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lChapter 1

General Introduction

1.0 Introduction

1.1 Fish Parasites

Parasitism is an obligatory association between two distinct species, in which one species parasite is dependent on its host for nutrients and shelter (Elmer & Glenn, 1961). The parasites can be divided into two groups which are ectoparasites and endoparasites. Ectoparasites are found on the external surfaces of the host such as skin, fins and gills, while endoparasites usually hide within the internal tissues or organs in the host (Cheng, 1986).

Fishes acts as hosts to a wide variety of parasites such as the protozoans, platyhelminthes, acanthocephalan, nematodes and crustaceans (Lim, 1987). Parasites are typically smaller than their host. A distinct host usually found to be infected by a group of parasites (Zander, 1998).

The chemical and physical factors which are either in micro-environment or macro-environment play an important role to determine the distribution and density of parasites (Cheng, 1986). Parasites show a preference among different age groups (Zander et al., 1993), sex of host (Lim, 1987) or according to host density and behaviour (Cheng, 1986).

Remarkably, parasites have a distinguished ability to deal with the normal physiological and structural changes of the host, for example, the peristaltic movement and intestinal mucosa (Cheng, 1986) for survival, and starting their parasitism phase.

Apart from that, the presence of parasites commonly irritates the host, causing immunological change (Lim, 1979) and may eventually cause death (Zander, 1998). Parasites penetrate the hosts' tissue, causing chronic body system problems to the host. Moreover, the irritation site will initiate secondary infection by micro-organisms to the host (Cheng, 1986).

1.2 Specific parasite

Gobies play a role as main host and transmitters (Zander, 1993) of parasites. Varieties of protozoan, helminthes, mollusks, crustaceans have been described as parasites of fish (Lim, 1987). They are able to reproduce either sexually or asexually. Some species of parasites have specific attachment to enable them to hold onto the host for food, transport (Cheng, 1986) or shelter (Elmer & Glenn, 1961).

The phylum Platyhelminthes consists of a large group of free-living invertebrates but most live as parasitic species on or in other organisms. There are 3 main classes under this phylum, namely, class Monogenea, class Trematode, and class Cestoda (Solomon et al., 2002).

Monogeneans are typical gillworms (Bunkley-Williams & E. H. Williams, 1994). Some species attach themselves on the body surface or invade into the inner organ of aquatic species (Reed, Francis-Floyd & Klinger, 1996). Monogeneans have a series of hooks that enable them to attach on the host (Reed et al., 1996). They usually inhabit the gill and feed on skin mucosa (Lim, 1987).

Reed et al. (1996) mentioned that a monogenean, Dactylogyrus sp., usually attaches itself to the gills of freshwater fish. It reproduces by laying eggs, while Gyrodactylus is typically found on the skin and fins of fish. This parasite produces live young or can be described as viviparous during its whole life.

Digenea was formerly described as digenetic trematodes and this species of flukes usually has at least two hosts in their life cycle (Smyth, 1962). The parasite usually has a terminal opening mouth with a sucker which is subterminal or ventral. It may or may not have an oral sucker (Yamagutti, 1958). They are particularly found as endoparasites in the digestive tract, but are sometimes found throughout the inner organ systems of vertebrates (Yamagutti, 1958). The adult digeneans are commonly hermaphroditic but reproduction still requires two worms. Digeneans have a complex life cycle with two to three intermediate hosts (Cheng, 1986).

Cestode is a parasitic flatworm or commonly known as tapeworm (Solomon, et al., 2002). An adult worm consists of a scolex which maybe armed with suckers and sometimes hooks for attachment on host (Solomon et al., 2002); and a strobila which is a linear series that be formed by one or more segments (also described as proglottides) which contain reproductive structures (Yamagutti, 1959). Most of the cestodes are monoecious and each proglottid has a male reproductive system and a female reproductive system. Both self and cross-fertilization will take place in cestode (Smyth, 1962).

Nematode is commonly called as round worm (Solomon et al., 2002) that can infect a vertebrate's eye, mouth, alimentary system, body cavity and the other parts (Smyth, 1962). This species can also be found as a parasite of many species of fishes. Some species parasitize plants and are described as an important agricultural pest (Campbell, 2002). It is smooth and consists of a cylindrical body, a pointed tail and cuticle layer surrounding its body (Solomon et al., 2002). Nematodes are typically dioecious. The female is generally larger than the male (Cheng, 1986). Apart from that, nematode life cycles differ depending on the species of the nematode (Smyth, 1962).

1.3 The Family of Gobiidae

The members which form the family Gobiidae are known as gobies. It is the largest family of marine fishes and consists of two hundred genera (retrieved from Wikipedia). The members of the Family Gobiidae have their own special characteristics. They have a cylindrical body and the pelvic fins usually form an adhesive disc (Maurice & Anthony, 1993).

This species usually inhabits the shore in shallow bays and estuaries and also freshwater swamps and lakes (retrieved from Wikipedia). Some species are restricted to coral reefs, rocky coast, sandy, mangrove swamp or muddy beaches (Murdy, 1989).

1.4 Oxudercine Gobies (Mudskippers)

The mudskippers are known as ‘ikan belacak', ‘ikan tembakul' or ‘ikan belukor' in Malay. The members of the goby family classified as mudskipper species based on body traits and behaviours (Murdy, 1989).

Oxudercine gobies are described as the members of mudskippers (Murdy, 1989). There are thirty four species from ten genera found throughout the world (Murdy, 1989). They are Apocryptes, Zappa, Pseudapocryptes, Apocryptodon, Parapocryptes, Oxuderces, Scartelaos, Boleophthalmus, Periophthalmodon and Periophthalmus. Only seven genera of mudskippers (Periopthalmodon, Periophthalmus, Boleophthalmus, Scartelaos, Oxuderces, Parapocryptes and Pseudopocryptes) were found along the Selangor coast (Khaironizam, 2004).

