7

1. Characteristics of Nemipterus furcosus

Placed in tropical zone of the Western Pacific Ocean, the South China Sea is a

global centre of marine biological diversity (Vo et al., 2013). The area is known for

high productivity and richness of flora and fauna with more than 3365 species of

marine listed fishes (Randall and Lim, 2000). Accordingly, the threadfin breams of

the genus Nemipterus are considered as economically important fish (Russel, 1990).

N. furcosus is a benthic species, belonging to genus Nemipterus, family

Nemipteridae, order Perciformes. It inhabits sand and muddy bottoms with depths of

8 to 110 m. It is natively distributed in the West Pacific from southern Japan to

northeastern Australia, and Indian Ocean including the Gulf of Mannar (Sri Lanka),

Andaman Sea, Strait of Malacca and northwestern Australia (Russel, 1990).

Figure 1. Nemipterus furcosus

The sex ratio of N. furcosus is related to its body sizes, in which males

predominate the larger size classes. There is some evidence of sequential

hermaphrodite in this species (Young and Martin, 1985). The investigation result of

population from the northwest shelf of Australia indicated that females with ripe egg

FISH, PARASITE, HEVAY MENTAL AND GENE EXPRESSION INTERACTION

Jacky Lee, University of Boras

8

are present all year round, but a higher rate was observed in November and December.

N. furcosus is a carnivorous species with day feeding behaviour. Its main foods include

crustaceans and small fishes (Russel, 1990).

Like other fish species in aquatic environment, N. furcosus also faces parasite

infection. There are a number of studies that identify parasites in this fish (Ho and

Kim, 2004; Justine et al., 2004, 2010, 2012; Moravec and Justine, 2005; Miller et al.,

2009; Rodney and Justine, 2009; Quilichini et al., 2009). Ho and Kim (2004) reported

parasitic infection of five copepod species from family Lernanthropidae in fishes of

the Gulf of Thailand. They discovered that N. furcosus was infected by Lernanthropus

nemipteri. Justine, et al. (2012) summarized the study on parasite diversity of coral

reef fish off New Caledonia. Through examination of 239 samples, 6 parasite groups

(Isopoda, Copepoda, Monogenea, Digenea, Cestoda, Nematoda) were reported in N.

furcosus.

Besides, N. furcosus was used to study heavy metal accumulation and its

potential effects on human health. Agusa et al. (2005) evaluated concentrations of

trace elements in marine fishes and its risk to consumers in Malaysia. The results

indicated that concentration of Hg in liver of N. furcosus was higher than in other fish

species. Ahmad et al. (2015) conducted a study to determine the concentration of total

mercury in the 46 edible species of marine fish collected throughout Peninsular

Malaysia. They found that mercury level in N. furcosus is relatively high (0.642 μg/g

of dry weight). The results for total mercury in demersal fish like N. furcosus are at

higher concentration, of nearly two times more than in the pelagic fish.

2. Heavy Metal Accumulation in Fish

Heavy metal contamination in aquatic ecosystems have received increased

worldwide attention, which triggers various research efforts related to this problem

9

(Mansour and Sidky, 2001). Metals may origin either from the natural environment or

from anthropogenic activities (Djedjibegovic et al., 2012). Global anthropogenic

emissions are larger than natural emissions for most trace elements (Agusa et al.,

2005).

The industrial development at the coastal areas may results in the discharge of

heavy metals into the coastal ecosystem, leading to both species diversity and

ecosystem damages (Dhaneesh et al., 2012). Fish species which largely represent the

highest trophic level in the aquatic food chain, may accumulate large amounts of

metals in their tissues from food and aquatic environment (Rajkowska and

Protasowicki, 2013). Many studies indicated that accumulation of heavy metals in the

tissue depends mainly on metal concentrations in water, as well as exposure period

although some other environmental factors, such as temperature, salinity, pH and

hardness may also play significant roles (Dhanees et al., 2012).

Heavy metals are taken up through different organs of the fish with different

concentrated levels among organs (Karadede et al., 2004). There are several metal

bioaccumulation ways in fish which includes ingestion of particulate material

suspended in the water, ingestion of food, ion-exchange of dissolved metals across

lipophilic membranes in the gill and adsorption through tissue membrane surfaces

(Squadrone et al., 2013). Metal distribution in different tissues can serve as a pollution

indicator (Alam et al., 2002). As metabolically active organs, liver and gills are target

organs for metal accumulation, while the accumulation in muscle tissue is lower

(Subotic et al., 2013).

