2.0 Research

2.1 Research Activities in 2007

In 2007 AKUATROP had given some money to lecturers in the Aquaculture

Research group to kick-start some projects. The lecturers have to apply for grants

namely FRGS (Fundamental Research Grant Scheme) and E-Science Fund to

supplement the projects. Some of the projects funded have been classified as flagship

projects of AKUATROP

Research Topics Researchers

Breeding and cultivation of horseshoe crab (Tachypleus gigas)

Dr. Zaleha bt Kassim

Captive culture of marine ornamental organism Assoc. Prof. Dr. Abol Munafi Ambok Bolong

Induce breeding of marble goby (Oxyeleotris marmorata)

Assoc. Prof. Dr. Abol Munafi Ambok Bolong

Fish disease on mariculture system of Terengganu, Malaysia

Prof. Dr. Faizah Shaharom

Inventorization of Bacterial Diseases and Experimental Inactivation of Selected Bacterial

Disease in Malaysian Ornamental Fish Assoc. Prof. Dr. Najiah Musa

Induce breeding of Malaysian Mahseer (Tor tambroides)

Assoc. Prof. Dr. Abol Munafi Ambok Bolong

Culture of Tiger Grouper (Epinephelus fuscoguttatus) in recirculating system

Assoc. Prof. Dr. Abol Munafi Ambok Bolong

Tank cultivation of Gracilaria species with enclosed water recirculation system and its agar evaluation

Prof. Dr. Faizah Shaharom Dr Siti Aishah Abdullah

Live feed culture Assoc. Prof. Dr. Anuar Hassan

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Title of project : Breeding and cultivation of Horseshoe Crab

Research fellow : Dr. Anil Chattergii, from Institute of Oceanography, India.

Project Leader : Dr. Zaleha Kassim

AKUATROP has been rearing 160 broodstocks of horseshoe crabs of two

species which are the coastal horseshoe crab (Tachypleus gigas) and mangrove

horseshoe crab (Carcinoscorpius rotundicauda). The broodstocks were bought from

Flexible Tech company based in Johor and also from local fisherman in Kelantan. The

broodstocks are reared in different tanks with circulation system in the hatchery. They

also produced approximately 600 eggs from July to October and there were only 5 eggs

which managed to hatch into larval stage in September and also in November. Presently

one master student is carrying out this research in Balok, Kuantan, Pahang.

Adult Horseshoe Crab hibernating in sand

New Hatchling Horseshoe Crab Horseshoe Crab fertilized egg

Mating Horseshoe Crab

Title of project : Validation of the biomedical potential in the amoebocyte

lysate of Malaysian horseshoe Crabs, Tachypleus gigas

(Muller) and Carcinoscorpius rotundicauda (Lattreili) and

their sustainable use for the society.

Research fellow : Dr. Anil Chattergii, from Institute of Oceanography, India.

Project Leader : Dr. Noraznawati Ismail

Apart from ecological studies UMT lecturers led by Dr. Noraznawati Ismail is

studying the bioactive compounds from Horseshoe Crab for biopharmacy and medicinal

products.

The main objective of this study is to develop potential Tachypleus Amoebocyte

Lysate (TAL) from T. gigas.

Horseshoe crab, namely Tachypleus gigas and Carcinoscorpius rotundicauda

are commonly found on Malaysia shores. In the East Coast of Peninsular Malaysia, the

coastal population concentrates in Kuantan, while the mangrove species are found in

Tok Bali, Kelantan and Setiu, Terengganu.

Researchers from AKUATROP and Institute Marine Biotechnology, University

Malaysia Terengganu will work together to carry out this project. A research network will

be established with other universities such as University Sains Malaysia and University

Darul Iman Malaysia, mainly on Chem-Informatics.

Title of project : Captive culture of marine ornamental organism

Project Leader : Assoc. Prof. Dr. Abol Munafi Ambok Bolong

Project 1: Broodstock production of False Clownfish (Amphiprion ocellaris) in captivity

E-Science grant : RM180,000.00.