According to Khaironizam (2004), Cantor (1849) had identified five species of mudskippers while Koumans (1953) had noted eight species in Malaysia. Apart from that, there were four species already reported by Macne (1968) and five species were described by Berry (1972). He also mentioned that Murdy (1989) stated 11 species of mudskippers in Malaysia while Takita (1999) had studied 12 species of mudskippers (refer to Appendix A5).

The mudskippers are euryhaline and semi-terrestrial species (Clayton, 1993). They have a rounded body and united pelvic fins. They also have a pair of protruding eyes and one to two rows of teeth in the upper jaws. Besides this, they can survive for a long time above water and move on land. Commonly, the males are smaller than the females. The mudskippers usually feed on crabs, insects, and other small organisms (Clayton, 1993).

There are several adaptations that allow the mudskippers to survive successfully out of the water. Their bronchial chamber can fill up with water which allows them to walk on land for up to several hours. They spend much of their lives out of water and are able to undergo osmoregulation as well as oxygen uptake via their skin and gills (Clayton, 1993). They are abundant in muddy areas or mangroves. Oxudercine gobies often build mud towers around their burrows as refuge from predators (Clayton, 1993).

The mudskippers have certain economic importance which provide substituted fishery for the period of torrential rain in some coastal countries. These fishes are commonly eaten in China, Taiwan, India and some parts of Malaysia. Nowadays, the mudskippers are also used as feed for aquarium fish (Clayton, 1993)

1.4.1 World Distribution of Mudskippers

Mudskippers can be found around the tropical world as well as in the coastal regions of Asia, from Indonesia to Borneo (see 3). A few species are also found in Africa and Australia, whereas none are found in the new world (refer to Appendix A1).

1.5 Literature Reviews on Gobiid Parasites (refer to Appendix A7)

There is too few research on parasitological studies of mudskipper in the Asian region. Most of the studies nowadays concentrate on the behavior and ecology of oxudercinae gobies (Khaironizam, 2004); taxonomic and cladistic of the oxudercine gobies (Murdy, 1989); and the nitrogen excretion of mudskipper in water and on land, the growth of mudskipper, habitat of mudskipper and osmoregulation system among this species (Clayton, 1993).

Mhaisen and Al-Maliki (1996) mentioned that Myxobolus pfeifferi (Sporozoa), Diplozoon sp. (Monogenea) and Neoechinorhynchus sp. (Acanthocephala) were found in dark-blotched mudskippers, Periophthalmus waltoni, from the mudflats of the Khor Al-Zubair estuary. A new record showed the heterophyid (trematode) had served Boleophthalmus pectinirostris and Scartelaos sp. as second intermediate host (Sohn et al., 2005).

Clayton (1993) indicated that cestode, agamofilarial and acanthocephalid cysts have been described as parasite in Periophthalmus koelreuteri (Pearse, 1933), Periphthalmodon schlosseri and Boleophthalmus boddarti (Khoo, 1966). Besides, the larval ascarid nematodes infected Periophthalmodon schlosseri (Khoo, 1966). Pseudapocryptes lanceolatus were found to be infected by parasitic copepod, Gnathia sp. A larval cestode was also described by Pearse (1932) in Periophthalmus modestus. A study by Choudhury and Nandi (1973) did not establish any parasitic infection on Boleophthalmus boddarti and Scartelaos histophorus. The intestinal flagellates were also noted by Pearse (1933) in Boleophthamus boddarti. The bacterial flora had been observed by Morii and Kasama (1989) to be in the intestine of Boleophthalmus pectinirostris and Periophthalmus modestus. In West Africa and Singapore, a new species of acanthocephalan was detected in Periophthalmus barbarus and Periophthalmus schlosseri.

Eighteen Gyrodactylus species were collected from gobies of the genus Pomatoschistus and the host-parasite relationship was discussed (Huyse et al., 2003). Geets et al. (1999) found that three gobies (Pomatoschistus minutus, P. lozanoi and P. pictus) were infected by Gyrodactylus arcuatus in the North Sea and noted that Gyrodactylus longidactylus n. sp. is host specific (Geets et al., 1998). This study showed that monogenea was found in Pomatoschistus lozanoi from the North Sea (Geets et al., 1998)

Longshaw et al. (2003) discovered that Gyrodactylus quadratidigitus n. sp. (Monogenea: Gyrodactylidae) was found on the on the gills and body surface of Thorogobius ephippiatus (Lowe). Neogobius fluviatilis, Neogobius kessleri and Neogobius melanostomus from Slovakia were investigated by Ondračková et al. (2003) and were found to be infected by a metazoan parasite.According to Garcia et al. (2004), Lepidogobius lepidus that has been newly recorded as being a host to thirty three cestode pleroceroides of Phyllobothrium sp.

Digenea (Aphalloides timmi, Apatemon gracilis, Podocotyle atomon, Cryptocotyle concavum, Cryptocotyle lingua) and nematode (Hysterothylacium sp.) are found to be present externally and internally in Pomatoschistus minutus, P. pictus, P.microps, Gobiusculus flavescens and Gobius niger (Zander, 2004). Pampoulie et al. (1999) noted that infection of Aphalloïdes cœlomicola has a bad impact on Pomatoschistus microps (Krøyer, 1838).

Gobies were described as main host and transmitters (Zander, 1993) of parasites. Zander (1998) noted that goby fishes serve the digenean, cestode, nematode and acanthoceplalan serve the goby fishes as their intermediate host. Pomatoschistus microps is the final host of Podocotyle atomon and Aphalloides timmi and these parasites had different abundance according to different seasons (Zander et al., 2002).

Malek (2001) mentioned that Labratrema minimus and Cryptocotyle concavum have infected gobies (Pomatoschistus microps and Pomatoschistus minutes). According to Charlebois et al. (1997), there were 144 collected round gobies parasitized by 7 species of parasites. They are Diplostomum sp.(digenean), Eustrongylides tubifex(nematode), Rhabdochona decaturensis, Spinitectus sp., Spiroxys sp.(nematode), Leptorhyncchoides thecatus (class Palaeacanthocephala) and glochidia (larvae of freshwater bivalves). Apart from that, another study showed that four of the 67 gobies had parasites and three of them were infected by Acanthocephalus dirus while the latter was infected by nematode.