Studies on bioaccumulation of pollutants in fish play key roles in determining

the effects of specific pollutants on fish, metal tolerance limits of fish species, and

biomagnification through food chains (Asuquo et al., 2004). This not only is

10

significant for the evaluation of ecosystem health, but also important for measurement

of human’s health risk through fish consumption. While essential metals, such as iron,

copper, zinc and manganese can produce toxic effects at higher concentrations, non-

essential metals such as mercury, lead and cadmium are toxic, even in trace amounts

(Subotic et al., 2013). The bioaccumulation of metals is therefore an index to measure

the pollution status of the relevant water body. It is also a useful tool to study the

biological role of the metals present at elevated levels in aquatic organisms, especially

fish (Tarrio et al., 1991). Fish has been considered as a valuable biomonitoring tool

to assess environmental pollution (Padmini et al., 2004).

Heavy metals may accumulate to toxic concentrations that cause ecological

damage and toxicity to living organisms (Guven et al., 1999; Ebrahimpour and

Mushrifah, 2009). Aquatic heavy metal pollution affects various physiological

processes in fish, including breeding and development (Jezierska et al., 2009). The

embryonic and larval stages of fish are generally considered to be most sensitive to

toxicity (Zhang et al., 2012). Embryonic developmental processes impacted by heavy

metals may results in the reduction of offspring quantity and quality (Sfakianakis et

al., 2015). Adult fish exposed to heavy metal contamination are not entirely risk-free.

Fish fertility may be reduced and spawners exposed to metal bioaccumulation would

further carry forward the contamination to their eggs and sperm (Jezierska et al.,

2009).

Heavy metal in fish related studies demonstrated that deformities in fish are

mainly caused by heavy metal pollution with the most common elements including

cadmium (Cd), copper (Cu), lead (Pb), zinc (Zn), mercury (Zn) and chromium (Cr)

(Sfakianakis et al., 2015). Cd which has no biological function in superior organisms

is also very toxic even at very low concentrations (Pretto et al., 2010). Regardless of

11

its function as an essential micronutrient, Cu is one of the most toxic metals for living

organisms (Paris-Palacios et al., 2000). Toxic effects of Pb in fish include disruption

of Na+, Cl- and Ca2+ regulation, while the chronic effects of exposure to fish are

hematological and neurological damages (Mager, 2011). Waterborne Zn toxicity may

disrupt the Ca2+ uptake in gills, leading to hypocalcemia and eventual death (Niyogi

and Wood, 2006). Hg exposure can affect the behavior, biochemistry, growth,

reproduction, development, and survival of fish (Crump et al., 2009). Cr may cause

various toxic effects in fish, including hematological, histological and morphological

alterations, inhibition/reduction of growth, production of reactive oxygen species and

impaired immune function (Reid, 2011).

2. Parasite Infection in Fish

Parasite is an organism that lives in or on another organism which is

physiologically dependent on the host and can causes some degree of harm to its host

(Marcogliese and Giamberini, 2013). Parasites may reduce their host’s growth,

resistance to other stressors, susceptibility to predation and reproductive ability

(Scholz, 1999). Parasites are able to modulate biomarker responses in organisms,

including some that are routinely employed in ecotoxicological studies, such as

metallothionein, cytochrome P450, oxidative stress enzymes, and heat shock proteins

(Marcogliese and Giamberini, 2013).

To date, 95 families of Isopoda have been recognized, in which only a few are

parasitic namely, Bopyridae, Cryptoniscidae, Cymothoidae, Dajidae, Entoniscidae,

Gnathiidae and Tridentellidae (Smit et al., 2014). The family Cymothoidae includes

40 recognized genera with more than 380 species worldwide (Ahyong et al., 2011).

Cymothoid isopods are obligate fish parasites that parasitize both marine and

freshwater fishes.

12

Figure 2. Parasitic isopod is attached in the mouth of Nemipterus furcosus

Members of the family Cymothoidae are among the largest fish parasites in

size and are highly host specific (Brusca, 1981). With the size of more than 6 mm,

these isopods are easy to be observed and collected. However, many aspects of their

biodiversity and biology are still unknown (Smit et al., 2014). Adult cymothoids are

usually parasitic to fish, which normally attached to the skin, gills and oral cavity of

their hosts, whereas their juveniles are often free living (Bunkley-Williams and

Williams, 1998). Their life cycle is holoxenic, meaning that they parasitize only a

single host (Holoxenic cycle) (Ravichandran, 2010). Like most isopods, cymothoids

are considered to feed principally on blood, but they may consume mucus, epithelium

and subcutaneous tissues of their host (Bunkley-Williams and Williams 1998).

