PHYLOGENETIC RELATIONSHIPS OF THE METAZOAN …

PHYLOGENETIC RELATIONSHIPS OF THE METAZOAN PARASITES

OF THE CLARIID FISHES OF LAKE VICTORIA INFERRED FROM

PARTIAL 18S rDNA SEQUENCES

CJ Mwita† and G Nkwengulila

2

† Departments of Aquatic Sciences and Fisheries and 2 Zoology and Wildlife Conservation,College of Natural and Applied Sciences, P.O. Box 35064, University of Dar Es Salaam

E-mail: [email protected],Telephone: 255 22 2410462/255 22 2650011, Fax: 255 22 2410480/2650244

ABSTRACTPhylogenetic relationships among twenty two metazoan parasites recovered from seven species ofclariid fish from Lake Victoria were analysed using partial 18S rDNA sequences. The 18S rDNAgene was amplified by polymerase chain reaction, directly sequenced, aligned and phylogeniesinferred using maximum parsimony. Heuristic bootstrap MP searches yielded one mostparsimonious tree (CI = 61%, HI = 39%), which showed that clariid parasites are monophyleticat higher taxonomic levels (Cestodea, Nematodea, Digenea and Crustacea). However the positionof two trematodes (Allocredium mazoensis and Clinostomum sp.) and a nematode (Contracaecumsp.) were not stable. Despite the present findings, the study utilised single species from eachtaxa, hence further analysis with additional sequences from a multitude of species isrecommended to further resolve the phylogeny of the parasites of the clariids in Lake Victoriaand elsewhere.

Key words: Clariid’s parasites phylogeny, Lake Victoria, Tanzania.

INTRODUCTIONThe Clariidae belong to a group of catfishesthat exploit a wide range of habitats fromstreams, rivers and freshwater lakes (Eccles,1992; Agnése and Teugels 2005). Due totheir catholic exploitation of habitats, theclariid catfishes are often infected by avariety of metazoan parasites (Mwita andNkwengulila, 2004; Barson et al. 2008;Mwita and Nkwengulila, 2008), inhabiting awide range of tissues and organs in theirhosts (Williams and Jones 1994, Mwita andNkwengulila, 2004). Among the clariids,the parasites of Clarias gariepinus are thebest studied (Mwita and Nkwengulila 2004)and those of other clariid species particularlyin Lake Victoria are poorly known, let alonetheir phylogenetic relationships.

With the advent of molecular systematicmethods, phylogenetic studies have beenundertaken in a number of fish parasitesparticularly from the Northern hemisphere(Jovelin and Justine 2001). However, little

agreement exists on the relationships amongthe different groups of parasites, partiallybecause of the poor representation of somegroups (Campos et al. 1998) and difficultiesin identifying parasites to species level at allstages of their life cycle (Chappell et al.1994; Niewiadomska, 1996; Galazzo et al.2002). The host-induced variations are madeworse when parasites for phylogeneticstudies are collected from a range of species(Campos et al. 1998), which occupy a widerange of geographical habitats (Hansen et al.,2003).

The present study is a link to the previousone (Mwita and Nkwengulila 2008) on theparasites of the clariid catfishes of LakeVictoria from which a total of 32 parasitespecies were documented from 658specimens of clariid fishes representingseven different species. Of the parasitesrecovered only 16 were identified to specieslevel, partially due to the reasons stipulatedabove as well as artefacts produced during

Mwita and Nkwengulila – Phylogenic Relationship of the Metazoan Parasite …

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fixation (Chappell et al. 1994). Studies haveshown that DNA-approaches provide waysof distinguishing between species whenmorphological criteria are ineffective(Mariaux, 1998, Galazzo et a l . 2002).Molecular characters also complementmorphological traits to reconstructphylogenetic relationships among organisms(Carreno and Nadler, 2003). The presentstudy thus aimed at utilizing the 18S rDNAgene to elucidate the phylogeneticrelationships of the common parasites of theclariid catfishes in Lake Victoria for twomajor reasons: (1) the 18S rDNA gene iswidely used in phylogenetic related worksparticularly those involving parasites, hencesuited for comparative studies, and (2) likecytochrome b, part of the 18S rDNA isknown to evolve fast enough to permit itsuse in resolving recent evolutionary history.

MATERIALS AND METHODSStudy AreaLake Victoria, the largest tropical lake in theworld, is shared between Tanzania, Ugandaand Kenya. The lake lies in a shallowcontinental sag between the two arms of theGreat Rift Valley, 1170 m above sea level.The lake has a maximum depth of 84 m, avolume of 2750 km3, and a surface area of68,800 km2. Primary inflows to the basininclude rivers such as the Kagera in the westand the Mara in the east. All outflows are tothe north along the Nile through LakeKyoga. The mean surface temperature isabout 25 oC while the temperature of deeperlayers is about 1 to 2 degrees lower (Witte&Van Densen 1995). The present studycovered the surroundings of Bukoba town,the Mwanza Gulf, Speke Gulf, parts ofUkerewe Island and the delta of Mara River(Fig. 1).

