Ihwan, M.Z.*, Shaharom-Harrisson, F.* and Kartini, M **.*

A Comparative Prevalence Study of Ectoparasites in Wild and

Cultured Grouper Before and After Transportation

*Institute of Tropical Aquaculture (AKUATROP)**Faculty of Maritime Studies and Marine Science (FMSM)

National Fisheries Symposium ( NaFiS ) 2008

Introduction • The fish will get stress easily if the management of

handling and transporting was not carried out carefully.

• Poor handling and non-systematic management of transportation will contribute to the stress factors (Bartelme, 2004).

• Environment has a major influence on every important parasite, especially in wild or cultured marine fish (Noga, 2000).

• This problem can lead to the reduction of the market value of live fish (Sholz, 1999).

•LiveLive FishFish TransportationTransportation– Problem in transportation can lead to lead to

the deaththe death of the fish (Lawler, 1993). – Several factorsfactors that cause the fish fish

diseases problemdiseases problem especially for the live fish;

• the presence of specific pathogen(s), stressful pathogen(s), stressful event(s), variations in temperatureevent(s), variations in temperature (Dror et.al., 2005) and also water qualitywater quality (Shaharom 1987, Shariff, 1984).

Objectives • To examineexamine and identifyidentify the

common ectoparasites that have infected the fish during transportation;

• To comparecompare the prevalence of ectoparasites in the wild and cultured grouper before and after transportation.

Grouper of the Study

Figure 1: Wild and Cultured Grouper that were used for this study (Information obtained from Heemstra and Randall, 1993)

Sampling Sitea.

b.

Figure 2: (a.) Maps from Endau, Johore to Aquaria KLCC, Kuala Lumpur and (b.) Maps from Setiu Wetland to University Malaysia Terengganu. (Maps obtained from Google Earth, 2007).

MethodologyMaterial • Alcohol 70%, 80%, 90% & 100%• Clove oil• Glycerin jelly• Saline water• Meyer Carmelum• Gris or cutex• Live fish Specimen• Dissecting set • Cavity Block• Glass Slaid and cover slip• Dissecting Microscope• Compound Microscope• Camera Lucida

• Camera digital• Petri disc• Rubber tray• Pointed needle

Methods

Identification

Sampling

Counting, Capture Photo,Collecting & Fixing.

Data Processing & Analyzes

Live Fish Examination(before)

Live Fish Examination(after)

Staining, Mounting & Drawing

Counting, Capture Photo,Collecting & Fixing.

Handling &Transporting (6 hours)

Live Fish Examination (Before and After)

Put the fish into the aquarium.

The lengths of fish were measure.

The weights of fish were measure.

The slide were observed under the

compound microscope.

Caught using net.

Smear using scalpel.

Put the mucous into the slide.

All the process cannot exceed than

three minutes (Untergrasser, 1989)

Data Processing & Analyses

The data collected were analyzed according to Margolis et al. (1982), the infection for each parasite species is defined as formula given below:-

Prevalence (%)Prevalence (%) = No. of Parasite Infected Fish No. of Fish Examined

Mean IntensityMean Intensity = No. of Parasite Found No. of Fish Infected

X 100

Result&

Discussions

Ectoparasites Species

• Four groups of ectoparasites were found:-

2. Ciliated Protozoan– Cryptocaryon Irritans (Ci)

– Trichodina sp. (Ts)– Unidentified Ciliates

(Uc)3. Monogenea

• Pseudorhabdosynochus sp. (Ps)

4. Leeches• Zeylanicobdella

arugamensis (Za)5. Copepod Parasite

• Caligus sp. (Cs)

1c.

1a.

4.

2.

3.

1b.

No. of SpeciesNo. of Species

• Ts show the second highest number in cultured grouper followed by Ci.

Table 2: Number of species found in wild and cultured grouper before and after transportation.

(Ci)

(Ts)

(Uc)

(Cs)

(Ps)

(Za)

• The highest number of species that were found in both species of grouper is Uc.

• The marine leech (Za) is only found in cultured grouper and parasitic copepod (Cs) is only found in wild grouper.