Cymothoid isopods are distributed in many different habitats, especially in

shallow waters in tropical or subtropical areas. Although the family is primarily

distributed in marine environment, it also inhibits in freshwater ecosystem. Their

diversity level is low in Africa and Asia and moderate in tropical South American

13

riverine systems (Smith et al, 2014). As obligatory parasitic species, they possess

several adaptation characteristics which include body shape with specific attachment

site on the host.

Among 40 genera of the family Cymothoidae, there are currently 48 species

recognized under genus Cymothoa (Hadfield et al., 2011). Numerous host attachment

sites are exhibited by Cymothoa spp., including the skin, fins, branchial, and buccal

cavities. Some species also burrow into fish’s musculature (Bakenhaster et al., 2006).

Despite their variability in attachment sites, cymothoids often show high host-

specificity (Smith et al., 2014). Species that are attached to the buccal cavity of hosts

are commonly referred as tongue-replacement. Of these species, the large female

almost always found attached to the host’s basihyal or also known as the ‘tongue’ with

bony structure (Hadfield et al. 2011). Cymothoids are generally protandrous

hermaphrodites (Bakenhaster et al., 2006) and enter the gills of the host as males after

a short infectious free-living manca stage. If the host’s buccal cavity is unoccupied,

the male migrates to the host’s basihyal, transforms into functional female, and awaits

the attachment of another male (Parker and Booth, 2013). The remainder of their life

cycle, including copulation, occurs within the host’s buccal cavity (Brusca and

Gilligan 1983).

Previous studies conducted on the effects of cymothoids are primarily

restricted to cultured fish (Horton and Okamura, 2001) and have shown that

cymothoids are sanguivorous and feed intermittently on the host’s blood vessels at the

site of attachment (Brusca and Gilligan, 1983; Colorni et al., 1997). In addition,

aquaculture studies have documented numerous pathogenic effects of cymothoid

infection, including mortality, tissue damage, decrease in amount of red blood cell

(anaemia), decline in mean weight and length, and inhibited growth (Parker and Booth,

14

2013). Sievers et al. (1996) studied the effects of the parasite Ceratothoa gaudichaudii

on the body weight of farmed Salmo salar in southern Chile. The findings showed that

salmon with less than three parasites weighed 4428 ± 949 g; those with three to eight

parasites weighed 4151 ± 983 g, and those with more than eight parasites weighed

3763 + 1056 g. The results indicated that high abundance of parasites significantly

reduced body weight of fish host.

Ecological studies on Cymothoa spp. are restricted to documenting parasite

prevalence and life history (Parker and Booth, 2013). Few studies have focused on the

interaction between cymothoids and their hosts within the natural environment, their

biological impacts on their hosts, and the subsequent ecological consequences (Brusca

and Gilligan, 1983; Colorni et al. 1997). However, there are several noticeable studies

of these aspects conducted recently. Through studying the effect of Cymothoid

Anilocra apogonae on the cardinal fish Cheilodipterus quinquelineatus, Ostlund-

Nilsson et al. (2005) found that parasitized fish lost more weight than non-parasitized

fish when held on a low-food regimen. This means that in harsh condition (lack of

food in this case), survival ability of parasitized fish is lower than that of non-

parasitized counterpart. The authors also observed that parasitized fish also had a

higher rate of oxygen consumption than non-parasitized fish. Results of Fogeman et

al (2009) showed Anilocra apogonae castrated its host, C. quinquelineatus. Parasitized

male failed to mouthbrood their young. The gonads of parasitized fish were smaller

and parasitized female fish have substantially fewer and smaller ova than the gonads

of non-parasitized fish. These isopods were relative large to the body size of their

hosts, averaging 3.8% of the host weight. In addition, despite the presence of other

potential hosts, A. apogonae only infested C. quinquelineatus.

15

3. Fish - Parasites Interaction under Heavy Metal Environment

The presence of organic and inorganic contaminants has led to the search for

improved monitoring methods that can express the biological and ecological

implications of pollution beyond the environmental chemical characterization (van

der Oost et al., 2003). However, due to very low concentrations of chemicals as well

as complex pollutant mixtures, chemical analyses alone are often insufficient to

determine environmental risks (Frank et al., 2012). This limitation leads to the

development and usage of biomarkers for the detection of contaminants in organisms

(Livingstone et al., 1994). Although various aquatic animal tissues have been widely

experimented for bioindicators, we are still in search of a more reliable and sensitive

bioindicators to monitor low levels of metal pollution in the environment (Sures,

2004). A number of intestinal parasites have demonstrated heavy metal

bioaccumulation capability (Marcogliese and Giamberini, 2013). Parasitic cestodes,

particularly acanthocephalans have been documented to display capacity in

accumulating heavy metals hundreds of times higher than the tissues of their fish host

(Sures, 2004). Indeed, intestinal parasites can be used as sensitive bioindicators of

heavy metal contamination in the environment (Marcogliese and Giamberini, 2013).