Figure: A map of Lake Victoria showing the study sites

METHODSFish collection, examination for parasitesand identification followed standardprocedures (Anderson 1992, Khalil et al.1994 and Gibson et al. 2002). Parasitesamples for DNA analysis were initiallysoaked in a 2 ml vial containing 95%

ethanol and left to stand for 1 hr, thentransferred into another similar vial withfresh 95% ethanol and stored at 4 oC untilrequired for DNA extraction. A total of 22parasites species (identified to genus orspecies level) with sufficient biomass wereconsidered for DNA analysis (Table 1).

Table 1: Parasite species analysed in the present study.

Family/Species Acc.no HostNematoda

Paracamallanus cyathopharynx DQ813445 C. werneriNeogoezia sp. DQ813444 C. liocephalus

Procamallanus laevionchus DQ813446 C. alluaudiSpinitectus petterae DQ813447 C. werneriQuimperia sp. DQ813448 C. liocephalusRhabdochona congolensis DQ813457 C. gariepinus

Contracaecum sp. DQ813456 C. gariepinusTrematoda

Diplostomum mashonense DQ813458 C. gariepinus

Tylodelphys sp.1 DQ813454 C. gariepinusTylodelphys sp.2 DQ813455 C. gariepinus

Eumasenia bangweulensis DQ813461 C. liocephalusAstiotrema reniferum DQ813459 C. gariepinusAstiotrema sp. DQ813460 C. gariepinusPhylodistomum folium DQ813462 B. docmac

Allocredium mazoensis DQ813450 C. gariepinusClinostomum sp. DQ813463 C. alluaudiMonobothrioides woodlandi DQ813449 C. werneriCestodaProteocephalus sp.1 DQ813465 C. gariepinusProteocephalus sp.2 DQ813451 H. longifilisPolyonchobothrium clarias DQ813464 C. liocephalusCrustaceanArgulus monody DQ813452 H. longifilisDolops ranarum DQ813453 C. gariepinus

DNA ExtractionDNA extraction was performed withone/individual specimen, for the largeworms such as nematodes, cestodes,crustaceans and leeches, and with severalspecimens for smaller worms like digeneans.Total genomic DNA was obtained with 20µl of proteinase K in 180 µ l of extractionbuffer (Qiagen Inc. Mississauga, Ontario)incubated for 4 to 5 hours in a heating blockat 56 oC with intermittent shaking. Sampleswere then heated to 70 oC for 10 minutesand total gDNA extracted by the Qiagencolumn (Qiagen Inc. Mississauga, Ontario)

according to manufacturer’s protocol andkept frozen until further analysis.

PCR AmplificationAmplification of 18S ribosomal DNAfragments from all parasites was carried outin an Eppendorf Mastercycler Thermal cyclerusing primers JLR24, 5’ –CGG AAT TCGCTA GAG GTG AAA TTC TTG G-3' andJLR25, 5’-CCG AAT TCC GCA GGTTCA CCT ACG G-3’ as described byCampos et al. (1998). The DNAamplification reactions contained 2.5 µl 10xPCR buffer (4.0 mM Mgcl2), 0.5 µ l of 10mM dNTPs, 1µl of each primer (10 mM),


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1.0 U HotStar Taq polymerase and 5 µlgDNA in a total of 25 µl reaction volume.

PCR conditions varied slightly dependingon the type of worms; annealing temperaturewas 48 oC for the nematodes, crustaceansand hirudineans, and 52 oC for theflatworms. Generally however, the PCRreactions were characterised by an initialdenaturation for 15 min at 95 oC, followedby 40 cycles at 93 oC for 30 sec, 48 oC/52oC for 90 sec, 72 oC for 2 min, with a finalextension at 72 oC for 5 min. Samples werethen held at 4 oC. The PCR fragments werethen run through an electrophoresis using2% agarose gel with ethidium bromide andvisualised under UV light. PCR productswere then purified using the QIAquick PCRpurification kit (Qiagen) following the

manufacturer’s protocols (Qiagen Inc.Mississauga, Ontario).

Sequencing and Sequence AnalysisDNA was sequenced with the dideoxy-termination method with fluorescent-labelledprimers using an ABI 377 Prism AutomatedDNA Sequencer. Both strands weresequenced using each of the two initial PCRprimers, JLR24 and JLR25. The sequencingproducts were then purified via isopropanolprecipitation and snap cooled before beingsubjected to sequencing electrophoresisreaction. The automated sequence data wereanalyzed using the Sequencher v. 3.0software (Gene Codes Corporation, Inc.).Chromatograms were visually inspected andconsensus sequences were aligned manuallyprior to further analysis (Plate 1).