Parasite infection of the male gobies was higher than female (Appleby, 1996). Most of the parasites were found from the gills, oral cavity and pharynx of gobies. Marianne et al. (2004) mentioned that Ellipsomyxa gobii (Myxozoa) infected goby, Pomatoschistus microps, as fish host. According to Lynda et al. (2004), there were four species of freshwater parasites were found in round goby, Neogobius melanostomus. These parasites included trematodes and crustaceans.

Schistocephalus solidus, Aphalloides timmi and Apatemon gracilis including a metacecaria had been found in the goby fishes (Zander, 2001). These gobies have a low richness, prevelance, mean intensity and mean abundance of parasite. The gobies are intermediate host of digenean which is Diplostomum sp., acanthocephala (Acanthocephalus dirus) and nematode, Eustrongylides sp. (Camp et al., 1999).

1.6 Objectives of Study

This is a general study on the parasites of oxudercine gobies (mudskippers) along the Selangor coastal area. The study is divided into two parts with the first part being a general survey of parasites on mudskippers while the second part discuss the relative factors affecting distribution of parasites. It also had an observation on the occurrences mudskippers along Selangor coast.

Chapter 2

Materials and Methods

2.0 Introduction

The present study focuses on the mudskippers found along the coastal area of Selangor. The fishes were caught from Jeram, Sementa (2 sites), Carey Island (3 sites) and Morib. A total of 127 specimens belonging to 7 species of mudskippers were collected and they included Boleophthalmus boddarti, Periophthalmus chrysospilos, Periophthalmus novemradiatus, Periophthalmus modestus, Periophthalmodon schlosseri, Pseudapocryptes elongates (Pseudapocryptes lanceolatus) and Scartelaos histophorus.

Fish parts including the body surface, gills and gut were observed. The weight and the size of each specimen (standard size and the length) were recorded. Apart from that, the diet of mudskippers also was documented according to their stomach contents. Next, the parasites found from different parts of fish specimens were identified and tabulated. ANOVA, Coefficient of Dispersion, the Chi Square Test and population parameters were used to analyze data.

2.1 Methodology

i) Fish collection site

The fish specimens were caught by having the sampling at Jeram, Sementa, Carey Island and Morib (see 4). The field works were carried out during spring tide because the low tide was the lowest level while the high tide had the highest point when compared to the normal time. The salinity and pH of water were recorded. Fishes were caught by using different sizes of scoop net (medium or small). Otherwise, the fish can be caught using the cast net or by angling. Some crustaceans or small fishes were used as bait to attract fishes.

The mudskippers were kept in water-filled aquarium to make sure that they may survive longer. (If the fishes die during sampling, some ice cubes can be added into plastic bag to freeze the dead fish). Species identification was done using a taxonomic characteristic key.

2.2 Fish Dissection

Each fish was killed by a blow on their head or by severing the nerve cord. The weight and the size of specimen (standard size and the length) were recorded. After that, the gill and alimentary system of the fish were taken out step by step for observations. The parts examined were placed in a petri dish (with some drops of seawater in it. The seawater possibly contained the protozoans, copepods, mud, sea grasses and the others)

- Gill removal

The operculum (a bony plate) was lifted and the gill parts were examined. Next, the operculum was cut across its base and the gill was being exposed. The gills were removed by cutting the upper and lower attachments of the arch. The gill apparatus were separated one by one into 8 pieces and the isolated gills were placed into a petri dish partially-filled with water. A pipette was used to suck and separate the parasite from the mucus, blood blot or tissues. A needle or a pair of fine forceps was usually used to scrape the mucus or tissue so as to separate the parasites from mucus or tissue.

- Alimentary system or gut part removal

The alimentary system is a canal which starts from the esophagus and ends below the rectum. The body of the fish was cut longitudinally from the operculum parts till the tail part of the fish. After getting out the gill apparatus, it was lifted out together with the alimentary system by using a pair of forceps. The alimentary tract was cut into 4 parts (stomach, small intestine, big intestine and rectum). Again, a needle or forceps was used to scrape the mucus or tissue to separate the parasites from the mucus or tissue. The food items from the stomach content of the fish were recorded because this could possibly gives us some informations on the life cycle of parasite (Mhaisen and Al-Maliki, 1996)

2.3 Parasite collection

Scalpel or dissecting needles were used to scrape gently the gill filaments and gut dermis of the fish. The parasite were sucked out by using a small pipette (if they were too small) or a pair of forceps (if they were bigger) and placed into a cavity block filled with some drops of seawater.

Then, the parasite was examined under a dissecting microscope. The parasite was preserved using different methods according to the species of parasites.

i) Fixation of the monogenean or any unknown species of parasite

A coverslip was placed over the parasite after it was placed at the centre part of a slide. The slide-fixed parasite would be examined with a dissecting microscope to make sure the slide contained the specimen.

After leaving the slide to be dry for a few minutes, a clear nail varnish was used to fix the four edges of the coverslip on the slide to prevent any movement of the coverslip. Ammonium picrate glycerin was added to the sides of the coverslip to allow it to seep under the coverslip, to fix and to clear the specimens.

ii) Preservation methods for different types of parasite

There were different parasite preservation methods used depending on the species and quantity of the collected parasites (see Table 1)

Table 1: Preparation methods for different types of parasite

Parasite

Preparation methods

Nematode

Warm 70% alcohol was poured over the worms and later the specimens were transferred to fresh 70% alcohol for storage

Trematode - Digenea

Warm 10% formalin was poured over the worms and the specimens were kept overnight in the fixative. They were transferred into fresh 70% alcohol for storage

Cestode

Warm 70% alcohol was poured over the worms and later transferred to fresh 70% alcohol for storage

2.4 Data analysis

Coefficient of Dispersion (C.D)

The coefficient of dispersion (C.D) was calculated to see how the population is dispersed, such as random, uniformed or clumped. It is calculated by dividing the variance over the mean and the relative relationship between mean and variance would establish the distribution pattern of the parasites in a population. If the C.D is close to or equals to one, the population is randomly dispersed. If the C.D is more than one, the population is clumped or over-dispersed. If the C.D is less than one, it shows that the population is uniformly dispersed or under-dispersed. The ratio of the mean to the variance is called the Coefficient of Dispersion (C.D) and the calculated formula is as below:

The Analysis of Variance (ANOVA)

The Analysis of Variance (ANOVA) (refer to Appendix A4) was used to test for significant differences among the variances. In this study, ANOVA was used to test for significant differences among the variances of parasites in the mudskippers sampled around the Selangor coastal area. In addition, the relative test had exhibited the interaction among the different factors affecting the occurrences and quantity of parasites. A test was calculated to show the correlation between the interaction of the different independent variables and the quantity of parasites on the gills and gut of the mudskipper. The significant result for fish survival in relation to the quantity of different parasite species under different variables was also documented.