Fish and other aquatic organisms serve as hosts to a range of parasites. These

parasites may influence their physiology which potentially affects their metal

regulation capacity. It is also documented that parasites modify the metal

accumulation in various fish tissues and organs and compete with their host for some

specific essential metals (Oyoo-Okoth et al., 2013). When metals are excessive in

living cells, they can bind to sensitive target molecules such as glutathione,

metalloenzymes, DNA, RNA or organelles (Wallace et al., 2003). Metals are usually

16

bound to specific metal-binding proteins such as metallothioneins (MT) or are

incorporated into crystals by isomorphic substitution (Lingard et al., 1992).

Metallothioneins play a central role in essential metal regulation in organisms (e.g. Zn

and Cu). They are also involved in detoxification of non-essential toxic metals such

as Cd and Hg (Roesijadi, 1996). Recent study indicated that the presence of digenean

parasites in Cd-exposed cockles Cerastoderma edule (L.) lead to a decrease of MT

concentrations in the infected individuals (Baudrimont et al., 2006). However, there is

a lack of information in fish MT induction. In a noticeable study, Fazio et al. (2008)

found a significant positive relationship between abundance of the swim bladder

nematode Anguillicola crassus and the expression level of the fish’s MT gene.

In the study of Frank et al. (2013), cestode Ligula intestinalis showed

significant effects of Heat shock protein 70 (HSP70) gene expression in roach (Rutilus

rutilus). After a two-year experiment under controlled condition, infected roach had

significantly lower HSP levels than uninfected conspecifics. As molecular chaperones,

HSPs are a super-family of highly conserved intracellular proteins in response to

various stressors (Feder and Hofmann, 1999). The synthesis of HSP increases when

organisms are exposed to adverse environmental conditions such as heat or different

forms of chemical pollution. Accordingly, increased levels of HSPs in organisms are

usually interpreted as a general sign of protein damage (Frank et al., 2013). Parasitized

organisms are at risk in polluted environments if the synthesis of these proteins is

inhibited by parasitism (Sures, 2008).

Parasites have the ability to moderate the levels of contaminants accumulation

in their host (Marcogliese and Giamberini, 2013). Fish infected with intestinal

acanthocephalans accumulated less heavy metals in their tissues than uninfected fish

(Sures and Siddall, 1999), thus potentially reduce adverse effects of heavy metals on

17

fish health. Grass shrimp Palaemonetes pugio parasitized by the isopod Probopyrus

pandalicol accumulated lower concentrations of mercury than their non-parasitized

counterparts (Bergy et al., 2002). These findings demonstrated that parasite infection

is not exclusively negative, especially when hosts are faced with polluted

environment. Parasites might be able to reduce pollutant levels in their hosts below a

critical value, leading to reduction of adverse effects (Sures, 2008).

However, the parasite’s beneficial effects on metal reduction in host tissues

can be outweighed by their harmful impacts. Although adult acanthocephalans do not

usually kill their definitive hosts, they could cause a certain degree of damage on host’s

intestine due to their attachment mode. It is well documented that Pomphorhynchus

laevis penetrates all layers of fish’s intestinal wall with its praesoma so that the bulbus

and the terminal proboscis project into fish’s body cavity (Taraschewski, 2000).

Despite the fact that parasites have been reported to reduce the

bioaccumulation level in fish host, there are studies which showed a contrast role of

fish parasites. Surprisingly, some parasites may strengthen the toxic effects of heavy

metals in fish host. First observations of this aspect were reported in 1977, when

independent authors found that fishes parasitized by cestodes were more susceptible

to waterborne metals than non-parasitized counterparts (Boyce and Yamada, 1977;

Pascoe and Cram, 1977). As a consequence, parasitized fishes had a shorter period of

survival. Several studies have focused on parasite-induced physiological stress in

relation to environmental pollution (Sures, 2008). These studies showed that negative

effects of parasites on host’s protection system make them less protected when they

are within polluted environment (Hoole, 1997; Bonga, 1997; Sures et al., 2006).

Immunosuppressive chemicals may damage host immune system, leading to higher

18

intensities of parasites. Because of this, parasites are considered be an additional threat

when hosts are confronted with contaminant environment (Sures, 2008).

