphatase for C. catla may be in favor of its higher susceptibility. These studies on
mucus contents are good assessment indicators of the possibility of parasitic attacks in
Chinese and Indian major carps.
Keywords: Fish, Lernaea, Pectin activity, Protein profile, Alkaline phosphatase
1-Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences,
Lahore, Pakistan
2-Department of Zoology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
3-Department of Zoology, Govt. Postgraduate Islamia College for Women, Cooper Road,
Lahore, Pakistan
4-Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan
*Corresponding author's Email: khalid.javed@iub.edu.pk1225 Abbas et al., Mucus properties of Chinese carp and Indian carp: Physical barrier to…
Introduction 2007; Tasawar et al., 2007a; Tasawar et
Fish depends upon its innate or non- al., 2007b) on level and intensity of its
specific immune mechanisms to fight attack and its variability from species to
against a variety of pathogens species (Kabata, 1985; Tasawar et al.,
(Subramanian et al., 2008). Fish mucus 2007b) the causes of this selective
layer helps in osmoregulation, attack have not yet been fully implicit.
reduction of friction and disease Earlier studies show that Leranaea
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resistance (Shephard, 1994). It provides remained ineffective till copepodid-I
mechanical, chemical and innate stage, and prior stages continue their
immunity against the pathogen further development off the fish host.
invasion. It reduces pathogen access This could be due to some degree of
due to its constant downward innate resistance or an immune
movement along lateral sides of the fish mechanism in fishes against the
and its various sprawling edges and parasitic copepodid-I, the basis for
sides. The presence of which may be morphological,
immunoglobulins, enzymes and lytic biochemical or immunological or a
agents ensure the neutralization of combination of all. Identification and
microorganisms (Ellis, 1990; Palaksha exploitation of this putative mechanism
et al., 2008) when they invade. Number (s) can pave the way for its application
of experiments has been performed on to control its invasion and subsequent
fish skin immunity and skin bacterial infections in susceptible species of fish.
infections (Lemaitre et al., 1996; Jones (2001) opined that the
Aranishi et al., 1998; Ebran et al., physicochemical characteristics of the
2000) however, little information is skin mucus, the presence of bio-reactive
available on the role of skin mucus as substances including lysozyme,
the first line of defense against various complement, C-reactive protein,
pathogens. haemolysins and lectins and the
Fish are vulnerable to many epidermal migration of inflammatory
ectoparasites present in living aquatic cells and their secretions may affect the
environments but the family Lernaeidae establishment and proliferation of ecto-
has been and is a common family parasitic copepods. Prem et al. (2017)
causing parasitic infestation in compared immunological and
freshwater fish all over the world biochemical properties of epidermal
(Tasawar et al., 2009). Members of mucus of three brackishwater fishes,
Cyprinidae fish family have been namely Lates calcarifer, Chanos
observed to be the most common hosts chanos and Mugil cephalus. Ghafoori et
of Learnaea -a persistent threat to fish al. (2014) observed significant changes
and fish culture. Although considerable in serum and mucus lysozyme in male
work has been done in various parts of and female Caspian kutum (Rutilus
the world and Pakistan (Bjorn et al., frisii kutum) under different seasonal
2001; Tasawar et al., 2001; Boxaspen, temperatures, gonadal growth and
2006; Oktener et al., 2007; Revie et al., reproductive migration. Irene et al.Iranian Journal of Fisheries Sciences 19(3) 2020 1226
(2011), Nigam et al. (2012), Rombout menace from attack. Mechanism of this
et al. (2014) and Salinas (2015) host-parasite relationship and
investigated the adaptive immune physiological role of mucus in this
system of teleost fish associated with complex will also be explored.
each of their mucosal body surfaces.
Evidence obtained from mucosal Materials and methods
vaccination and mucosal Experimental site
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infection studies reveal that adaptive Studies were conducted in fish pond
immune responses take place at the complex of Fisheries and Aquaculture
different mucosal surfaces of teleost. Department at Ravi Campus Pattoki.
The main mucosa-associated lymphoid
tissues (MALT) of teleosts are the gut- Experimental animal
associated lymphoid tissue (GALT), Grass carp (Ctenopharyngodon idella),
skin-associated lymphoid tissue silver carp (Hypophthalmicthys
(SALT), the gill-associated lymphoid moiltrix), rohu (Labeo rohita), mrigal
tissue (GIALT) and the recently (Cirrhinus mrigala), thaila (Catla catla)
discovered nasopharynx-associated and common carp (C. carpio) were
lymphoid tissue. Similar studies related selected as experimental fish species for
to comparative analysis of innate the planned studies.
immune parameters of skin mucous
secretions like alkaline phosphates, Experimental procedure
lysozyme total protein contents etc in A series of experimentation was
different fish species were studied by a conducted for the determination of
number of investigators like Loganatha possible causes of selective Lernaea
et al. (2013) in Channa striatus, attack in the Indian and Chinese carps.