The objectives of the project are to study:

Sexual Dimorphism of A. ocellaris morphometric characteristics

Histological studies on the gonad of A. ocellaris.

Identification of male and female A. ocellaris sex marker.

Twenty families of A. ocellaris were collected at Pulau Bidong and Pulau Rhu,

Terengganu. The fish was then weight and morphometric measurements were done by

following Park et al. (2003). Gonads from male, female and non breeders were taken out

before being fixed in Bouin’s solution. Standard histology protocols were done to

observe the gonad structure of each fish according to sex. The gonads were then

observed under microscope equipped with Motic Camera for identification according to

sex.

For genetic work, tissue and fins of 20 male and 20 female fish were dissected.

They were then preserved in vials containing absolute ethanol and was stored under -20

0C prior to DNA extraction. DNA was extracted using commercial DNA extraction kit.

OPERON primers will be used to amplify genomic DNA and purification will be

conducted using commercial PCR purification kit. The PCR amplification will be

conducted following Dinesh et al. (1993). The bands of DNA that represent each sex

specific fragments and the primers that amplified the bands will be identified at this point.

Clownfish gonad in bouins fixative. Tissue and fin sample for DNA extraction

Project 2 : Study on the mechanism of sex determination of Protandrous

Anemone fish (Amphiprion ocellaris)

FRGS Grant : RM15,080.00

The objectives of this project are:

To design specific primer for sex identification of A. ocellaris.

To study sexual determination mechanism in A. ocellaris.

This project involves amplification and isolation of the DNA fragments of interest.

Then, DNA fragments will be inserted into vectors by using commercial cloning kit to be

transformed. The transformed DNA will then be incubated in ice for 20 minutes followed

by heat shock for 30 second at 42 ˚C. Then it will be incubated in ice again for 2-5

minutes. SOC medium will then be added and they are incubated in it for 90 minutes at

37 ˚C. The transformation culture will be spread on an LB agar containing ampicillin (50

µm ml-1), IPTG (40 mg ml-1) and X-gal (50 mg ml-1). The plates will be incubated at 37 ˚C

overnight. White colonies from the agar plate will be transferred to LB broth containing

ampicillin (50 µm ml-1). The broth will be incubated overnight at 37 ˚C at 250 rpm. The

culture medium with cloud water will be extracted using commercial plasmid extraction

kit following the protocols provided by the manufacturer.

Confirmation of the sex specific markers will be determined using designed

primers. The amplification products will then be analyzed by gel electrophoresis and the

presence or absence of a single band of sex-specific fragments will be identified.

Embedding process for gonad study. Histological study- gonad tissue processing.

Project 3 :Sexual differentiation and reproduction aspect of Tomato Anemone

fish (Amphiprion frenatus)

FRGS Grant : RM11,848.68

The objectives of this project are:

To identify feeding behaviour between sexes

To propagate A. fernatus in captivity

For feeding behavior study, 10 pairs of fish will be put in an aquarium each where

they will be acclimatized for 2 weeks. Fresh cockles and spirulina will be mixed and

blend together before being fed to the fish two times a day; morning and evening. The

Sampling activities.

Female fish (above), male fish (below) Tissue and fin sample for DNA extraction.

Tanks for hormone treatment. Sampling activities.

behavior of both sexes such as the aggressiveness and amount of food taken will be

observed before, during and after being fed.

For propagation experiment, the broodstock will be fed twice daily. Different

types of food will be tested to see which food can stimulate the broodstock to spawn.

Large PVC pipes will be provided as a breeding substrate in each aquarium. The water

quality will be monitored and maintained daily.

Project 4 : Identification and propagation of copepod for aquaculture

FRGS Grant : RM11, 848.68

This project was conducted to evaluate the effects of different feeding type

towards the size and population of marine copepod (Acartia sp.).

This one month feeding study consist of four microalgae diets (Isochrysis sp.,

Tetraselmis sp., Nannochloropsis sp.,Chlorella sp.), two non algae diets (rice bran and

yeast) and a control (no food given). For each treatment the density of copepods were

set at seven individual ml-1 of culture medium. They were fed once a day (20 x 104 cells

ml-1 for microalgae and 0.6-0.8 gL-1 for yeast and rice bran). The size was measured

once a week and the population was counted every two days.

