Neobenedenia melleni Parasite of Red Snapper, Lutjanus ...

Research ArticleNeobenedenia melleni Parasite of Red SnapperLutjanus erythropterus with Regression Statistical Analysisbetween Fish Length Temperature and Parasitic Intensity inInfected Fish Cultured at Jerejak Island Penang Malaysia

Rajiv Ravi and Zary Shariman Yahaya

School of Biological Sciences Universiti Sains Malaysia Minden 11800 Penang Malaysia

Correspondence should be addressed to Rajiv Ravi rajiv ravi86yahoocom

Received 15 January 2016 Revised 4 March 2016 Accepted 15 March 2016

Academic Editor Bernard Marchand

Copyright copy 2016 R Ravi and Z S Yahaya This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The fish parasites collected from Lutjanus erythropterus fish species showed a correlation with parasitic intensity fish size andtemperature and statistical model summary was produced using SPSS version 20 statistical software Statistical model summaryconcluded that among the variables which significantly predict the prevalence of Neobenedenia melleni parasites are fish lengthand water temperature both significant at 1 and 5 Furthermore the increase in one unit of fish length holding other variablesconstant increases the prevalence of parasite by approximately 1 (07asymp1) unit Also increasing the temperature from 32∘C to 33∘Cwill positively increase the number of parasites by approximately 032 units holding other variables constant The model can besummarized as estimated number of Neobenedenia melleni parasites = 82 + 07 lowast (fish length) + 032 lowast (water temperature) Nextthis study has also shown the DNA sequence and parasitic morphology of Neobenedenia melleni Nucleotide sequence for 18sribosomal gene RNA in this study showed 99 similarity with N melleni EU7078041 from GenBank Finally all the sequence ofNeobenedenia melleni in this study was deposited in GenBank with accession numbers of KU843501 KU843502 KU843503 andKU843504

1 Introduction

Information and quantitative data on cultured fishes arelimited in Southeast Asia However the existing data explainsclosely that similar species of fishes are cultured throughoutthe Southeast Asian region and the dominant parasites foundinfecting each species of these cultured marine fishes aresimilar [1ndash5] Numerous studies on the parasitic fauna ofmarine fishes have indicated that the dominant parasitesin each fish species are the same regardless of the wild orcultured [3 6] The main difference between the wild andcultured diseased marine fishes is that the number andvariety of parasites in both groups of cultured fishes greatlyexceed those found in the wild fishes [7]

Monogenean parasites have been recognized as seriouspathogens of fish in sea cage aquaculture [8ndash10] Monogeneaparasites have no intermediate host predominantly parasitise

the external surfaces of fish and display two distinctivediets that traditionally divide them into two subclasses theblood feeding polyopisthocotylea and the epithelial feedingmonopisthocotylea [11] These are sometimes named Het-eronchoinea and Polyonchoinea respectively [12]These sub-classes are united by various morphological synapomorphiclarvae with three ciliated zones adults and larvae with twopairs of pigmented eyes one pair of ventral anchors (hamuli)and one egg filament [13] Inference about the Monogeneaparasite is monophyletic which has been ubiquitous fordecades [13ndash18] Neobenedenia melleni (MacCallum 1927)Yamaguti 1963 a capsalid monogenean of the subfamilyBenedenia sp is disreputable as a widespread pathogen ofmany teleost species in aquaculture [19] This parasite feedson epithelial cells mucus of host fish which gives increasedeffects towards irritation and mucus hyperproduction oftheir hosts [20] Like most of other monogenean groups

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2016 Article ID 1946283 8 pageshttpdxdoiorg10115520161946283

2 Journal of Parasitology Research

benedenids have traditionally been identified to species onthe basis of morphological characters such as the shape ofposterior hamuli the type of anterior attachment organ andthe length of uterus vitelline reservoir and the type andrelative size of testes [21]Though it has been argued for a longtime that morphological characters based identification ofparasite can be affected to a large extent by extrinsic factorssuch as the age of parasite environmental temperatureand even artifacts caused by various dealings for specimenprocessing as discussed by Li et al [22] most monogeneanscould be appropriately distinguished because of their highlevel of host specification However Neobenedenia mellenidoes not obey the rule because it has been reported frommore than 100 teleost fish species belonging to more than 30families with worldwide distributions [23]

To date there is no reference yet that has been done onthe correlations ofNeobenedenia melleni parasite infestationsto the fish size temperature and salinity factors in MalaysiaThis is an important aspect of research as it will benefit fishfarmers for aquaculture industry to predict any fish parasiteinfestation in their farm and to take initiatives to preventparasitic infections Thus the objective of this study is toshow the prevalence and statistical analysis of Neobenedeniamelleni parasite to the fish size and water temperature inLutjanus erythropterus fish species sampled from cage cultureJerejak Island Penang Peninsular Malaysia Furthermorewe have successfully identified the parasite species usingmorphology and molecular approach