Bensussan et al. (2012), in C. carpio) In the first trial Abbas et al. (2014)
Easy and Ross (2010) in Atlantic investigated Lernaea susceptibility,
salmons and Palaksha et al., (2008) in infestation and treatment. After
olive flounder. However an complete eradication of the parasite and
investigation of the possible causes for on completion of the growth period,
variability in the susceptibility to healthy fishes under similar
Lernaea in different carp species in the environmental conditions were
present experiments require further collected from commercial rearing
explorations to ensure that whether it is ponds and transported alive in the same
due to the biochemical, immunological, water to the fish hatchery for further
morphological and genetic commissioning the subsequent
characteristics of the host. experimental steps. Each fish weighed
The present study was planned to 830±316 g on the average. Prior to
explore the conceivable reasons and initiation of mucus collection all the
factors or bio-active molecules in the fishes were bathed in potassium
epidermal mucus of Indian and Chinese permanganate (KMnO4) solution (8.0
major carps which attract or repel this ppm) to remove any microbial or fungal1227 Abbas et al., Mucus properties of Chinese carp and Indian carp: Physical barrier to…
infection/infestation to alleviate the and protein concentrations in different
possibility of any type of contamination samples were then calculated and
in mucus samples. Then the fishes were determined by comparing their readings
acclimatized in cemented circular tanks with the standard curve of BSA solution
in the hatchery building under a stress (Bradford, 1976; Zaika, 1988).
free environment for 24 hours for
normalizing bodily functions of fishes. Polyacrylamide gel electrophoresis
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Five samples of each experimental fish (SDS-PAGE)
species were collected and every Electrophoresis was carried out as
sample was repeated three times. described by Laemmli (1970) with
slight modifications. 15% separating
Mucus collection and 4% staking buffer were used to run
Fishes were anesthetized with MS-222 the SDS- PAGE under constant voltage
(100 mg L-1) and laid on the smooth of 120. The FermentasPageRuler™
surface of a table with towel support for protein ladder was used as the standard
safe handling. A well immersed plastic marker for non-reducing protein. The
spatula was gently wiped on the gel was stained with PageBlue™
dorsoventral surface of fish which (Fermentas) stain for identification of
scraped off mucus from the skin protein bands for molecular weight
surface. During the collection of mucus, determination.
maximum precautions were adopted to
keep it free of common contaminants Lectin activity
like blood, scales or intestinal fluids. Lectin activity was determined
Samples were centrifuged at 12000×g according to the Ewart et al. (2001)
at 4 ˚C for 10 minutes and stored at -40 method. 50 µl mucus was diluted in
˚C in a biomedical freezer. Prior to two-fold serial dilutions in 50µl
analysis, mucus samples were thawed at phosphate buffer (PBS) (0.05 M, pH
room temperature for smooth and 6.2) in U bottom microtiter plates. 50µl
hassle free operation of the equipment. rabbit RBCs solution (1%) was mixed
in the dilutions and incubated at room
Total protein contents temperature for one hour. RBCs in PBS
Bradford Micro Assay technique was were used as the control. The plate
applied to determine protein contents surface was read to determine
using Bovine Serum Albumin (BSA) as hemagglutination (HA) titer. The HA
standard. BSA solution and mucus titer, defined as the reciprocal of the
dilutions were made in de-ionized water highest dilution exhibiting
in flat-bottom microtiter plates. hemagglutination, was computed as one
Bradford protein solution (50 μl) was hemagglutination unit. (Specific
added to each well and absorbance was activity is the number of
-1
recorded at 595nm. A standard curve heamagglutination unit’s mg lectin)
was drawn from various but (Wang et al., 2001).
consecutive dilutions of BSA solutionIranian Journal of Fisheries Sciences 19(3) 2020 1228
Alkaline phosphatase test Range Test was applied to compare
Mucus was incubated with 4 mM p- means from different treatment groups
nitrophenyl phosphate in ammonium to identify group differences.
bicarbonate buffer (100 mM) with 1
mM MgC12 (pH 7.8) at 30°C. The Results
increase in optical density (OD) was Protein profile of mucus
measured continuously for 2 to 3 hours The protein profile of mucus extracts of
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at 405 nm using a micro plate reader. all the fish species was conducted on
The initial rate of the reaction was used SDS-PAGE gel. PAGE showed
to calculate the activity (One unit (U) of molecular weight ranges of various
activity is defined as the amount of proteins from 12.59 to 100 kDa. L.
enzyme required to release 1µmol of p- rohita has the most diverse group of
nitrophenol product in 1 min) (Fletcher, proteins (12.59 to 89.13 kDa) whereas
1986). C. mrigala has the lowest number of
bands (31.62 to 89.13kDa) marking less
Statistical analysis protein diversity than L. rohita. Proteins
Statistical analysis was performed by below 50 kDa were very common in all
using SAS software (9.1 Version). the fish species tested. High molecular
Data were analyzed using ANOVA proteins were more dominant in C.