Chi Square Test

The Chi Square Test was done to test whether a sample from a population follows a specified distribution or not. The Chi Square value was calculated as follow:

Where as:

= The observed number of cases which be written off as i row number of j column number

= The expected number of cases which under the H0 to be characterized with i row number of j column number

= It is run over all cells

r = The number of rows in Chi-square table

k = The number of columns in Chi-square table

The calculated value was tested at a significance level of 95%. The value was yielded by the formula which was approximated by distribution with (r-1) (c-1). If the calculated value exceeded the tabulated one, the null hypothesis would be rejected at p = 0.05.

Population Parameter

The basic parasitological parameters which are prevalence, abundance and intensity were calculated for each parasite. According to Cox (1982), prevalence is defined by the percentage of host. Formula calculation of each basic parasitological parameter is as below:

Prevalence is to look at how common a host (mudskipper) is infected in a population.

Prevalence = Number of infected host X 100%

Number of host examined

Intensity is the quantity of parasite found in the host samples (mudskippers).

Intensity = Total number of parasites

Total number of infected host

Abundance is the total number of parasites found in all the examined hosts.

Abundance = Intensity

Total number of host examined

Chapter 3

Results and Discussions

3.0 Introduction

This study focuses on parasites of oxudercine gobies (mudskippers) along Selangor coast. The fishes were caught (see Chapter 2) from Jeram, Sementa (2 sites), Carey Island (3 sites) and Morib (refer to 4). There were totally 127 mudskippers caught for study. The collected fish were identified by using taxonomic keys and cladistic analysis of family Gobiidae. 127 mudskippers consist of 7 species from 5 genera were collected. They were 27 Boleophthalmus boddarti, 36 Periophthalmus chrysospilos, 54 Periophthalmus novemradiatus, 1 Periophthalmus modestus, 1 Periophthalmodon schlosseri, 7 Pseudapocryptes elongates (Pseudapocryptes lanceolatus) and 1 Scartelaos histophorus (refer Appendix A3).

In this study, parasites of the gills, the gut and the body surface of fish were investigated (see Chapter 2). There was found nothing from the body surface of fish. Five groups of parasites found from the whole study. They were monogenean, digenean, nematode, cestode and some unknown parasite cysts. Apart from that, the environmental factors (pH and salinity of water) also were discussed (see Table 2; Appendix A2).

3.1 Parasites of oxudercine gobies (mudskippers)

Table 2 showed the summarized parasite data of oxudercine gobies (mudskippers) (refer to Appendix A2). Parasites species differ among oxudercine gobies (mudskippers) based on studied sites.

Five groups of parasites were detected from this present study (see Table 3; 5-11). Identification is not easily to be done based on general main beliefs about parasite. There are varies in morphology and biological characters among parasites (see Satyu Yamaguti, 1953). The collected parasite consists of digeneans (see section 1.2; 5-8), monogenean (see section 1.2; 9), nematode (see section 1.2), cestode (see section 1.2; 10), and unidentified parasite cysts (see 11).

Digeneans inhabit the gills and the gut of mudskippers (see Table 3). In this study, three types of digeneans were found in the gut of mudskippers. However, two of these three types of digeneans were also found in the gills. These digeneans are probably regurgitated from the gut as they have the same morphology with the gut's digeneans.

Gyrodactylus sp. (Monogenea, Gyrodactylidae) was described from the gills of mudskippers (see 8). The gills are the preferred part for monogenean. Table 3 showed that cestode and nematode inhabit the gut of fish specimens. Most of them found as encysted form in the mudskippers.

In this study, the unidentified parasites cysts were found in the gills and gut of the mudskippers (see Table 3; 11). The unidentified parasites found in the gut of the mudskippers are probably cysts of cestode or nematode. However, some unknown parasites cysts which were found in the gills are considered as myxosporid.

Table 2 - Excel

Table 3: Parasite record of the examined fish

Parasite species

Number of Species

Microhabitat

i) Digenea (see 5-8)

3

Gut and gill

ii) Monogenea (see 9)

1

Gill

iii) Cestode (see 10)

1

Gut

iv) Nematode

1

Gut

v)Unidentified parasite cysts

3.2 Parasites and their hosts

Table 4 showed the presences of parasites on oxudercine gobies (mudskippers). Eight monogeneans were found limited to Pseudapocryptes lanceolatus (see Table 4 & Table 5). Monogenean seems to be host specific and it prefers to inhabit the gills of Pseudapocryptes lanceolatus.

Next, digeneans occurred in Periophthalmus chrysospilos and Periophthalmus novemradiatus (see table 4). There were 136 digeneans found in Periophthalmus chrysospilos while ten digeneans were detected from Periophthalmus novemradiatus (see Table 5). It is possibly because digeneans are able to adapt well in Periophthalmus chrysospilos.

Periophthalmus chrysospilos, Periophthalmus novemradiatus and Periophthalmodon schlosseri were infected by cestodes (see Table 4). Most (twenty eight cestodes) were found in Periophthalmus novemradiatus (see Table 5). According to Table 5, there were twenty five nematodes found from Periophthalmus novemradiatus. Nematodes also occurred in Periophthalmodon schlosseri, mostly in the encysted form in the gut of mudskippers.