At the end of the experiment, Acartia sp. fed with Isochrysis sp. had significantly

(P<0.05) bigger size (591.15 ± 5.33 µm) (Figure 1) than other treatments. The

Tomato clownfish, Amphiprion frenatus Broodstock tanks

population of Acartia sp. was also significantly (P<0.05) larger (313 individual ml-1)

(Figure 2) when fed with Isochrysis sp. compared to other treatments. Unfortunately, for

rice bran and yeast treatment, all the Acartia sp. died at beginning of the experiment.

This shows that Isochrysis sp. is the best and most reliable diet to produce Acartia sp.

for marine fish larvae culture compared to other microalgae diets while the non-algae

diets are not suitable for culturing Acartia sp.

Title of project : Induce breeding of marble goby (Oxyeleotris marmorata)

Project Leader : Assoc. Prof. Dr. Abol Munafi Ambok Bolong

AKUATROP is now renting a few earthen ponds in Maras to study the Marble goby

culture in various aspects. The objectives of this flagship project are:

The broodstock management to produce extensive larvae for culture;

To study the growth rate, survival and diet requirement for broodstock and larvae

reared in captivity hatchery and earthen ponds;

To produce extensive live feed culture for larvae, fry and fingerlings of marble

goby.

A total of 51 broodstocks (19 males: 32 females) were separately placed in two earthen

ponds in Maras, Terengganu and together with tilapia in a poly-culture system. The

brood stocks were left for natural breeding in substrates made of 40 – 50cm length and

18cm diameter PVC pipes. These substrates also function as egg collectors.

591.15

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Isochrysis sp.

Tetraselmis sp.

Chlorella sp.

Nannochloropsis sp.

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Isochrysis sp.

Tetraselmis sp.

Nannochloropsis sp.

Chlorella sp.

Figure 1: Size of Acartia sp. at the end of study period (µm).

Figure 2: Population of Acartia sp. at the end of study period (ind ml-1)

AKUATROP officers will come and check for eggs every two days. The substrate which

have eggs, were placed in small cages made of 200µm net to study the growth and

survival rate of the fertilized eggs. This project is being carried out by a masters student

from Malaysia.

Checking the fertilized eggs Larvae which hatched

Integrated culture with tilapia Washing fertilized net

Adult of Marble Goby

Title of project : Parasitic Crustaceans in Marine Cultured Fish: Prophylaxis

And Treatment Studies for Marine Cultured Fish against the

Marine Parasite Crustacean.

Research Fellow : Prof. Patrick Woo from University of Guelph, Canada

Project leader : Prof. Dr. Faizah Shaharom

There has been a large epidemic and huge mortalities of the sea bass Lates

calcarifer cultured in cages in Tok Bali, Kelantan. These sea bass were heavily infected

with the crustacean parasite Lernantrophus species which are blood feeders and cause

acute respiratory failure of fish.

With the increasing use of risk analysis for disease prevention and the

development of precautionary management measures, generating information to support

bio-security assessments should be given high priority. Research to support aquaculture

bio-security should be focused, for instance, on pathways of pathogen spread, methods

for inactivation of infectivity, and development of vaccination strategies.

There is still lack of information about the parasitic crustacean and their life cycle

from Malaysia, the crustacean parasite may require a different life cycle period due to

different climatic condition. Due to that reason stated above, research in taxonomy and

life cycle of this parasite is important in order to understand their biology. The knowledge

of their characteristic and life cycle will help in developing a new methodology to prevent

the infection of this parasitic crustacean in local marine cultured fish. By understanding

their life cycle we can try to culture this parasite for further studies such as vaccination

work. The mass culture of the crustacean parasites will lead to new studies and

biotechnological approaches of culturing the parasite. This will be the first study on

taxonomy and life cycle of parasitic crustacean in Malaysia.