The capability of parasites in pollutant regulation has strong implications in

ecotoxicological studies and environmental monitoring programs. Heavy metal levels

in sentinel organisms can be reduced by parasites. Accordingly, measurement may fail

to indicate correct degree of contamination. Therefore, biomonitoring programs

should take into account the influence of parasite infections on the levels of metal

pollutants in sentinels (Sures, 2008).

4. Gene Expression and Molecular Biomarkers

Physiological mechanisms, which have evolved in fish, permits metal

regulation in their body tissues by limiting the uptake or active elimination of excess

metals (Giguère et al., 2006). In response to pollutants, a cell can manipulate its

metabolism, normally by activating or synthesizing specific stress-related proteins. In

general, protein concentration reflects the relative level of the corresponding mRNA,

while transcription rate is determined by the presence and the effective concentration

of stressors (Pina et al., 2007). Therefore, changes of gene expression levels in sentinel

species may serve as biomarkers for environmental pollution assessment (Tom and

Auslander, 2005). Recently, with advanced achievements in molecular biology, a new

family of biomarkers based on the transcription analysis of stress related genes has

been established. Levels of environment-affected transcripts and proteins are widely

used as environmental biomarkers (Yudkovski et al., 2008).

A biomarker is defined as a change in a biological response (ranging from

molecular through cellular and physiological responses to behavioral changes), which

can be related to exposure to, or toxic effects of, environmental chemicals (Peakall,

1994). Biomarkers measured at the molecular or cellular level have been proposed as

19

sensitive, early warning tools in environmental assessment (Pathiratne, 2010).

Traditionally, heavy metal level is measured from the whole organism or specific

organs. However, this conventional method of metal pollution monitoring does not

provide any information about the effects of heavy metal at subcellular level

(Stankovic et al., 2014). Therefore, the evaluation of biomarkers like metallothioneins

(MTs), phytochelatins (PCs), and antioxidant enzymes (catalase, superoxide

dismutase, glutathione S-transferases and glutathione peroxidases, lipid peroxidation),

may be useful in evaluating metal exposure and predicting effects caused by

environmental pollutants (Stankovic and Stankovic, 2013).

Molecular biomarker are also used to study the effects of parasites on their

hosts. In the last decade, the influence of parasites on host physiology has increasingly

been studied in the context of polluted environments (Gismondi et al., 2012).

Proteomics and genomics are considered useful tools to investigate host-parasite

interaction (Biron et al., 2005). Recently, differential gene expression in non-

parasitized fish and parasitized fish has been used to estimate the effects to parasitic

infections on the fish host (Lindenstrom et al., 2004; Collins et al., 2007; Fazio et al.,

2008; Frank et al., 2013). In this case, gene expression appeared to be a powerful tool

to analyze physiological response of fish host against parasitic infection (Fazio et al.,

2008).

Biomarkers are categorized into specific group or non-specific group. The use

of metallothionein – a metal specific biomarker has been widely employed to indicate

the presence of heavy metals (Giguère et al., 2003). Certain metals entering the

nucleus can induce the synthesis of RNA that encode MTs (Stankovic et al., 2014). As

a universal biomarker, heat shock protein (HSP) expression is commonly used for

20

cellular stress (Sherman and Goldberg, 2001). HSP functions to maintain protein

integrity in the presence of stress such as heat or chemicals (Iwama et al., 1998).

5.1. Metallothionein

Metallothionein (MT) was first discovered in 1957 by Margoshes and

Vallee via purification of a Cd-binding protein from horse (equine) renal cortex

(Margoshes and Vallee, 1957). Thereafter, numerous investigations have been

conducted to determine the function of MT in Cd toxicology (Klaassen et al., 1999).

While molecular biomarkers are considered to be one of the most sensitive and earliest

responses to pollutants (Rodríguez-Ortega et al., 2009), MTs are suggested as core

biomarkers for heavy metal assessment, because their induction indicates biochemical

response to heavy metal levels in the environment (Amiard et al., 2006).

MTs are low molecular weight (6000–7000 Da), cysteine-rich (33%), heat-

resistant cytosolic proteins containing sulphur-based metal clusters (Atli and Canli,

2008). MTs constitute a protein superfamily of 15 families, found in all animal phyla

examined to date and also in certain fungi, plants and cyanobacteria (Carpene et al.,

2007). Until now, there are four known isoforms of MT (MT-I–IV) in vertebrates

(Coyle et al., 2002). Of these four isoforms, MT-I and MT-II are the most commonly

studied MTs in aquatic species (Andrews, 2000; Coyle et al., 2002). MTs are widely

distributed in aquatic animals, and two types of MT have been identified in teleosts.