2 Materials and Methods

21 Sampling Locality and Parasite Collection The experi-ment was carried out with 400 fish specimens of cultured Lut-janus erythropterus fish species from Jerejak Island PenangPeninsular Malaysia (5320097 longitude 1003189185 lati-tude) The length (cm) of each fish was measured priorto parasite examination Fresh water medium was used asanesthetics to reduce the stress as well as for easy handlingAfter the fish has been anaesthetized presence of ectoparasitewas examined via external fish body examination and directobservation under light microscope [24] The site specificityof parasite was obtained from head body and both sides ofinner operculum

First morphological identification of parasite was doneby first staining the parasite with a few drops of lactophenolsolutions (200mL lactic acid 200 gL phenol 400mL glyc-erol and 200mLdeionizedwater) Upon staining slides wereobserved under the compound microscope (Leica USA)Parasite found was taken out carefully from the infected areaand then the number of parasites obtained from each fish wasrecorded preserved with 70 ethanol solution in universalbottle for further examination After the pictures of parasiteshad been taken identification of parasites collected was doneby morphological observation using identification keys assuggested by Kua et al [25 26]

22 Morphological Method Using Scanning Electron Micro-scope Second morphological identification was done using

the Supra 50vp ultra high resolution LEO analytical Fesemscanning electron microscope Electron microscopic samplepreparation was done as suggested by protocol of Supra 50vpultra high resolution LEO analytical Fesem scanning elec-tron microscope guide manual Firstly suspended samples inethanol were put into serial dilution of 90 80 and 70ethanol Then a droplet of the suspension was placed on acarbon film coated 400-mesh copper grid for 1ndash3 minutesThe droplet is then dried using pieces of filter paper Thegrid was then placed in a filter paper lined Petri dish forpreservation in desiccator Finally imaging would be carriedout after 3 days of preservation

23 Molecular Method Using DNA Identification Thegenomic DNA extraction and purification of the parasitewas performed using the procedures provided by QiagenDNeasy Blood and Tissue Kit (Qiagen Inc Valencia CAUSA) Purified genomic DNA was eluted by adding 100 120583Lof buffer AE to the same spin column in a new Eppendorftube and centrifuged at 5200 g for 1min The centrifuge stepwas repeated again for a total of 200120583L sample volume DNAsample was stored at minus20∘C and concentration measuredwith ACT-Gene NanoDrop spectrophotometer (ASP 2680Taiwan)

Ribosomal RNA 18s partial sequences were amplifiedfrom purified genomic DNA using the specific primers 18sF(51015840-GCG CGA GAG GTG AAA TTC AT-31015840) as forwardprimer and 18sR (51015840-AGT TTA CCC AGC CCT TTC GA-31015840) as reverse primer as discussed by Dang et al [27]synthesized by MyTACG Bioscience (Malaysia) Polymerasechain reaction (PCR) was carried out using a total volumeof 25 120583L master mix solutions (14 120583L of ddH

2O 25 120583L of

Promega PCR buffer 3 120583L of Promega MgCl2solutions 1 120583L

of Promega dNTP 1 120583L of each forward primer and reverseprimer 2120583L of DNA template and 05120583L of Promega Go TaqDNA polymerase) Standard cycle conditions for PCR wereset accordingly by initial denaturation for 10min at 95∘Cfollowed by 35 cycles of 30 s at 95∘C 30 s at 50∘C 60 s at 72∘Cand final elongation of 7 minutes at 72∘C The whole PCRwas carried out in MyCycler thermal cycler Bio-Rad PCRsystems (USA) Purification of PCR product was performedusing the procedure and materials provided in a QIAquickPCR Purification Kit (Qiagen Inc) Amplification productswere sequenced in both directions by MyTACG BioscienceCompany (Malaysia)

24 DNA Alignment and Phylogenetic Analysis Alignmentanalysis of nucleic acid sequences was performed usingClustalW2MEGA 5 Distance-based tree approach to speciesidentification was conducted using MEGA 5 software ABLAST search was conducted with DNA sequence that wasamplified Using MEGA 5 as discussed by Tamura et al[28] a distance-based tree approach to species identificationwas carried out by neighbour-joining the 18s sequences ofrecorded species from the BLAST search and those analyzedin this study Pairwise distance calculation is done usingMEGA 5 analysis tools and the Kimura 2-parameter [29]method serves as the substitution model In addition the

Journal of Parasitology Research 3

Figure 1 Ventral view of whole in SEM Neobenedenia melleni

bootstrap method was deployed as test of phylogeny using1000 bootstrap replications Finally all the sequences weresubmitted in GenBank according to submission protocols