Hence AKUATROP together with NAFISH (National Fish Health Centre) and Marine

Finfish Production and Research Centre (PPPIL) have embarked on the study of these

crustacean parasites and developed methods of treatment and control.

National Fish Health Centre (NaFish)

Collaborations based on Sampling Site

Setiu, Terengganu

Marine Finfish Production

and Research Centre

(PPPIL)

Penang

AKUATROP, UMT

+ +

Tok Bali, Kelantan

Sg. Pinang

Species of parasitic crustacean have been observed and collected.

Caligus sp.Lernantropus sp.

Female Male

Title of project : Inventorization of Bacterial Diseases and Experimental

Inactivation of Selected Bacterial Disease in Malaysian

Ornamental Fish

Project leader : Assoc. Prof. Dr Najiah Musa

The objectives of the project are to isolate, identify and characterize bacterial

diseases found in marine ornamental fish in Malaysia and to investigate the

effectiveness of chemicals in treating selected bacterial disease. The project ran

smoothly according to schedule for phase 1 i.e. June till December 2007 where they

managed to isolate, identify and characterize bacteria from ornamental fish. The 2nd

phase (Jan 2008 until June 2009) will be continued under new funding. Two papers

were presented at SEAFDEC International Workshop on Emerging Fish Diseases in

Asia held in Bangkok, Thailand from 6-7th December 2007.

Title of project : Induce breeding of Malaysian Mahseer (Tor tambroides)

Project leader : Assoc. Prof. Dr. Abol Munafi Ambok Bolong

AKUATROP has developed recirculating system for keeping of Kelah (Tor

tambroides) which now house 30 broodstocks of red Malaysian mahseer and 450 of

Tor juveniles. The broodstocks and juvenile were bought from local farmer in Kenyir,

Terengganu and also were given by Kelah World Sdn. Bhd. The weights of the

broodstocks were between 700g – 2400g each and the juvenile were between 60g –

120g each. AKUATROP has also a joint research project with Kelah World Sdn. Bhd

to carry out induce breeding of Kelah. Dr. Abol Munafi and his research team has

managed to induce bred the fish to produce fingerlings.

Hormone Injection process Sperm stripping from male broodstock

Incubation of fertilized egg in aquarium Mixing sperm to fertilize the Kelah eggs

Recirculating System Tank in UMT Separating Tank in Kelah World

Sieve used to protect incubated eggs Newly hatched larvae

Newly hatched larvae view under profile projector

Title of project : Culture of Tiger Grouper (Epinephelus fuscoguttatus) in

recirculating system

Project leader : Assoc. Prof. Dr. Abol Munafi Ambok Bolong

This project on grouper culture is a joint collaboration with Marine Finfish

Production and Research Centre, Tanjung Demong. Tiger grouper (Epinephelus

fuscoguttatus) will be cultured in Recirculating Aquaculture System (RAS) at the

AKUATROP Marine water Hatchery. The RAS system has already been set up and

grouper culture activities have already started. One PhD student has already registered

under AKUATROP to run this project.

The growth and feeding of wild-caught young grouper Epinephelus sp in a

recirculation aquaculture system (RAS) was studied. The following is the flow chart of

nursing wild caught young grouper in RAS.

Flow Chart Of Nursing Wild-Caught

Young Grouper in RAS

Flow Chart Of Nursing Wild-Caught

Young Grouper in RAS

CULTURE SYSTEM

MAINTENANCE

WATER QUALITY REQUIREMENT

CULTURE MANAGEMENT

FRY NURSERY FEEDINGWATER

QUALITY

FISH

DISEASE

CAUGHT FRY FROM THE WILD

ACCLIMATION

GROW OUT

HARVESTING

MARKETING

Schematic diagram of the recirculation system used to rear wild-caught grouper

Wild grouper were kept in the marine hatchery system at AKUATROP. Orange

spotted grouper has been used in this project because of its high demand in Malaysia

and also it is an indigenous species. The fry of wild grouper were collected at Bukit

Keluang Besut, Terengganu. Fish were acclimatized for 14 days before running the

experiment so that fish are not highly stressed.