There were 402 unidentified parasite cysts (see section 3.1) observed in Boleophthalmus boddarti, Periophthalmus modestus and Periophthalmus novemradiatus (see Table 4 & Table 5). Most were found in Boleophthalmus boddarti. .

Table 4: Parasites from Different Mudskippers Species

Parasite Species

Boleophthalmus boddarti

Periophthalmus modestus

Periophthalmus

novemradiatus

Host Species

Periophthalmus chrysospilos

Periophthalmodon schlosseri

Pseudapocryptes

Lanceolatus

Scartelaos hisphorous

Monogenea

-

-

-

-

-

+

-

Digenea

-

-

+

+

-

-

-

Cestode

-

-

+

-

+

-

-

Nematode

-

+

+

-

+

-

-

Unknown parasite cysts

+

-

+

-

-

-

-

+ = Parasite detected; - = Parasite not detected

Table 5: The Quantity of Compound Parasite Community Present in the Different Mudskipper Species

Host Species

Parasites

Digenea

Monogenea

Nematode

Cestode

Unidentified cysts

Boleophthalmus boddarti

0

0

0

0

395

Periophthalmus chrysospilos

136

0

0

1

0

Periophthalmus novemradiatus

10

0

7

28

6

Periophthalmus modestus

0

0

0

0

1

Pseudopocryptes lanceolatus

0

8

0

0

0

Periophthalmodon schlosseri

0

0

25

4

0

Scartelaos histophorus

0

0

0

0

0

3.3 Parasite and their habitat

The salinity and pH of the water from the specimen collection areas were recorded (see table 2). Table 6 showed that site Sementa 2 with salinity reading from 23‰ to 25‰ and pH of water between 7.35 and 7.58 had the highest parasitic infection rate. There were 29 out of 40 samples found to have parasitic infection. Morib recorded the lowest parasitic infection rate (6%), only one out of 18 mudskippers was infected with parasite cysts. The parasitic infection rate of the mudskippers may perhaps vary with a distinct study area. The abundance of parasite was probably affected by the salinity of the macro-habitat (Zander & Reimer, 2002).

Monogenean prefers to inhabit in water with pH 7.44 (see Table 7; 13). Digeneans can be found in water with pH ranging from 5.71 to 8.71 (Table 7). Most digeneans were found in water with pH 7.35.

Cestode and nematode occurred individually in the inner body of the host. Cestodes were found from the site Carey Island 2, site Sementa 2 and Morib (see Table 2). Table 7 showed that twenty-eight cestodes were found from site Carey Island 2 (see Table 2) with water pH measured as 5.71. Nematodes could be found in water with a wide range of pH ranging from 5.71 to 8.71 (Table 7). With reference to Table 7, the unidentified parasite cysts were found more abundantly in water pH 7.35 (Table 7; 14). The abundance of parasites in a distinct area differs with pH of water based on their own optimum pH and tolerance to the environment.

Table 6: The Parasitic Infection of Mudskipper in Different Locations along Selangor Coast

Locations

Total of Samples

pH water(‰)

Salinity

Parasitic infections

Infection Rate

Jeram

5

N.A

N.A

2

40%

Carey Island 1

5

7.44

11

3

60%

Carey Island 2

36

5.71

17

14

39%

Carey Island 3

3

8.17

11

2

67%

Sementa1

20

6.81

28

5

25%

Sementa2

40

7.35-7.58

23-25

29

73%

Morib

18

7.21

25-30

1

6%

2

Table 7 : Parasites in different pH range.

pH of water

Parasite

5.71

6.81

7.21

7.35

7.44

7.58

8.17

Digenea

10

0

1

135

0

0

1

Cestode

28

0

1

4

0

0

0

Monogenea

0

0

0

0

8

0

0

Nematode

1

4

0

25

0

0

2

Unidentified cysts

0

8

0

390

1

2

0

3.4 Compound Parasite Community

Table 8 listed 402 unidentified parasite cysts recorded from the entire study. There were 146 digeneans found from the oxudercine gobies (mudskippers) and thirty two nematodes, mostly in encysted form. Monogenean was found in a small quantity from the oxdercine gobies. There were totally 8 monogeneans detected from the specimens.

Table 9 showed a variation in prevalence, intensity and abundance of gobiid parasites. Monogenean had a lower prevalence which is around 2.36%. This suggests that mudskippers are not the preferred hosts of monogeneans. Digeneans have 15.75% prevalence in this study. The prevalence of unidentified parasites' cysts was noted as 17.3%. The prevalence results obtained suggest that the level of parasitic infection is different among oxudercine gobies on Selangor coast (see Table 9). A distinct mudskipper species hardly ever found with parasitic infection under the natural condition.

The intensity of unidentified parasite cysts and digeneans were considerably higher than the other parasites. Both of them were recorded respectively as 18.3 and 7.3 intensity. The monogeneans and cestodes achieved a similar intensity at around 2.7. The low intensity indicates that the number of parasite found from a distinct host is relatively small. The compound parasite attained a low abundance in the mudskippers, in the range of less than 0.2.

Table 8: The Quantity Parasites Present in the Mudskipper from Study Sites

Parasite species

Quantity

Digenea

146

Monogenea

8

Nematode

32

Cestode

33

Unidentified parasite cysts

402

Table 9: The Total, Prevalence, Intensity and Abundance of Compound Parasite Community in Mudskippers

Parasite Species

Total

Prevalence

(100%)

Intensity

Abundance

Monogenea

8

2.362

2.667

0.021

Digenea

146

15.748

7.300

0.057

Cestode

33

7.874

2.750

0.022

Nematode

32

3.150

8.000

0.063

Unidentified Parasite cysts

402

17.323

18.273

0.144

2

3.5 Statistical Analysis

3.5.1 Distribution Patterns and Aggregation of parasites

The distribution pattern of parasite can be estimated using Coefficient of Dispersion (C.D) and scheming the relationship between the mean (X) and the variance (S2). The coefficient of dispersion (C.D) predicts the aggregation of parasites is neither random, uniform nor clumped (see Table 10 & 11).