25 Statistical Analysis andWater Parameters Records Statis-tical analysis in this study was performed using the StatisticalPackage for Social Sciences software SPSS version 20 Themultiple regression analysiswas employed and in all cases thesignificance level is set at 5 as discussed by Field [30] Thewater parameters were measured in sea cage using SaltwaterMaster Test Kit Aquarium Pharmaceuticals Index (API)USA The procedure for each test was done according tomanufacturerrsquos instructions

3 Results and Discussion

31 Morphological Analysis of Neobenedenia melleni Usingmorphological key as described by Lawler [31] and Bullard etal [32 33] In revising the generic diagnosis forNeobenedeniaWhittington and Horton [23] noted a variety of forms whichwere more than 80 specimens attributed to Neobenedeniamelleni from various host species We are able to identifythe parasite collected as Neobenedenia melleni according toFigures 1ndash6 AS accessory sclerite 40 120583m T testis organsAO anterior attachment organ P pigmented eye MA maleaccessory gland reservoir G gland of Goto V vitellinereservoir A anterior hamulus 150 120583m P posterior hamulus40 120583m Total length of a sample specimen Neobenedeniamelleni in this study was recorded as 1050 120583m The widthlength is recorded as 700 120583m

32 DNA and Phylogenetic Analysis Based on the resultsobtained upon gel electrophoresis analysis of the DNA tem-plate and PCR product clearly visible bands were detectedaround 700 bp sequence whereby this analysis was referredto Lucigen 1 kb DNA marker (USA) (Figure 7) Besidesthat the optical density (OD) ratio of DNA was 20 withconcentration of 135 (ng120583L) for genomic DNA The DNAsequence was further analyzed using Clustal W BioeditSoftware The 18s sequence was successfully analyzed recov-ered from all Neobenedenia melleni individuals NucleotideBLAST sequence for 18s ribosomal RNA gene from this studyhas shown 99 similarity with N melleni EU7078041 from

AS AS

Figure 2 Ventral view of Neobenedenia melleni in SEM ASaccessory sclerite 40 120583m

T T

Figure 3 Ventral view of Neobenedenia melleni in SEM T = testisorgans

200120583m

Figure 4 Ventral view of wholeNeobenedeniamelleni in compoundmicroscope

AO

AOP

P VMA G

200120583m

Figure 5 Ventral view of Neobenedenia melleni in compoundmicroscope AO anterior attachment organ P pigmented eyeMA male accessory gland reservoir G gland of Goto V vitellinereservoir


P

A

A

P

200120583m

Figure 6 Ventral view of Neobenedenia melleni in compoundmicroscope A anterior hamulus 150 120583m P posterior hamulus40 120583m

1 2 3 4 5 6

Lane 2-5 Neobenedenia melleniLanes 1 6 DNA ladder1kb

700bp

Figure 7 Gel electrophoresis of PCR 18s ribosomal RNA gene

GenBank dataset as shown in Figure 8 Meanwhile Figure 9shows the constructed phylogenetic tree which shows twoclosely related clades between species

All the individuals of Neobenedenia melleni recorded inthis study showed a close relationship between species thatwas recorded fromNCBINeobenedenia melleni EU7078041as 96bootstrap value followed by 76of similarity betweenAllobenedenia epinepheliEU7078001The least similarity wasrecorded with Encotyllabe chironemi AJ2287741 Benedeniaepinepheli EU7078021 and Neobenedenia girellae AY551326Finally all the sequence of Neobenedenia melleni in thisstudy was deposited in GenBank with accession numbersKU843501 KU843502 KU843503 and KU843504 All thissequence is available as public database in GenBank

Figure 8 Nucleotide BLAST sequence for PCR product

Table 1 The table shows the descriptive statistics of dependent andpredictor variables

Characteristics Frequency 119899 Prevalence () Mean SDDependent variablesNeobenedenia melleni 379 948a 2524 28

Predictor variablesFish length 375 938a 243 37

Categorical variablesWater temperature320 154 544330 129 456

Water salinity320 168 594330 115 406

Note aTotal percentage is not 100 due to missing values SD standarddeviation

33 Statistical Modeling for Neobenedenia melleni A totalof all 379 fishes were infected by Neobenedenia melleniparasite out of 400 examined fishes in natural sea culturecage environment Table 1 shows the descriptive statisticsof dependent variable and predictor variables involved inthis study The average number of the Neobenedenia melleniparasite found in examination of fishes is approximately 25with a standard deviation of 28Themean value of fish lengthis 243 cm with a standard deviation of 37 Meanwhile thebinary coded variables water temperature (0 = 32∘C 1 =33∘C) and salinity (0 = 32 ppt 1 = 33 ppt) both have a higherpercentage of low temperature (544) and salinity (594)compared to its counterpart