The RAS system consisted of a culture tank (6 x 3 x 2 x 1.5F), sedimentation

cabinets (4 x 4 x 3 x 2.5F), mechanical filter cabinets (12 x 4 x 3 x 2.5 F), biology filter

cabinets (4 x 4 x 3 x 2.5 feet) and holding tank (6 x 4 x 3 x 2.5) & 1000L.

Fiber Glass tank used for grouper culture Newly hatched larvae view under profile projector

Piping and aeration tube/wires

Tanks Filter system

Basket for culturing seaweed Cleaning seaweed

Title of project : Tank cultivation of Gracilaria sp with enclosed water recirculation system and its agar evaluation

Project Leader : Dr Siti Aishah Abdullah

This project is studying the growth rate and productivity of selected Gracilaria sp

using tank culture method and also enclosed water recirculation system. The agar yield

and agar quality of selected Gracilaria sp will be evaluated before and after cultivation.

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Specific Growth Rate (%) vs Week

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Conc. 1 Conc.2 Conc.3 Conc. 4 Control

Fig. 2. Specific growth rates (SGR, presented as % increment of biomass per week) of Gracilaria changii in different concentration of ammonium sulphate and disodium phosphate solution.

Tank set-up for seaweed cultivation

Title of Project : Live Feed Culture Project Leader : Assoc. Prof. Dr. Anuar Hassan

This project is to grow and maintain the important marine and freshwater

plankton in the institute, for various research and culture activities. Brown and green

algae stock cultures were carried out in 15ML test tube. After 7 days, the algae were

transferred into two litre Elenmayer flasks after 5 days old. This stock was used in mass

culture

Algae on Agar Plate Plankton culture room in AKUATROP

Green Algae Brown Algae

Title of Project : Development of Cyst From Marine Harpaticoid Copepods.

Project Leader : Dr. Zaleha Kassim

Current research carried out by the researcher is on the trial culture of marine

harpacticoids on different substrates in laboratory condition and generation time study.

This study was conducted to compare the growth of three harpacticoid species which

are Schizopera knabeni, Paradactylopodia oculata, and Robertsonia knoxi cultured in

laboratory condition and also to observe their generation time and morphological

changes (nauplii, copepodite, and adult). The objectives for this project are to determine

the environmental condition to induce cyst development in marine harpacticoid

copepods, to determine the period of encystment of harpacticoid copepods under

manipulated laboratory culture condition, to determine the spatial and seasonal pattern

of cyst production in natural environment of Malaysian Coast and to study the

ultrastructure of harpacticoid during encystment using Scanning Electron Microscopic

(SEM) method

As a conclusion, this study is done especially to investigate the seasonal

distribution and stages of harpacticoid cysts found in natural environment along the

coastal area of The Malay Peninsula. The cysts found from the wild will be stocked for

laboratory culture. The sediment for substrates and benthic diatom as food will be

prepared. The replicates of culture plates will be used to grow the cyst, nauplii,

copepodids and adults of selected harpacticoid species up to 24 weeks (6 months).

Besides that the role of salinity, temperature or food will be determined in the process of

cyst development. After the cysts have been produced, it will be undergo SEM study and

will be cultured under the threshold limit found from the previous experiment. The results

will indicate the lower and upper limit of temperature, salinity and food requirement for

the encystment period of harpacticoid cyst and stages of cyst development.

A

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B

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Picture of Harpaticoid. A. Staining Specimen; B. and C., morphology of Harpaticoid

2.2 Research Fellow

Dr. Nandana Gunawickrama served AKUATROP from 2006 to September 2007

as a Research Fellow. During his service at the AKUATROP he has managed to publish

two books with the help of UMT Publisher entitled ‘Fish Cytochrome P4501A: a

biomarker for aquatic environment health’ and ‘Controlled Laboratory

Experiments: design and data analysis - an aquatic Toxicology perspective’.

Fish Cytochrome P4501A: a biomarker for aquatic environment health

Controlled Laboratory Experiments: design and data analysis - an

aquatic Toxicology perspective