The result of Coefficient of Dispersion (S2 > X) showed that the distribution of the parasites in a study site was clumped (see Table 10). The phenomenon may be affected by environmental factors. Table 11 showed that the parasite distribution was more clumped than expected in the oxudercine gobies. The parasites usually are considered to be host specific. They need certain host to complete their life cycle. The host provides them with a suitable environment and nutrition.

Table 10 : Distribution of Infected mudskippers in Study Sites

Number of infected fish

Number of study sites

Mean, X

56

7

8.0

Variance, S2

632

7-1=6

105.3333

C.D

13.1667

Table 11 : Distribution of Parasites in Oxudercine Gobies (mudskippers)

Number of parasites

Number of fish species

Mean, X

621

7

88.7143

Variance, S2

123209.4

7-1=6

20534.9

C.D

231.4723

3.5.2 ANOVA Test

Correlation analysis was conducted using a S-PLUS statistical method (S-Plus 6.2 Academic Site Edition). The analysis of variance (one way ANOVA) was used to determine samples differences (refer to Appendix A4). Correlation analysis determined the linear relationship among the measured responses. A one-way analysis of variance (ANOVA) on each measured factors indicate that the significance between the occurrences of parasite with a distinct host species (oxudercine gobies) in a study area.

Hypotheses:

: The occurrence of certain parasite is independent with host species in a studied site

HA : The occurrence of certain parasite is dependent with host species in a studied site

3.5.2.1 ANOVA testing for 3 measured factors on parasites of the oxudercine gobies (mudskippers)

ANOVA test was done using 3 factors which are the host species, the sex of host, and the study site. This test may determine how the factors interact with gobiid parasites along the Selangor coast. Table 12 showed that the host species and environmental factors (macrohabitat) significantly affect the occurrence of parasite in the mudskippers. The sex of host (female or male) may or may not affect the occurrence of parasites on mudskippers.

Table 12: The three main factors affecting the occurrences of parasites on mudskippers

(P-values for main effect)

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

Effect df SS MS F P

Host species 6 4.75 0.79 4.69 0.00027 (S)

Host Sex 1 0.57 0.57 3.38 0.06843 (S/NS)

Study Site 6 5.73 0.96 5.65 0.00004 (S)

Residuals 113 19.13 0.17

* Total parasites, X = Log(X+1)

3.5.2.2 ANOVA testing for 3 measured factors on the gill parasites and gut parasites

ANOVA test was used to determine the relationship among the host, the sex of host and the study site with reference to the gill parasites (see Table 13) and gut parasites (see Table 15). The four genera of fish specimens were Boleophthalmus, Periophthalmus, Pseudapocryptes and Periophthalmodon.

i) Gill Parasites

Table 13 showed a significant effect between the host genera and the occurrences of parasites. The parasites prefer to serve certain mudskipper species as their host. The study site with a probability value of 0.06 indicates that there were some interactions between the environment of study site (macrohabitat) and the parasites on the gill of mudskippers (see Table 13).

There were 3 groups of parasites found from the gills. They were digenean, monogenean and unidentified parasite cysts. Digeneans and monogeneans interact significantly with the host and environment (see Table 14). Both parasites were suggested to have host specificity.

Table 13: The Three Main Factors Affecting Gill Parasites on Mudskippers

(P-values for main effect)

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

Effect df SS MS F P

Host genera 3 1.668436 0.5561453 7.812235 0.0000854 (S)

Study Site 6 0.887823 0.1479705 2.078559 0.0609336 (S/NS)

Host Sex 1 0.000172 0.0001717 0.002412 0.9609126 (NS)

Residuals 116 8.257925 0.0711890

* Total parasites, X = Log(X+1)

Table 14: Result of ANOVA Test for Gill Parasite against Host Species, Host Sex and Study Site. (P-values for main effect)

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

P - value

Parasites

Host species

Host Sex

Study Site

Digenea

0.003838 (S)

0.758516 (NS)

0.004617 (S)

Monogenean

0.000000 (S)

1.000000(NS)

0.000000 (S)

Unidentified Parasite cysts

0.238282 (NS)

0.981684 (NS)

0.812846 (NS)

ii) Gut Parasites

The existences of a distinct gut parasite maybe affected by the host species and the environmental factors (see Table 15). The host genera and the environment of the macrohabitat have a significant effect (in the range of P<0.05) on the existence of the gut parasites. There were four types of parasites were found from the gut of the mudskippers. They were digenean, cestode, nematode and unidentified parasite cysts. Table 16 showed that the host fish species and environment of the macrohabitat could affect the quantity of digeneans. Digenean was specific to host and environment of macrohabitat. The other gut parasites showed significant interaction with host species.

Table 15: The Three Main Factors Affecting Gut Parasites on Mudskippers

(P-values for main effect)

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

Effect

df SS

MS

F

P

Host genera 3 1209.362 403.1208 6.136085 0.0006587(S) Locality 6 1028.375 171.3958 2.608893 0.0208333 (S)

Sex 1 108.130 108.1295 1.645889 0.2020990 (NS)

Residuals 115 7555.125 65.6967

Table 16: Result of ANOVA Test for Gut Parasite against Host Species, Host Sex and Study Site (P-values for main effect)

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

P

- value

Parasites

Host species

Sex

Locality

Digenea

0.000000 (S)

0.000488 (S)

0.0000000 (S)

Cestode

0.028748 (S)

0.134147 (NS)

0.290563 (NS)

Nematode

0.000000 (S)

0.406793 (NS)

0.123248 (NS)

Unidentified Parasite cysts

0.012161 (S)

0.965951 (NS)

0.506758 (NS)

3.5.3 Chi Square Test

Hypotheses:

: The parasites do not distribute by preferred part of a specific host species

HA : The parasites distribute by preferred part of a specific host species

Chi square statistics is used to test how a sample from a population follows a specified distribution. The Chi square statistics result show that a significant effect at the probability (P) value is less than 0.05. The host species and environmental factors (microhabitat and macrohabitat) independently played an important role in affecting the number and type of parasites (see Table 17). Parasites distribution was clumped among the mudskippers. They were host specific and have their own preference to inhabit the particular part of the mudskippers.