Table 2 shows that the variables are positively correlatedwith one another and are significant at 1 A large correlationof 092 is observed between fish length and prevalence ofNeobenedenia melleni (increase in fish length will increasethe prevalence of Neobenedenia melleni parasite) and 0437


Table 2 Correlations between variables using bivariate analysis

Variables Prevalence of Neobenedenia melleni Fish length Water temperature Water salinityPrevalence of Neobenedenia melleni 1Fish length 0917lowastlowastlowast 1Water temperature 0298lowastlowastlowast 0270lowastlowastlowast 1Water salinity 0437lowastlowastlowast 0428lowastlowastlowast 0384lowastlowastlowast 1Note lowastlowastlowastsignificant at 1

Neobenedenia melleni 18s_1Neobenedenia melleni 18s_2Neobenedenia melleni 18s_3Neobenedenia melleni 18s_4Neobenedenia melleni 18s (EU7078041)

Allobenedenia epinepheli 18s (EU7078001)

Encotyllabe chironemi 18s (AJ2287801)

Benedenia epinepheli 18s (EU7078021)

Benedenia sp 18s (AJ2287741)

Neobenedenia girellae (AY551326)

Neobenedenia sp (HM2225361)

Caligus clemensi (BT0806981) OUTGROUP

02

96

38

76

45

43

100

Figure 9 Constructed phylogenetic tree for Neobenedenia melleni

Table 3 Variance inflation factor values of predictor variables

Variables Tolerance statisticsVariance

inflation factor(VIF)

Fish length 0751 1332Temperature 0778 1286Salinity 0659 1518

between water salinity and prevalence of Neobenedenia mel-leni parasite (increase in water salinity increases the preva-lence of Neobenedenia melleni parasite) Finally a moderatecorrelation of 03 is observed between water temperature andprevalence of Neobenedenia melleni parasite [30]

According to Table 3 the variance inflation factor (VIF)values are less than 100 or 20 and the tolerance statisticsare above 02 [30] The tolerance statistics is the reciprocalof VIF or 1VIF Multicollinearity issues are negated becausethe values met more than the requirement of VIF varianceinflation factor and tolerance statistics

Table 4 shows the multiple correlation coefficients 119877 thecorrelation among all the independent variables (tempera-ture fish length and salinity) and the dependent variablewhich is at value 0912 The 119877-Square value shows that all thepredictors account for 845 of variation in the prevalence of

parasiteThe adjusted119877-Square value (0843) is similar to thatof 119877-Square indicating that if these data were collected fromthe population rather than a sample it would have a similarresult Therefore the result from this sample is generalizedto the entire population of Neobenedenia melleni parasiteinfesting in fishes as discussed in Field [30]

Table 5 shows that themodel is a significant fit to the dataat less than 5 Thus the model is significantly improved tothe ability to predict the dependent variable prevalence ofNeobenedenia melleni parasite infesting in fishes [34]

Table 6 shows the parameter estimates of multiple regres-sionmodeling Among the variables that significantly predictthe prevalence of Neobenedenia melleni parasite are fishlength and water temperature both significant at 1 and 5however salinity is not a significant predictor ofNeobenedeniamelleni in this analysis Furthermore the increase in one unitof fish length holding other variables constant increases theprevalence of parasite by approximately 1 (07asymp1) unit Alsoincreasing the temperature from 32 to 33 degrees Celsiusincreases the number of parasite by approximately 032 unitsholding other variables constantThe 95 confidence interval(CI) conforms to the results obtained by observing theparameter estimate for fish length and temperaturewithin theconfidence interval and within a positive confidence intervalbound


Table 4 The model summary

Multiple correlation coefficient 119877 119877-Square Adjusted 119877-Square Standard error of the estimate0912 0845 0843 1124

Table 5 The model fit values

Sum of squares df Mean square 119865-statistics 119875 valueRegression 1829444 4 457361 361868 0000Residual 334930 265 1264Total 2164374 269

Table 6 Fitted values of the predictor variables via multiple regression analysis

Variables Parameter estimates 120573 Standard error (SE) 95 Confidence interval (CI)Lower bound Upper bound

Constant 8244 0465 7329 9159Fish length 0669lowastlowastlowast 0021 0628 0710Temperature 0319lowastlowast 0156 0013 0625Salinity 0124 0172 minus0214 0462Note Significant at lowastlowastlowast1 and lowastlowast5 significance level

The model can be rewritten as

Estimated number of 119873119890119900119887119890119899119890119889119890119899119894119886 119898119890119897119897119890119899119894 parasites

= 82 + 07 lowast (Fish Length) + 032

lowast (Water Temperature)