statistical test. Significance level was catla and were primarily of 100 kDa
fixed as p1229 Abbas et al., Mucus properties of Chinese carp and Indian carp: Physical barrier to…
observed in C. carpio (Table 1). The carpio did not show any type of
protein concentrations in mucus of C. infestation throughout the whole study
mrigala, L. rohita and H. molitrix fell period.
in-between these two extremes and
were quite similar to each other but Alkaline Phosphatase Activity
different from other experimental fish Alkaline phosphatase activity is another
species (Table 2). stress indicator and varies with the level
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of stress and type of fish species
Lectin activity exposed. C. catla has the highest
Lectin activity is the measure of the activity (184.6 U L-1) and C. carpio the
heamagglutination titer (HA). The C. lowest (31.1±0.1) (Table 2). Alkaline
idella showed maximum HA value (29) phosphatase activity increased with
or the lectin activity hence showed the intensity of Lernaea infestation. Totally
lowest resistance and the highest free of parasite infestation C. carpio
susceptibility to pathogen invasion. H. showed the minimum ALP value and C.
molitrix showed the lowest titer value catla with highest infestation displayed
(21) indicating fish was healthy with no the highest value for ALP and proved to
or minimum pathogen infestation. The be the preferred host of Lernaea. ALP
HA value for C. mrigala and C. carpio values (35.8±0.4) in the skin mucus of
was recorded as 27. L. rohita showed H. molitrix were very close to C. carpio
the 28 HA value (Table 2). C. mrigala (31.1±0.1) hence depicted no parasite
and L. rohita showed very little infestation.
infestation by the pathogen however, C.
Table 2: Protein concentration, hemagglutination (HA) titer value and alkaline phosphatase (ALP)
activity in mucus of experimental fish species.
Protein
HA Titer
Sr. NO. Fish species Concentration ALP (U L-1)
-1 value
(mg ml )
1 L. rohita 2.63±0.48a 28 87.5±0.7a
2 C. mrigala 2.88±0.07a 27 61.3±0.4b
3 C. catla 3.02±0.57b 26 184.6±0.1c
4 C. idella 3.29±0.13b 29 152.5±0.7d
5 H. molitrix 2.87±0.18a 21 35.8±0.4e
6 C, carpio 1.80±0.09c 27 31.1±0.1f
Figures with different superscripts in column are significantly different (pIranian Journal of Fisheries Sciences 19(3) 2020 1230
and acquired immune systems (Yano, protein value of 3.02±0.57 mg ml-1. The
1996). Mucus of fish skin is a dynamic lowest protein value was observed in C.
biological interface between fish and carpio (1.80±0.09 mg ml-1) while
their environment and is composed of values of other three species fell in
biochemically-diverse secretions from between these extremes. Comparing the
epidermal goblet and epithelial clavate protein concentrations with level of
(immune) cells (Pickering, 1974; Ellis, Lernaea infestations it appears that a
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2001; Powell, 2000). These cells higher protein concentration might have
secrete water and mucins like gel attracted Lernaea because it was a
forming macromolecules (Negus, 1963; convenient food and drastically
Shephard, 1994) ultimately proliferate its population. Arifa et al.
transforming into mucus. This epithelial (2011) reported protein contents in L.
mucus has to face potential pathogen rohita (0.55 mg ml-1) which is lower
attacks and it retreats and deters than the values observed in our studies.
possible parasitic attacks. It prevents Stosik et al. (2001) observed protein
colonization of various parasites. concentration values (3.06 mg ml-1) in
Many members of the fish family C. carpio which are comparatively
Cyprinidae are the intermediate and higher than ours which may be due to
definitive hosts of this bacteria and environmental differences and methods
fungi (Ebran et al., 2000) due to the of collection of mucus. Contradictory to
inherent presence of certain enzymes, previous as well as our studies
lytics and immunoglobin parasites. Mozumder (2005) observed quite
Baur (1962) and Hoffman, (1967) higher values of mucus proteins in cod
reported that Lernaea cyprinacea (38.28 mg ml-1) and salmon (9.84 mg
parasitizes Carissus auratus, Anguilla ml-1). These broader variations can be
japonica, Carassius vulgaris, Gobio due to species differences and treatment
fluviatilis and C. carpio. They further protocols. Ghafoori et al. (2014) also
claimed that parasite lacks host observed the variations in the protein
specificity to an extent that it can infect contents of the male and female
all freshwater fish and even frog Caspian kutum (R. frisii) and reported
tadpoles and salamanders. Our studies that the values can be different due
contradict the previous findings and seasonal temperature, reproductive
confirm that this parasite does have host activity and migration. Nevertheless
specificity during infestation. Probable these observations can hardly be
reasons of this specific attack were compared with our current species
explored on two Indian and three which are herbivorous while former are
Chinese major carps. Primarily we used carnivorous in nature. Moreover
three indicators- an important factor in carnivorous species do not face such
host defense mechanism. During the parasitic challenges at least in our
current studies, protein concentration environment which demands further
was the highest in C. idella (3.29±0.13 investigation to resolve these
mg ml-1) and then came C. catla with a contradictory observations. Variations1231 Abbas et al., Mucus properties of Chinese carp and Indian carp: Physical barrier to…
in the mucus proteins due to handling immune system against bacteria
stress were also investigated by the because fish remained bacteria free
Easy and Ross (2010) in the Salmo during studies (Ramasamy et al., 2011).