• Monogenea Ps show the lowest number in both of the grouper species.

No. of Species

• All the fish have a specific parasite on its body but it cannot affect the fish until the changes of environment factor that can contribute to the outbreaks of disease.

Table 2: Number of species found in wild and cultured grouper before and after transportation.

(Ci)

(Ts)

(Uc)

(Cs)

(Ps)

(Za)

• Among the groups, ciliated protozoa shows the higher number of species because of its short and direct lifecycle.

• The increasing number of species is depend on the environmental factor.

0

20

40

60

80

100

120

Before After

Pre

vale

nce

(%)

Wild Cultured

Figure 3: Prevalence of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

05

1015202530354045

Before After

Mea

n In

tens

ity

Wild Cultured

Figure 4: Mean Intensity of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

Prevalence & Mean Intensity

• The prevalence of ectoparasite in culture grouper (100%) is higher than wild grouper (65%) before transportation.

• The prevalence increase after transportation in both species which is cultured grouper still 100% infected compared to wild grouper that only 95%.

• The mean intensity after transportation is 37.4 for cultured grouper and 7.1 for wild grouper.

• Values for mean intensity of ectoparasites before transportation are 3.2 for wild and 5.1 for cultured grouper.

• All cultured grouper was infected before and after transportation; and about 65 % and 95 % of wild grouper was infected before and after transportation.

• The infections of cultured grouper was about 3x greater before transportation and 8x greater after transportation than the wild grouper.

0

20

40

60

80

100

120

Before After

Pre

vale

nce

(%)

Wild Cultured

Figure 3: Prevalence of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

05

1015202530354045

Before After

Mea

n In

tens

ity

Wild Cultured

Figure 4: Mean Intensity of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

Prevalence & Mean Intensity

• The prevalence of ectoparasites during transportation is depend on the procedure of transportation technique.

• Problems in procedures of transportation techniques can contribute disease problems especially after transportation.

• So, better procedure of handling new arriving fish, after a long transport is needed.

0

20

40

60

80

100

120

Before After

Pre

vale

nce

(%)

Wild Cultured

Figure 3: Prevalence of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

05

1015202530354045

Before After

Mea

n In

tens

ity

Wild Cultured

Figure 4: Mean Intensity of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

Prevalence & Mean Intensity

• The high infection of ciliated protozoan in fish cultured appear to be common occurrence.

• The ectoparasites can be abundant if the ectoparasites has a direct lifecycles and short generation time.

• Quarantine is one of the procedure that can ensure the transportation is clear from transmittable ectoparasites before and after the fish release into the water.

• This procedure can contribute to the decreasing of the total species number of ectoparasites.

Conclusion • There are four groups of ectoparasite four groups of ectoparasite

(6 species)(6 species) that infect the fish during transportation.

• The increasing number of ciliated protozoanciliated protozoan after transportation in both of grouper species shows that

this parasite is a dominant species during dominant species during transportationtransportation..

• Cultured grouperCultured grouper is more easily affectedeasily affected fishes compared to the wild grouper.

• Further study is neededFurther study is needed to determine the factors that may contribute to the prevalence of ectoparasites during transportation.

Acknowledgement

a and to all members who is involved directly or indirectly

throughout my study. All the kindness and hard work is well appreciated.

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THANK YOUQuestion & Answer

Counting, Photo Capturing,Collecting and Fixing.

• Counting

– By using this Direct counting methods, all the cells (parasite) are counted whether dead or alive (Abedon, 2003).

• Captured Photo

– Digital Camera

• Collecting & Fixing– Dissecting Set

• All the ectoparasite are collected using dissecting microscope except Ciliated Protozoan and fix into 70% alcohol.

Methodology

Water Quality

No. of Species

0100200300400500600700

Ci Ts Uc Ps Za Cs Ci Ts Uc Ps Za Cs

wild cultured

No

. o

f sp

ecie

s

before after

Results

• Ts show the highest number in cultured grouper and followed by Ci.

• The highest number of ectoparasites that were found in both species of grouper is Uc.