(1)

Figure 10 shows the histogram of the residuals data whichhas a bell shaped curve indicating that the residuals arenormally distributed This is further verified by visualizingthe normal P-P plot in Figure 11 which also shows that thepoints lie along a diagonal line indicating that the residualsare normally distributed Figure 12 shows that the points arerandomly and evenly dispersed throughout the plot and con-cur with the assumptions of linearity and homoscedasticity ofthe residuals has been met as discussed in Field [30]

In this study we have deployed multiple regressionanalysismethod to observe biotic and abiotic factors that haveinfluenced themiscellany of parasites in hosts like fish lengthwater temperature parasites count and salinity [9] Thesemultiple variables are predicted to influence cultured fish andto come across rates with parasites and with the number ofparasites that can endure in populations A positive relation-ship is predicted among fish length temperature salinity andparasite diversity because larger fish represent larger infectionsurface area for parasitic colonization [34 35] Besides thattemperature is mainly important as an environmental factorwhich merely controls the development period of parasiticcopepods Parasites growth rates egg production survivalrate and conscription are reported to be high at higher watertemperatures [21 23] The multiple regression analysis isintegrated with the objective to produce a model that wouldbest predict the optimal number of parasitic infestation basedon observed values of three independent variables whichwere the length of fish mean temperature and salinity

20minus2

60

4 6

20

0

80

40

minus4

Dependent variable number of Neobenedenia melleniHistogram

Freq

uenc

y

Mean = 829E minus 16

Std dev = 0993

N = 270

Regression standardized residual

Figure 10 Histogram of the residuals data shows that the histogramhas a bell shaped curve indicating that the residuals are normallydistributed

Several monogenean species exhibit short life cycles inwarm temperatures [9 23] Accelerated parasitic life cycleswill increase the metabolic and development rate associatedwith warm conditions [8 14 15] Presently the reason for theunpredictable and irregular nature of Neobenedenia melleniinfection is unknown Steps that can be implemented inreducing this rapid parasitic infestations are to have moreattentive frequent fish stock monitoring during warm hightemperature water conditions [36] The major role of this


060402

06

08 10

02

00

08

10

04

00

Normal P-P plot of regression standardized residualDependent variable number of Neobenedenia melleni

Observed cum prob

Expe

cted

cum

pro

b

Figure 11 P-P plot shows that the points which represent theresiduals lie along the diagonal line showing that the residuals arenormally distributed

0minus1minus2

0

1 3

minus4

2

minus2

minus3

ScatterplotDependent variable number of Neobenedenia melleni

Regression standardized predicted value

Regr

essio

n st

anda

rdiz

ed re

sidua

l

4

6

2

Figure 12 The figure shows that the points are randomly andevenly dispersed and do not have any specific pattern indicating thatthere is homoscedasticity or homogeneity of variance and linearityassumption is met

temperature factors has been previously described by studiesof life cycle of Neobenedenia melleni Ogawa and Yokoyama[10] explained thatNeobenedenia melleni took only 10 days tocomplete at 30∘C as opposed to 20 days at 20∘C in seawater

Accordingly toGrau et al [8] the hatching survival rate ofNeobenedenia melleni eggs was less than 12 when incubatedat salinity less than 18 ppt for 4 days

4 Conclusion

In summary this study has established an overview withstatistical analysis for correlations of fish length and tem-perature that influences the number of fish parasites presentin Lutjanus erythropterus fish species Furthermore mor-phology and DNA sequence identification were shown forNeobenedenia melleni parasite found in this cultured fishfrom Jerejak Island Penang Malaysia

Competing Interests

All the authors declare that there are no competing interestsin this paper

Acknowledgments

Funding for the study was provided by Universiti SainsMalaysia Research University Grant (1001PBIOLOGI811259) The authors acknowledged GST group of companiesfor allowing them to perform these experiments at their fishfarm Furthermore MalaysianMyBrain MyPhD ScholarshipProgram is acknowledged Mr Johari at the SEM UnitSchool of Biological Sciences Universiti Sains Malaysia isacknowledged for assisting with the photography sessionsand Ms Azirah Akbhar Ali Ms Yanie Zain and Ms FatynnAmirah Firuz are acknowledged for assisting in fish samplingworks

References

[1] Y C Chong andTMChao ldquoQuarantine treatment of importedEpinephelus tauvina fryrdquo Singapore Veterinary Journal vol 8pp 2Gndash26G 1984

[2] Y C Chong and T M Chao Common Diseases of Marine FoodFish Fish Handbook Primary Production Department of theMinistry of National Development Singapore 2011

[3] T S Leong Parasites and Diseases of Cultured Marine Finfishesin Southern Asia Universiti Sains Malaysia 1994