The MT-I isoform was isolated from Pleuronectes platessa, Cyprinus carpio,

Oncorhynchus mykis, Esox Lucius and Takifugu obscurus, while the MT-II isoform

was reported from Siniperca chuatsi, Carassius cuvieri and Danio rerio (Kim et al.,

2012).

Typically, MTs have high metal content comprising predominantly Zn, Cu or

Cd, highly conserved 18–23 cysteine residues and no aromatic amino acids or histidine

21

(Coy et al., 2002). Generally, MTs include two binding domains (α, β) that are

assembled from cysteine clusters called metal-thiolate clusters (Petrlova et al., 2007).

Each domain contains a ‘mineral core’ enclosed by two large helical turns of the

polypeptidic chain. The N-terminal right-handed β domain binds three bivalent metal

ions. The C-terminal α domain is left-handed and binds four bivalent ions. Zinc is

preferentially located in the β domain and cadmium in the α domain (Vergani et al.,

2005). Therefore, while the α domain may play a central role in heavy metal

detoxification, the β domain is able to regulate zinc and copper homeostasis, (Zhou et

al., 2000). The tertiary structure of MT is dynamic, and Zn and Cd exchange rapidly

within the β domain, more slowly in the α domain, and may also exchange with other

ions bound to intracellular ligands (Coyle et al., 2002). While MT is mainly found in

association with zinc, it also binds a wide range of other metal ions, including Cd2+,

Hg2+, Cu1+, Cu2+, Ag1+, Au1+, Bi3+, As3+, Co2+, Fe2+, Pb2+, Pt2+, and Tc4+ (Bell and

Vallee, 2009). Zn plays an important role in regulation of MT genes. The binding of

Zn to metal transcription factor (MTF-1) allows the protein to bind to metal response

elements (MREs) in the promoter region which, in turn, initiates MT gene

transcription (Coyle et al., 2002).

Although at the transcriptional level, MT genes can be induced by various

physiological and toxicological stimuli, MTs in aquatic organisms is proposed as

suitable biomarkers for heavy metal exposure, including Cd, Cu, and Zn since their

expression levels correlate positively with these metals (Kim et al., 2012). The

chemistry of the thiol (–SH) group determines the behaviour of MTs (Amiard et al.,

2006). The metal–thiolate clusters within the MT molecules allow rapid exchange of

metallic ions between clusters and with other MT molecules. These characteristics of

22

binding and transference of metals appear to be unique to MT and fundamental to their

biological role (Monserrat et al., 2007).

Figure 3. Metallothionein (MT) structure. Model of two binding sites of MT. Red big

beads are metal atoms (e.g., Zn), and small yellow beads are sulfur atoms (Ruttkay-

Nedecky, 2013).

To clarify the structure and function of MTs in fish, their genomic

characteristics are studied. Kim et al. (2008) reported full-length cDNA sequence of

MT gene from anadromous river pufferfish, Takifugu obscurus (family:

Tetradontidae). MT gene in T. obscurus included 183 base pairs encoding 60 amino

acids of a putative protein. It had 20 cysteine residues among the 60 amino acids

(approximately 33.3%). MT sequence of olive flounder, Paralichthys olivaceus was

identified by An et al. (2008). The full-length MT cDNA also consisted of 183 base

pairs, including an open reading frame and encoding a protein of 60 amino acids.

Aquatic organisms express MTs in association with heavy metal resistance

which was caused by rapid fluctuations of the metal content in water. Remarkable

efforts have been focused on the use of MT for monitoring metal pollution in

23

environment (Gao et al., 2009). Studies on MTs in both marine and freshwater fish

are mainly targeted to determine their functional role and validate their potential to be

powerful biomarkers (Kim et al., 2008). The main functions of MTs include

antioxidant defence and detoxification of both essential and non-essential metals

(Monserrat et al., 2007). In metal homeostasis, MTs participate in a number of

biochemical processes by providing a reservoir of Cu2+ and Zn2+ in the biosynthesis

of metalloenzymes and metalloproteins within the cells (Gao et al., 2009). MTs play

a key role in metal detoxification by strongly binding to metals and reducing metal

availability in ionic form in the cytoplasm (Wang and rainbow, 2010). The expression

of these proteins induced by heavy metals has been studied in both laboratories and

fields, although most studies have been restricted to laboratories where fish were

exposed to heavy metals (Knapen et al., 2007). Exposure to sub-lethal levels of heavy

metals may result in the production of MTs (Klaverkamp et al., 1984), e.g., in the

Cyprinus carpio, Carassius auratus gibelio (De Boeck et al., 2003), Salmo trutta

(Hansen et al., 2006), Carassius auratus (Choi et al., 2007), Gobio gobio (Knapen et

al., 2007), Sparus sarba (Man and Woo, 2010), Pelteobagrus fulvidraco (Kim et al.,

2012).