• The marine leech (Za) is only found in cultured grouper and parasitic copepod (Cs) is only found in wild grouper.

• Monogenea Ps show the lowest number in both of the grouper species.

Table 2: Number of ectoparasites found in wild and cultured grouper before and after transportation.

Results

Table 3: Prevalence and Mean Intensity of Ectoparasite in Wild and Cultured Grouper before and after Transportation.

Prevalence & Mean Intensity

• The conditions within the delivery containers can be quite different to that of natural seawater, better procedure of handling new arriving fish, after a long transport is needed.

• Water quality should be closely monitored to prevent the disease problem.

• Temperature, pH and salinity are the main concerns for the arriving fish.

a.

b. c.

d.

e.

f.

g.

h.i.

j.

1 mm

• Lunules are present.• Second antenna have

segmented. • Mouth cone is short,

blunt.

• Second maxillae is subchelate.

• Sternal furca is present.

• First leg with vestigal endopod.

• Second leg with both rami well developed, three-segmented.

• Fourth leg has three- or four-segmented.

Figure 7: Parts of Caligus sp. ; a. Second Maxillae, b. First Leg, c. Sternal Furca, d. Fourth Leg, e. Prominent maxillipeds, f. Second Antenna, g. Mouth Cone, h. Abdomen and Caudal Rami, i. Second Leg and j. Lunules.

Results

      number % abundance Prevalence(%)

Mean intensity

Ci Before wild 18 43.9 35 2.6

Ci Before Cultured 42 29.6 70 3

Ts Before wild 4 9.8 5 3.1

Ts Before Cultured 76 53.5 80 4.8

Uc Before wild 16 39.0 40 2

Uc Before Cultured 15 12.7 25 3.6

Ps Before wild 2 4.9 5 2

Ps Before Cultured 3 2.1 0 1.5

Za Before wild 0 0 0 0

Za Before Cultured 3 2.1 15 1

Cs Before wild 1 2.4 5 1

Cs Before Cultured 0 0 0 0

     number % abundance Prevalence

(%)mean intensity

Ci After Wild 18 43.9 35 2.6

Ci After Cultured 40 5.3 70 2.9

Ts After Wild 44 45.8 70 3.1

Ts After Cultured 167 22.3 100 8.4

Uc After Wild 15 15.6 25 3

Uc After Cultured 543 71.4 90 30.2

Ps After Wild 0 0 10 0

Ps After Cultured 3 0.4 15 1

Za After Wild 0 0 0 0

Za After Cultured 4 0.5 20 1

Cs After Wild 0 0 0 0

Cs After Cultured 0 0 0 0

Literature Review

• Parasite Host – Barber (2004) stated that parasites depend on host-

derived energy for growth and development and able to acquire nutrients from the host.

– The prevalence and spread of infectious disease are influenced by a variety of factors including host density, parasite transmission mode and the spatial structure of host population (Altizer et. al. 2000, Poulin 2006).

Literature Review

• Ectoparasites– Ectoparasite is a parasite which lives on the surface

of a host and dependent on at least one gene or its product from that host to complete their cycle (Anwar, 2005).

– Ectoparasite is also known as a parasite that lives on the outer surface of its host and also found on the skin and gill of the body and normally sticks to the mucus or cling to the gill filament (Schmidt and Roberts, 2000).

– The number of parasite found depends on how bad the infection is.

– Common ectoparasite that infect the fish are protozoan, helminthes and parasitic copepod.

• Grouper – Moderate-to-large sea basses found on the reef, top level

predators characterized by bass-like bodies, small scales, big mouths and big appetites.

– Most economically important marine fish in Asia, particularly in Thailand, Malaysia, Philippines, Singapore, Indonesia and Taiwan (Kanchanakhan, 1996).

– A lot of study about the disease of cultured grouper from FAO 1988, Leong and Wong 1988, Kanchanakhan 1996, Yambot and Song 2006, Cruz-lacierda et. al. 1999 and Chong and Chao 1986; but there have not much study for the wild grouper.

Literature Review

Wild Grouper

CulturedGrouper

CulturedGrouper

Sampling LocationTable 1: Specific location for sampling site.