[4] T S Leong and S Y Wong ldquoParasites of wild and culturedgolden snapper Lutjanus johni (Bloch) in Malaysiardquo TropicalBiomedicine vol 6 pp 73ndash76 1989

[5] T S Leong and S Y Wong ldquoParasites of marine finfishes cul-tured in ponds and cages in Indonesiardquo in Tropical Fish HealthManagement in Aquaculture J S Langdon G L Enriquez andS Sukimin Eds vol 48 of Special Publication Biotrop BogorIndonesia 1992

[6] T-S Leong and S-YWong ldquoA comparative study of the parasitefauna of wild and cultured grouper (Epinephelus malabaricusBloch amp Schneider) inMalaysiardquoAquaculture vol 68 no 3 pp203ndash207 1988

[7] T S Leong and S Y Wong ldquoParasites of healthy and diseasedjuvenile grouper (Epinephelus malabaricus Bloch amp Schneider)and seabass (Lates calcarifer Bloch) in floating cages in PenangMalaysiardquoAsian Fisheries Science vol 3 no 3 pp 319ndash327 1990

[8] A Grau S Crespo E Pastor P Gonzalez and E CarbonellldquoHigh infection by Zeuxapta seriolae (Monogenea Heterax-inidae) associated with mass mortalities of amberjack Serioladumerili Risso reared in sea cages in the Balearic Islands


(Western Mediterranean)rdquo Bulletin of the European Associationof Fish Pathologists vol 23 no 3 pp 139ndash142 2003

[9] L A Tubbs C W Poortenaar M A Sewell and B K DigglesldquoEffects of temperature on fecundity in vitro egg hatching andreproductive development of Benedenia seriolae and Zeuxaptaseriolae (Monogenea) parasitic on yellowtail kingfish Seriolalalandirdquo International Journal for Parasitology vol 35 no 3 pp315ndash327 2005

[10] K Ogawa and H Yokoyama ldquoParasitic diseases of culturedmarine fish in Japanrdquo Fish Pathology vol 33 no 4 pp 303ndash3091998

[11] D T J Littlewood K Rohde R A Bray and E A HerniouldquoPhylogeny of the Platyhelminthes and the evolution of para-sitismrdquo Biological Journal of the Linnean Society vol 68 no 1-2pp 257ndash287 1999

[12] W A Boeger and D C Kritsky ldquoPhylogenetic relationships ofthe Monogenoideardquo in Interrelationships of the PlatyhelminthesD T J Littlewood and R A Bray Eds pp 92ndash102 Taylor ampFrancis London UK 2001

[13] A E Lockyer P D Olson and D T J Littlewood ldquoUtility ofcomplete large and small subunit rRNA genes in resolving thephylogeny of the Neodermata (Platyhelminthes) implicationsand a review of the cercomer theoryrdquo Biological Journal of theLinnean Society vol 78 no 2 pp 155ndash171 2003

[14] B E Bychowsky Monogenetic Trematodes Their Systematicsand Phylogeny Academy of Sciences USSR Moscow RussiaEnglish translation by W J Hargis Jr and P C OustinoffAmerican Institute of Biological Sciences Washington DCUSA 1957 (Russian)

[15] J-L Justine ldquoCladistic study in the Monogenea (Platy-helminthes) based upon a parsimony analysis of spermio-genetic and spermatozoal ultrastructural charactersrdquo Interna-tional Journal for Parasitology vol 21 no 7 pp 821ndash838 1991

[16] J-L Justine ldquoNon-monophyly of the monogeneansrdquo Interna-tional Journal for Parasitology vol 28 no 10 pp 1653ndash16571998

[17] J Llewellyn ldquoTaxonomy genetics and evolution of parasitesrdquoThe Journal of Parasitology vol 56 no 4 pp 287ndash504 1970

[18] I Mollaret B G M Jamieson and J-L Justine ldquoPhylogenyof the Monopisthocotylea and Polyopisthocotylea (Platy-helminthes) inferred from 28S rDNA sequencesrdquo InternationalJournal for Parasitology vol 30 no 2 pp 171ndash185 2000

[19] D T J Littlewood R A Bray and K A Clough ldquoA phylogenyof the Platyhelminthes towards a total-evidence solutionrdquoHydrobiologia vol 383 pp 155ndash160 1998

[20] T S Leong and S Y Wong ldquoParasites of grouper Epinephelussuillus from Pulau Langkawi and Kelantan Malaysiardquo JournalBioscience vol 6 article 5457 1995

[21] S Yamaguti Systema Helminthum Volume IV Monogenea andAspidocotylea Interscience Publishers New York NY USA1963