Hypothesis Tested

X² statistic

P- value

Host species X Parasite compound community

1932.09

0.00 (S)

Host Species X Location of parasite on fish

(Gill and Gut)

110.5

0.00 (S)

Table 17: Result of the X² statistic on the association analysis between host species and parasite compound community; the host species and infection site of parasites.

* Significant (S) = P<0.05

* Non-Significant (NS) = P>0.05

Chapter 4

General Discussions

4.0 Introduction

The compound parasite community which was identified from different species of mudskippers consist of digenea, monogenea, cestode, nematode and unknown parasite cysts (see Table 3). Seven species of mudskippers [Boleophthalmus boddarti, Periophthalmus chrysospilos, Periophthalmus novemradiatus, Periophthalmus modestus, Periophthalmodon schlosseri, Pseudapocryptes elongates (Pseudapocryptes lanceolatus) and Scartelaos histophorus] were collected along Selangor coast. The environmental factors may affect the density and distribution of the parasite (Cheng, 1986) based on the host species.

4.1 Parasites of oxudercine gobies (mudskippers)

4.1.1 Parasite compound community

Varieties of parasites serve the fish as host in nature (see Chapter 1). Most parasites have special attachment and they depend on host metabolically (see section 1.2). Parasites suck foods and nutrients from host and set off their new stage of life cycle in the host. Besides, they able to move frequently to deal with the typical physiologic changes of the host's inner body (see section 1.2).

The parasite species differs among mudskippers in a same studied region (Marcogliese, 2002). Digenean, monogenean, cestode, nematode and unidentified parasite cysts were found internally from oxudercine gobies in the present study (see Table 3). The previous study noted that the parasitic protozoa, copepod, bacterial flora and larval helminth parasite infected different species of the mudskipper (Clayton, 1993; Table 18).

Digeneans were more widely found in Periophthalmus chrysospilos than in Periophthalmus novemradiatus. Periophthalmus chrysospilos maybe served as main host by digeneans (see section 3.2). The parasites have specific attachment organs enabling them to hold onto the hosts for nourishment and transport (see section 1.2). Digeneans cysts could be found in the gills of the fish. Moreover, the gills chamber of the gobiid fishes is important macrohabitat for digeneans to complete their life cycle (see Zander, 2004; c.f section 3.1). The adult of digeneans (migrate from first intermediate host) hide within the abdominal cavity of the gobiid fishes and the attachment of these parasites may induce the mortality of their host (Pampoulie et al., 1999).

Monogenean (Gyrodactylus sp.) has a direct life cycle in the fish (Cheng, 1986). Monogeneans are gill-inhabiting parasites (Bunkley-Williams & E. H. Williams, 1994). This species parasite also can be found on the fins, in the alimentary tract or on other parts of fish (Lim, 1979). The gills are important to monogenean. This parasite feeds on blood which is accessible in the gills (Lim, 1987). Monogenean seems to be host specific since only Pseudapocryptes lanceolatus harboured these parasites in the study area (see Table 4). Pseudapocryptes lanceolatus have longer gill filaments rather than other mudskipper specimens. The function of the gills almost similar among fishes, whereas the morphology of the gills differs based on fish species (Lim, 1979). Monogenean has a low parasitic infection among the mudskipper species along the Selangor coast (see section 3.4).

A huge quantity of unidentified parasite cysts could be found from Boleophthalmus boddarti (see section 3.2; Table 5). Boleophthalmus boddarti may be the main host for the unidentified parasite cysts if it is compared with Periophthalmus novemradiatus. Periophthalmus novemradiatus was probably served as occasional host by these unknown cysts because of its lower infection rate (see. Zander & Reimer, 2002). The parasite cysts found in oxudercine gobies probably were considered as the myxosporid (in the gills) or the cysts of cestode or nematode (in the gut) (see Chapter 3). These cysts are apparently not digested by the host (Zander & Reimer, 2002). No current study shows that theses parasites could obviously have impact on the biology of the mudskippers.

Cestode and nematode could be found in Periophthalmus novemradiatus and Periophthalmodon schlosseri (see Table 2; Table 3). Most were found in encysted form from the gut dermis of the mudskippers. Instead, the adult cestode has attachment hooks (see section 1.2), and the gut (see Table 3) is probably acted as a preferred site by them. Their attachment on the gut dermis of the host may be related to the nourishment that the gut provides (Cheng, 1986).

The unidentified parasite cysts, digenea, nematode and cestode were recognised as gut parasites (see Table 3). The oxudercine gobies (mudskippers) may serve as their intermediate host. The 5 groups of parasites showed different prevalence, intensity and abundance (see. Section 3.4). The gobiid fishes have a low prevelance, intensity and abundance of parasite (Camp et al., 1999). The low abundance indicates that such a parasite could not obviously bring the host to death (Pampoulie et al., 1999).

Parasite distribution is more clumped than expected due to the host species and the environmental effects (see section 3.5.1). They do not follow a specified distribution in a macrohabitat (see section 3.5.3). Most parasites attach on hosts by using their distinctive adhesive organs (see section 1.2). Different species of parasites may metabolically depend on a host at different levels (Cheng, 1986). They may bring bad effects or damages to the host (Zander, 1998). Further experimental study on the effect of parasites on mudskippers is needed.

4.1.2 Host species and their occurrences

Mudskippers are euryhaline organisms and they typically inhabit in mudflat and mangrove areas (Clayton, 1993). According to a previous study (from year of 1984 to 2004), 17 species of mudskippers which distribute along the Selangor coast were collected for taxonomic research. In this present study, 7 mudskipper species were caught and examined for parasites (see Appendix A5).

A total of 127 mudskippers were collected at random, along the Selangor coast (Table 2). The length (L) of specimens principally ranged between 3.90 and 16.00 centimetres. The weight (W) was noted to be from 0.40 to 108.10 gram. It is suggested that the specimens consist of the mudskippers from different generation stages.