Choi et al. (2007) found that Carassius auratus injected with CdCl2 had a

significant increase of MT mRNA levels in the brain, liver, kidney and intestine tissue

in a dose-dependent manner over all tested time (6, 12, 24 and 36 h). In another

experiment, Cho et al. (2008) evaluated MT expression of eight somatic tissues (brain,

heart, gill, intestine, kidney, liver, muscle, and spleen) and two gonadic tissues (ovary

and testis) under metal-exposed condition. They observed that transcription of MT

levels was highly induced by exposures to waterborne cadmium, copper or zinc.

However, cadmium stimulated MT transcripts stronger than copper and zinc, while

24

liver was more responsive to heavy metals than gill and kidney. Knapen et al. (2007)

determined hepatic MT expression to assess the applicability of MT as an

environmental biomarker in natural fish populations. Also, significant correlations

were found between heavy metal accumulation levels with levels of MT protein and

mRNA. MT proteins are also significantly correlated to MT transcription level. The

results showed that both gene expression and MT protein had the potential to be

sensitive biomarkers for metal exposure.

5.2. Heat shock protein 70

A severe problem caused by heavy metals and organic pollutants in the marine

environment has prompted numerous studies on the effects of these pollutants on the

biological functions of aquatic organisms (Padmidi and Rani, 2008). When exposed

to chemical stress, the cell would respond by inducing a number of stress proteins that

include predominantly family of Heat shock protein (HSP) (Sanders, 1993). The

synthesis of HSPs is increased in response to a variety of physical and chemical

stressors (Yamashita et al. 2010). Due to their sensitivity to even minor assaults, HSPs

are suitable as an early warning bioindicator of cellular hazard (Gupta et al., 2010).

The HSPs were discovered by Ritossa (1962), who observed focal swellings

on polytene chromosomes in Drosophila salivary glands after exposure to a sudden

elevated temperature (Roberts, et al., 2010). They are categorized into several groups

according to their molecular mass and function. The families primarily include

HSP100, HSP90, HSP70, HSP60, HSP40 and several smaller HSP groups (Gupta et

al., 2010). Many members of HSP families have counterparts which are referred as

Heat shock cognates (HSCs). HSCs are expressed within the cell under normal non-

stress conditions (Robert et al., 2010).

25

HSPs are highly conserved molecular chaperones that can be found in diverse

organisms from bacteria to mammals (Feder and Hofmann, 1999). They are named

based on their functional role, in which they prevent inappropriate protein

aggregations; HSPs also play other vital roles in folding and unfolding, assembly and

disassembly, transport and degradation of misfolded/aggregated proteins (Benarroch,

2011). Generally, members of this family are distributed in the cytosol, mitochondria

and endoplasmic reticulum (Gupta et al., 2010). However, under stress conditions,

HSPs migrate into the cell nucleus to repair or protect the nuclear proteins and

minimize protein aggregation to prevent genetic damage (Rhee et al., 2009). HSPs act

as cellular responses to other noxious stimuli, including starvation, chemotherapeutic

agents, inflammation, anoxia ischemia, and viral and bacterial infection (Cha et al.,

2013). As a consequence, the term ‘stress protein’ is more preferably used compared

to HSP (Whitley et al., 1999). Among the HSPs, HSP70s are one of the most conserved

and important protein families (Li et al., 2015) and also the most thoroughly studied

(Rhee et al., 2009). The HSP70 family includes Heat shock proteins with molecular

weights from 68 to 75 kDa (Gupta et al., 2010).

Structurally, HSP70s possess three functional domains: (i) ~44 kDa amino-

terminal adenine nucleotide-binding domain (NBD) or ATPase domain, which binds

and hydrolyzes ATP and, provides energy for substrate binding and releasing; (ii) ~18

kDa substrate binding domain, which consists of two-β sheets that form a pocket

structure where substrate-interaction occurs; (iii) ~10 kDa carboxyl-terminal domain,

which is rich in a-helices and adopts a lid-like structure that is positioned over

substrate binding domain (Dang et al., 2010). Based on flexible structure, HSP70

group shows unique versatility, demonstrating the ability to assist in a large number

of protein-folding processes, from de novo polypeptide folding to the translocation of

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proteins across membranes (Morris et al., 2013). As molecular chaperones, HSP70s

are involved in plenty of key cellular processes as protein synthesis, translocation,

assembly, and degradation (Sharma and Masison, 2009). By producing reactive

oxygen species (ROS), heavy metals can induce oxidative stress in aquatic organisms

(Lushchak, 2011). ROS can attack cellular macromolecules such as proteins, lipids,

and DNA (Kim et al., 2014). Heavy metals mainly target structural and enzymatic

proteins (Chang and Suzuki, 1996). Under stress condition, HSP gene is

transcriptionally activated to repair damaged protein, and improves cell survival (Zhu

et al., 2014).