[22] A-X Li X-Y Wu X-J Ding et al ldquoPCR-SSCP as a moleculartool for the identification of Benedeniinae (Monogenea Capsal-idae) from marine fishrdquo Molecular and Cellular Probes vol 19no 1 pp 35ndash39 2005

[23] I D Whittington and M A Horton ldquoA revision of Neobene-denia Yamaguti 1963 (Monogenea Capsalidae) including aredescription of N melleni (MacCallum 1927) Yamagutirdquo Jour-nal of Natural History vol 30 no 8 pp 1113ndash1156 1963

[24] Z Kabata Parasitic Copepoda of British Fishes The Ray SocietyLondon UK 1979

[25] B C Kua S Z AbdullahM F Abtholuddin N FMohd andNN Mansor ldquoMarine leech isolated from cage-cultured sea bass(Lates calcarifer) fingerlings a parasite or Vectorrdquo in Abstractin Programme Book of the 15th Scientific Conference ProceedingTerengganu Malaysia December 2006

[26] B C Kua and H Faizul ldquoScanning electron microscopy ofthree species of Caligus (CopepodaCaligidae) parasitized oncultured marine fish at Bukit Tambun Penangrdquo MalaysianJournal of Microscopy vol 6 pp 9ndash13 2010

[27] B T Dang A Levsen C Schander and G A BristowldquoSomeHaliotrema (Monogenea Dactylogyridae) from culturedgrouper (Epinephelus Spp) with emphasis on the phylogeneticposition of Haliotrema cromileptisrdquo Journal of Parasitology vol96 no 1 pp 30ndash39 2010

[28] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[29] Kimura ldquoEstimation of evolutionary distance betweennucleotide sequencesrdquo Molecular Biology and Evolution vol 3pp 269ndash285 1984

[30] A Field Discovering Statistics Using SPSS 2nd edition 2005[31] A R LawlerZoogeography andHost-Specificity of the Superfam-

ily Capsaloidea Price 1936 (Monogenea Monopisthocotylea) AnEvaluation of the Host-Parasite Locality Records of the Superfam-ily Capsaloidea Price 1936 and Their Utility in Determinationsof Host-Specificity and Zoogeography Special Papers in MarineScience no 6 1981

[32] S A Bullard G W Benz and J S Braswell ldquoDionchuspostoncomiracidia (Monogenea Dionchidae) from the skinof blacktip sharks Carcharhinus limbatus (Carcharhinidae)rdquoJournal of Parasitology vol 86 no 2 pp 245ndash250 2000

[33] S A Bullard G W Benz R M Overstreet E H Williams Jrand J Hemdal ldquoSix new host records and an updated list ofwild hosts forNeobenedeniamelleni (MacCallum) (MonogeneaCapsalidae)rdquo Comparative Parasitology vol 67 no 2 pp 190ndash196 2000

[34] R Ravi and Z S Yahaya ldquoRelationship between size of fishtemperature and parasitic intensity in snakehead fish speciesfrom Kepala Batas Penang Peninsular Malaysiardquo PertanikaJournal of Tropical Agricultural Science vol 38 no 2 pp 295ndash307 2015

[35] R Ravi and Z S Yahaya ldquoDNA barcoding of marine leeches(Zeylanicobdella argumensis) from crimson red snapper (Lut-janus eryhtopterus) peninsularMalaysia and their phylogeneticanalysisrdquo in Proceedings of the 9th Regional IMT-GT UNINETConference Penang Malaysia November 2014

[36] K L Main and C Rosenfeld Eds Culture of Highvalue MarineFishes in Asia and the United States The Oceanic InstituteHonolulu Hawaii USA 1995

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Enzyme Research



Microbiology


benedenids have traditionally been identified to species onthe basis of morphological characters such as the shape ofposterior hamuli the type of anterior attachment organ andthe length of uterus vitelline reservoir and the type andrelative size of testes [21]Though it has been argued for a longtime that morphological characters based identification ofparasite can be affected to a large extent by extrinsic factorssuch as the age of parasite environmental temperatureand even artifacts caused by various dealings for specimenprocessing as discussed by Li et al [22] most monogeneanscould be appropriately distinguished because of their highlevel of host specification However Neobenedenia mellenidoes not obey the rule because it has been reported frommore than 100 teleost fish species belonging to more than 30families with worldwide distributions [23]

To date there is no reference yet that has been done onthe correlations ofNeobenedenia melleni parasite infestationsto the fish size temperature and salinity factors in MalaysiaThis is an important aspect of research as it will benefit fishfarmers for aquaculture industry to predict any fish parasiteinfestation in their farm and to take initiatives to preventparasitic infections Thus the objective of this study is toshow the prevalence and statistical analysis of Neobenedeniamelleni parasite to the fish size and water temperature inLutjanus erythropterus fish species sampled from cage cultureJerejak Island Penang Peninsular Malaysia Furthermorewe have successfully identified the parasite species usingmorphology and molecular approach