Mudskippers are restricted to different environments (Khaironizam & Norma-Rashid, 2004). Boleophthlmus, Pseudapocryptes and Scartelaos are more recognised as an aquatic species than Periophthalmus and Periophthalmodon (Clayton, 1993).

Periophthalmus novemradiatus is a vast parasitic-infected species with various parasites being found from it. These species of mudskippers are commonly found along the straits of Malaysia (see Appendix A1). Periphthalmus modetus is rarely found on Selangor coast (see Appendix A1). This species is often found in East Pacific region (see Appendix A1).

The food content of fish may provide information on the life cycle of the parasite (Mhaisen and Al-Maliki, 1996). Unexpectedly, the diet of the mudskipper specimens is almost similar among each others. In this case, it is not an important clue to determine the parasitic infection on mudskippers. The mudskippers commonly take fish plankton, seaweeds, crustaceans and copepods as foods, while some of them prefer to eat seashells or insects (see Table 2). Apart from that, the preferences of each mudskipper species differs based on their own behaviour and food accessibility in a region. Periophthalmodon sp are recognised as carnivorous while Boleophthalmus sp is categorised as an herbivore (Clayton, 1993).

The abundance or richness of a mudskipper species in an area or habitat is determined by preferences of a mudskipper on the substrate circumstance or the presence of muddy substratum and accessibility of food supply (Khaironizam & Norma-Rashid, Y., 2005).

4.2 Factors affecting species distribution of parasites

The environmental factors may restrict the intensity and distribution of parasites on a host (Zander, 2002). The quantity of parasite is possibly influenced significantly by pH and the salinity of water from different study areas (refer to section 3.5.2). The existence of a distinct parasite is probably affected by the environment of macrohabitat (ecosystem) and microhabitat (host's inner condition) (Pampoulie et al., 2004).

Parasites are able to inhabit in extreme environments (Cheng, 1986). The host species obviously restrict the occurrence and distribution of certain parasites in a particular area (see Chapter 3). Moreover, the occurrences of parasites also depend on the tolerance of their host as well as in brackish water (Zander & Reimer, 2002).

Monogenean is host specific (see section 3.1). This parasite was only found from site Carey Island 1 which was recorded to have 11‰ and 7.44 pH of water. It has a low abundance along the Selangor coast (see Table 9). Besides this, cestode, nematode and digenea are less specialised to oxudercine gobies and the environment in a study site (see section 3.1-3.3). These parasites are more generalist to pH and salinity of water because they are widespread in a wide range of pH (see table 7). They develop their life cycle based on their own optimum condition (Cheng, 1986) and the occurrences of their distinct host (Zander & Reimer, 2002).

In this case, the occurrences and abundance of parasites are determined by environmental changes. The parasites may tolerate into different characteristics, such as the type of water or environment. The pressure of the environment such as the salinity and pH of water would affect the intensity and abundance of parasites (Zander & Reimer, 2002). The parasites will clump in a favourable environment. Thus, the occurrences of parasite is determined by the adaptation of the parasite as well as its host in a ecological system (Zander & Reimer, 2002; section 3.5.2)

The preference of a parasite towards the different species of hosts (see Table 9) differs with each other, based on the study areas. The 7 study areas have ecological differences. Site Carey Island 1 is situated near an oil palm estate and estuarine area where the water level is always constant. Site Carey Island 2 and site Carey Island 3 are in a river bank area. The fishes from Jeram were collected from mud flat or mud bank. Sementa is located in the mixed forest zone of mangrove area. Whereas the field area in Morib where fish collection was carried out is a vast sand mud beach site. The area is situated nearby the mangrove forest.

4.3 Limitations

It was not easy to obtain a complete mudskippers collection from Selangor coastal area for parasitological study in such a limited time. The best time to catch the mudskipper as during spring tide. Not all the mudskipper species from Selangor coastal area were included into the statistical analysis. The samples sizes were not in a sufficient number for data analysis because the specimens were collected randomly from different studied areas. Thus accuracy of the data analysis maybe affected.

4.4 Suggestions for further study

It is important to have a sufficient number of mudskippers to observe, so as to ensure a good study. Investigations should be carried out throughout the whole coastal area in Malaysia. The research areas may include the area along the West and East Strait of Peninsular Malaysia, Sabah or Sarawak. Diversity of oxudercine gobies and parasite species of mudskippers could be discussed between Strait of Peninsular Malaysia or East and West Malaysia. A complete study should be formed for different species of oxudercine gobies.

Gobies play an important role as a transmitter of a parasite. Parasites may capitalize within the food webs in an ecosystem. Dietary studies may give information about the life history of gobiid parasite. A histological work could be carried out to study the damage of the host by the parasite. Different types of parasites have different preferences on the host species and the attaching site. The effect on the host due to the attachment of parasites as well as certain species of parasites could be investigated.

Species identification of parasites should be done. This will be useful for parasite taxonomy work (parasites were stained for morphological study), histological work and further study of gobiid parasites. This project already sets up a good baseline data for gobiid parasites. Various literatures and information sources on gobiid parasites have been cited, and a general introduction about oxudercine gobies and their distribution has been done. A database about parasites on oxudercine gobies (mudskippers) should be set up to share this information with other people. .

Table 18: Parasite Record of Previous Study and Present Study Based on Oxudercine Gobies (Mudskipers).

Host Species

Parasite Record

Previous Study

Present Study

Boleophthalmus boddarti

Intestinal flagellate, agamofilial and

Unidentified parasite cysts

acanthocephalid cysts, cestode

Pseudapocryptes lanceolatus

Parasitic copepod

Monogenea

Periophthalmus novemradiatus

Cannot be found

Digenea, cestode, Nematode,

Unidentified parasites cysts

Periophthalmus chrysospilos

Cannot be found

Digenea, cestode

Periophthalmus modestus

Bacterial flora

Cannot be found

Periophthalmodon schlosseri

Larval ascarid nematode, cestode,

Nematode, Cestode

agamofilial and acanthocephalid cysts,

acanthocephalan species, parasitic

Copepod

Scartelaos histophorus

Parasitic protozoan, myxosporidian

Cannot be found

sporozoan, heterophyid fluke

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