salar mucus. They suggested that there A band with a molecular weight of
were correlations of some mucus 31.62 kDa was present in the L. rohita,
enzyme/protein profiles with stress; C. mrigala and H. molitrix whereas
however, the variability in S. salar 29.51 kDa in C. idella may indicate the
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mucus enzyme levels and actin presence of carbonic anhydrase that has
fragmentation patterns suggested other a molecular weight of 31.0 kDa (Broad
triggers for inducing changes in mucus Range BioRad SDS-PAGE Molecular
protein composition. Weight Standards, Catalog Number
When various proteins were resolved 161-0317).
on Polyacrylamide Gel Electrophoresis Lectins are the proteins present in all
(SDS-PAGE), C. catla was the only living organisms. They recognize and
fish species which showed the protein bind to specific carbohydrates of the
band with the highest molecular weight cell surface (glycoproteins and
(100 kDa) (Table 2) which was not glycoconjugates) and agglutinate them
present in any other species. (Lis and Sharon, 1998). Lectins play a
Contradictory to all other species C. variety of physiological roles especially
carpio showed a distinct protein band in the innate immune system as a means
of 14.13 kDa size which is quite close of defense against invading pathogens
to lysozyme which has antibacterial (Hoffmann et al., 1999). Ng et al.
property against pathogens (Fleming, (2003) observed lower lectin activity
1922). It contributes to the innate (27) in grass carp than we did (29).
immune system and is a natural form of Sahoo et al. (2005) determined
protection from pathogens. The enzyme haemagglutination titer (lectin activity)
functions by attacking peptidoglycans in three Indian major carps to
(found in the cell walls of bacteria, investigate normal physiological non-
especially Gram-positive bacteria) and specific immune response under culture
hydrolyzing the glycosidic bond that conditions. Haemagglutination titre
connects N-acetylmuramic acid with values for C. mrigala (64 or 26), and L.
the fourth carbon atom of N- rohita (512 or 29) are in line with the
acetylglucosamine. The catalytic role of present study, but for C. catla (512 or
lysozyme in various pathogens in fish is 29) values are significantly higher than
prominent except in bacteria. So it can ours (26). Dash et al. (1993) also
be claimed that 100 kDa protein in C. observed haemagglutination titre
catla attracted Lernaea while 14.13 variation (32 to 512) in two catfish
kDa proteins in C. carpio completely species, viz. Heteropneustes fossilis and
deterred Lernaea attack. The catfish Clarias batrachus in different seasons.
skin mucus also contained peptide of Manihar et al. (1991) reported similar
13.3 kDa and 13.9 kDa band suggesting trends in HA activities in different
that it played a role in the innate species of the family Channidae.Iranian Journal of Fisheries Sciences 19(3) 2020 1232
Higher values for agglutination titer can (69.57±11.16 and 85±25.1 U L-1,
be used as an indicator of either a respectively) that are quite higher than
previous exposure to disease or contact that in the skin mucus of fish in the
with pathogen contaminated water present study due to the difference of
(Troast, 1975). As the fish utilized in studied medium. However, ALP
present study were from the same increased with the increase of
culture environment, the variation in infestation that is consistent with our
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agglutination titer observed during the studies. Thus our results support the
current study may be due to variations phenomena that the different fish
in their immune responses. species have different resistances to the
Alkaline phosphatase (ALP) is a environmental stresses like temperature,
potential stress indicator in Atlantic growth periods and disease causing
salmon (Salmo salar) that plays a agents and were in comparison to the
protective role in wound healing (Ross findings of Kumar et al. (2010) whose
et al., 2000). Iger and Abraham (1990) investigation indicated that mucus of
were unable to know the cause of ALP C. chanos has stronger innate immune
activity differences in the mucus of properties as compared to that of other
rainbow trout however; they suggested two fishes and therefore, polyculture of
that this variation may be related to this fish with other fish or shrimp
stressors including acidity, thermal species may have beneficial effects for
elevation, polluted water and the disease prevention.
species response to these stressors.
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