2 Materials and Methods

21 Sampling Locality and Parasite Collection The experi-ment was carried out with 400 fish specimens of cultured Lut-janus erythropterus fish species from Jerejak Island PenangPeninsular Malaysia (5320097 longitude 1003189185 lati-tude) The length (cm) of each fish was measured priorto parasite examination Fresh water medium was used asanesthetics to reduce the stress as well as for easy handlingAfter the fish has been anaesthetized presence of ectoparasitewas examined via external fish body examination and directobservation under light microscope [24] The site specificityof parasite was obtained from head body and both sides ofinner operculum

First morphological identification of parasite was doneby first staining the parasite with a few drops of lactophenolsolutions (200mL lactic acid 200 gL phenol 400mL glyc-erol and 200mLdeionizedwater) Upon staining slides wereobserved under the compound microscope (Leica USA)Parasite found was taken out carefully from the infected areaand then the number of parasites obtained from each fish wasrecorded preserved with 70 ethanol solution in universalbottle for further examination After the pictures of parasiteshad been taken identification of parasites collected was doneby morphological observation using identification keys assuggested by Kua et al [25 26]

22 Morphological Method Using Scanning Electron Micro-scope Second morphological identification was done using

the Supra 50vp ultra high resolution LEO analytical Fesemscanning electron microscope Electron microscopic samplepreparation was done as suggested by protocol of Supra 50vpultra high resolution LEO analytical Fesem scanning elec-tron microscope guide manual Firstly suspended samples inethanol were put into serial dilution of 90 80 and 70ethanol Then a droplet of the suspension was placed on acarbon film coated 400-mesh copper grid for 1ndash3 minutesThe droplet is then dried using pieces of filter paper Thegrid was then placed in a filter paper lined Petri dish forpreservation in desiccator Finally imaging would be carriedout after 3 days of preservation

23 Molecular Method Using DNA Identification Thegenomic DNA extraction and purification of the parasitewas performed using the procedures provided by QiagenDNeasy Blood and Tissue Kit (Qiagen Inc Valencia CAUSA) Purified genomic DNA was eluted by adding 100 120583Lof buffer AE to the same spin column in a new Eppendorftube and centrifuged at 5200 g for 1min The centrifuge stepwas repeated again for a total of 200120583L sample volume DNAsample was stored at minus20∘C and concentration measuredwith ACT-Gene NanoDrop spectrophotometer (ASP 2680Taiwan)

Ribosomal RNA 18s partial sequences were amplifiedfrom purified genomic DNA using the specific primers 18sF(51015840-GCG CGA GAG GTG AAA TTC AT-31015840) as forwardprimer and 18sR (51015840-AGT TTA CCC AGC CCT TTC GA-31015840) as reverse primer as discussed by Dang et al [27]synthesized by MyTACG Bioscience (Malaysia) Polymerasechain reaction (PCR) was carried out using a total volumeof 25 120583L master mix solutions (14 120583L of ddH

2O 25 120583L of

Promega PCR buffer 3 120583L of Promega MgCl2solutions 1 120583L

of Promega dNTP 1 120583L of each forward primer and reverseprimer 2120583L of DNA template and 05120583L of Promega Go TaqDNA polymerase) Standard cycle conditions for PCR wereset accordingly by initial denaturation for 10min at 95∘Cfollowed by 35 cycles of 30 s at 95∘C 30 s at 50∘C 60 s at 72∘Cand final elongation of 7 minutes at 72∘C The whole PCRwas carried out in MyCycler thermal cycler Bio-Rad PCRsystems (USA) Purification of PCR product was performedusing the procedure and materials provided in a QIAquickPCR Purification Kit (Qiagen Inc) Amplification productswere sequenced in both directions by MyTACG BioscienceCompany (Malaysia)

24 DNA Alignment and Phylogenetic Analysis Alignmentanalysis of nucleic acid sequences was performed usingClustalW2MEGA 5 Distance-based tree approach to speciesidentification was conducted using MEGA 5 software ABLAST search was conducted with DNA sequence that wasamplified Using MEGA 5 as discussed by Tamura et al[28] a distance-based tree approach to species identificationwas carried out by neighbour-joining the 18s sequences ofrecorded species from the BLAST search and those analyzedin this study Pairwise distance calculation is done usingMEGA 5 analysis tools and the Kimura 2-parameter [29]method serves as the substitution model In addition the








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Competing Interests


Acknowledgments


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








AS AS


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4 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P

A

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Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












02

96

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43

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4 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













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4 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


060402

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4 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Neobenedenia melleni Parasite of Red Snapper, Lutjanus ...

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