Figure 4. Structure of the ATPase fragment of a 70K heat-shock cognate protein

(Flaherty et al., 1990)

In eukaryotic organisms, several types of HSP70 have been identified, which

differ in expression pattern and cellular function (Li et al., 2015). There are two genes

encoding for the HSP70 protein family, a cognate HSC70, and an inducible, HSP70

gene (Deane and Woo 2006). While HSC70s are constitutively expressed in unstressed

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cells in relation to developmental progress, HSP70s are known to be induced by stress

factors such as high temperature, microbial infection, and heavy metals (Deane and

Woo 2006; Dang et al., 2010). cDNAs encoding for HSC70 and HSP70 have been

described for several fish species, such as Oncorhynchus mykiss (Zafarullah et al.

1992), Oryzias latipes and O. celebensis (Arai et al., 1995), Danio rerio (Graser et

al., 1996), Megalobrama amblycephala (Ming et al. 2010), Sebastes schlegeli (Mu et

al., 2013), Schizothorax prenanti (Li et al., 2015). Ming et al. (2010) isolated and

cloned two cDNAs of HSC70 and HSP70 from the liver of Megalobrama

amblycephala. The results determined that cDNAs included 2336 (HSC70) and 2224

(HSP70) base pairs (bp) in length and consisted of 1950 and 1932 bp of open reading

frames, which encoded proteins of 649 and 643 amino acids, respectively. Using the

same technique, Li et al. (2015) identified HSC70 and HSP70 cDNAs from the liver

of Schizothorax prenanti. They found that cDNAs were 2344 and 2292 bp and

contained 1950 and 1932 bp open reading frames, encoded proteins of 649 and 643

amino acids, respectively.

Although stress defence mechanism for most organisms is species-specific,

stress protein production is the only known universal response (Gross, 2004). Of

which, HSPs, in particular the HSP70 family have been commonly studied and used

widely as biomarkers in aquatic toxicology (Clark and Peck, 2009). To meet certain

criteria, effective stress biomarkers must be: quantifiable, universal within the study

group, sublethal, and reliable for interpretation (Morris et al., 2013). As a ubiquitous

and highly conserved family protein in nearly all organisms, genes encoding HSP70

are easy to isolate and identify within a genome. HSP70 is responsive to a large variety

of stresses and generally much more sensitive to stress than traditional markers

(Sorensen et al., 2010). HSP70 has unique versatility in terms of its activity range in

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comparison with all other stress proteins (Mayer, 2010), therefore, in most cases, it is

the easiest to detect.

To date, a number of laboratory and field studies have been performed to

evaluate the effects of heavy metals on HSP70 expression of fish. For instance,

increased levels of HSP70 have been measured in tissues of fish exposed to heavy

metals such as copper, zinc, mercury, nickel, cadmium and lead (Sanders et al. 1995;

Williams et al. 1996; Duffy et al. 1999; Boone and Vijayan, 2002; Ali et al., 2003;

Deane and Woo, 2006; Fulladosa et al., 2006; Padmidi et al., 2009; Rajeshkumar et

al., 2013). Boone and Vijayan (2002) exposed rainbow trout hepatocytes to CuSO4,

CdCl2 and NaAsO2 to investigate the effects of heavy metal on HSC70 and HSP70

expression. The results indicated that while HSP70 level was induced, HSC70 level

showed no change over the treatment. This suggested that HSC70 was not modulated

by sublethal acute stressors in trout hepatocytes. Fulladosa et al., (2006) collected

blood cells from silver sea bream (Sparus sarba), then treated them with different

sublethal concentrations of cadmium (II), lead (II) or chromium (VI). As a

consequence, HSP70 was remarkably overexpressed after exposure to metal

concentration as low as 0.1 M. Rajeshkumar et al. (2013) estimated HSP70 expression

in milk fish (Chanos chanos) at sites polluted with Cu, Pb, Zn, Cd, Mn, Fe. Compared

to less polluted sites there was an increased level of HSP70 in tissue of fish from

polluted sites. Relative expression of HSP70 in different tissue showed that the

expression intensity was high in fish sampled from polluted sites. They concluded that

HSP70 is a useful biomarker for heavy metal induced oxidative stress.