Methodology

• First sampling-The wild grouper is transferred from Endau to Aquaria KLCC.

• Second Sampling-The cultured grouper is transferred from Setiu Wetland to UMT.

Sampling Technique • Wild Grouper

– Fishing– By Lorry

• Cultured Grouper– Netting – By Van

Methodology

Staining & MountingMethodology

Take some picture or make a good drawing. Take some picture or make a good drawing.

Make another smear of the gill and let it air dry. Make another smear of the gill and let it air dry.

Then fix the slide with methanolThen fix the slide with methanol

The dried slide will be submerged into 1% silver nitrate. Then rinse The dried slide will be submerged into 1% silver nitrate. Then rinse with distilled water and put in the Petri dish which is filled with with distilled water and put in the Petri dish which is filled with

distilled water. Under the Petri dish put a piece of white paper and distilled water. Under the Petri dish put a piece of white paper and place the dish under the sunlight for 3-4 hour. The sunlight will oxidize place the dish under the sunlight for 3-4 hour. The sunlight will oxidize

the silver nitrate to change into a black colour.the silver nitrate to change into a black colour.(Berland,2005)

Ciliated Protozoan e.g. : Trichodina, Crytocaryon Irritans and Unidentified Ciliatese.g. : Trichodina, Crytocaryon Irritans and Unidentified Ciliates

Staining & MountingMethodology

(Berland,2005)

Monogenea

Monogenean will be picked out using pointed needle.Monogenean will be picked out using pointed needle.

Then placed it in a drop of ammonium picrate or glycerin (semi Then placed it in a drop of ammonium picrate or glycerin (semi permanent mount) permanent mount)

Seal the cutex around the edge of the cover slip.Seal the cutex around the edge of the cover slip.(permanent mount).(permanent mount).

e.g. : e.g. : Pseudorhabdosynochus sp

Staining & MountingMethodology

(Berland,2005)

Parasitic Copepod e.g. : e.g. : Caligus sp

Collect the parasite and put into Cavity Block containing 70% alcohol.

Label the bottle with name of collector, date and the place where parasite was found.

Permanent Mount : the parasite is placed in a few drops of glycerin jelly and covered with cover slip. The edge of the cover

slip will be sealed with nail varnish to make it permanent mount.

Staining & MountingMethodology

(Berland,2005)

Leeches e.g. : e.g. : Zeylanicobdella arugamensis

Collect using pointed needle and placed in cavity block Collect using pointed needle and placed in cavity block filled with saline.filled with saline.

Clean the parasite from mucous in several changes of Clean the parasite from mucous in several changes of saline.saline.

Straighten the leech in Berland’s fluid before fixing in 70% Straighten the leech in Berland’s fluid before fixing in 70% alchohol. alchohol.

Relax the leech by putting them for several hours or Relax the leech by putting them for several hours or overnight in the fridge.overnight in the fridge.

Drawing Process Methodology

• Drawing using Lucida Microscope.– Curved

specimens/organs are traced on paper.

– Stage micrometer scale is also drawn.

(Berland,2005)

Identification • Important organs for identification were drawn

and counted. • The digital pictures facilitated identification

easily. • Reviews from Kabata 1979, Moller and Anders

1986 and many sources like previous journal, books and also information from the expert were used for the identification of these parasites by morphology and taxonomy.

Methodology

Handling & Transporting

Catch the fish Separate into quantity in aquarium

Transferred into tank

Packing and ready for transported

Percentage Abundance

020406080

100120140

Ci Ts Uc Ps Za Cs Ci Ts Uc Ps Za Cs

wild cultured

Ab

un

da

nc

e (

%)

before after

• Percentage abundance of ectoparasite shows that the ciliated protozoan is the dominant parasite infecting all the fish examined.

• Uc is increase (71.4%) in the cultured grouper after transportation compared to wild grouper that decreased after transportation; from 39.0% to 15.6%.

• Ts shows the increased of percentage abundance after transportation in wild grouper which 36% more than before transportation different for cultured grouper.

Figure 6: Percentage Abundance of Species; Before and after Transportation.