Plate 1: Chromatogram for the clariid parasites DNA sequences from automated DNASequencher

Phylogenetic relationships were inferredusing the neighbour-joining method (NJ)based on the Hasegawa, Kishino and Yano(1985) [HKY85] distances that estimate atransition/transversion ratio and basefrequencies. The minimum evolutionary

(ME) rate assumed gamma distribution witha shape parameter of 0.5. Starting tree(s)were obtained via neighbour joining withtree-bisection-reconnection (TBR) branchswapping algorithm. Maximum parsimony(MP) searches were performed using

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heuristic method with TBR. Gaps weretreated as missing, multistate taxainterpreted as uncertainty and starting treeobtained via stepwise addition with additionsequences obtained randomly in a 10xreplicates. All the analysis were performedas implemented in PAUP* v.4.b10 forMacintosh (Swofford 2000). Reliability ofeach clade was assessed statistically using1000 replicates of bootstrap resampling(Felsenstein 1985).

RESULTSThe 18S rDNA fragments analyzed consistedof 530 nucleotide characters long of which133 characters were constant. 180 variablecharacters were parsimonious uninformativeand 217 characters were parsimoniousinformative. Nucleotide frequencies wererelatively equal in distribution such that A(25.2%), C (22.3%), G (28.4%) and T(24.1%), the lowest sequence divergencesobserved among the nematodes was 0.95%and Monobothrioides woodlandi wasarbitrarily used as an outgroup due todifficulties in obtaining a proper outgroupfor a study involving all groups of parasites.

Heuristic MP searches yielded one mostparsimonious tree (CI = 61%, HI = 39%)(Fig. 2), the topology of which did notconflict much with that of the NJ tree (Fig.3). In both the MP and NJ trees all theparasites analyzed formed monophyleticclades at the higher taxonomic level suchthat cestodes, nematodes, digeneans andcrustacean were each recovered as a separateclade supported by high bootstrap valuesabove 50% (Figs. 2 and 3).

The MP tree (Fig. 2) excluded A.m a z o e n s i s , C l i n o s t o m u m sp., D.mashonense and Tylodelphys sp. 1 and 2from the clade containing the trematodes,and Contracaecum sp. from the clade withthe nematodes. On the other hand, NJsearches yielded a tree that separated eachparasite into its respective higher taxon i.e.nematodes, cestodes, trematodes andcrustaceans. However, this tree slightlydiffered from the MP tree on the position ofClinostomum sp. (Trematoda), which isexcluded from a clade containing thetrematodes (Fig. 3).


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Figure 2: Consensus tree based on maximum parsimony (MP) with 1000 replicates bootstrapvalues (numbers in branches).


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Figure 3: A consensus tree based on the Neighbour Joining (NJ) method with bootstrap valuesindicated in branches (1000 replicates).

DISCUSSIONThe relationships among the parasitesinfecting fish of the family Clariidae of theLake Victoria basin were deciphered bypartial 18S rDNA gene sequences. The useof the 18S rDNA gene was based on itsconserved nature and potential to providemany informative characters for comparativeanalysis (Carreno and Nadler 2003). Based

on the said gene the present analysis wasable to separate the four major parasite taxainfecting the clariid fish species from theLake Victoria basin. The cestodes,trematodes, crustaceans and nematodes eachemerged as an independent group.

Despite the rise in the number ofphylogenetic studies on the invertebrates


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groups (Brooks 1979, Brooks andMcLennan 1993, Olson et al. 2001), veryfew have investigated groups comprising offlatworms (Platyhelminthes), roundworms(nematodes) and crustaceans in one study(Campos et al. 1998). Few still analyzedsuch groups from closely related hostspecies such as fish from the familyClariidae as investigated in the present study(Barson et al. 2010). The majority ofstudies analyzed the so called compositecommunities of parasitic and free-livinginvertebrates from different forms of lifesuch as birds, mammals and fish (Olson etal. 2001). Nevertheless, despite such a widevariation the results of the present studyclosely agreed with previous investigatorswhom utilized the 18S rDNA to resolve thephylogenetic relationships of various formsof parasites (Ehlers, 1986; Campos et al.,1998) at least at the higher organizationallevel.

The results of the present study showed thattrematodes infecting the clariids in the LakeVictoria basin are not monophyletic butcomprise of three independent groupsnamely; clinostomidae, digeneans anddiplostomids. In the NJ and MP treesClinostomidae and Allocreadiidae (A .mazoensis) are placed close to the cestodes.This is not surprising as both cestodes andtrematodes belong to the same phylum – thePlatyhelminthes (Campos et a l . 1998,Olson et al. 2001, Littlewood 2006).

Most of the digeneans analyzed in thepresent study share a common body formwith regard to arrangement of internalstructures such as position of the oralsucker, ventral sucker, posterior location ofthe egg filled uterus and the presence of thevitelline follicles (Gibson et a l . 2002,Mwita 2006). These structures and theirrespective arrangements form an importantfeature in the classification of digeneans,hence therefore, may also indicate thepossibility of common or closely relatedancestors among the trematodes as revealedby the results in this study.

The above body form however, slightlydiffers from that of diplostomids, in that thebody of a diplostomid is divided into thedorsoventrally flattened anterior region andthe conical hind body. In addition to theventral sucker, the diplostomids have atribocytic or Brandes organ located in theforebody, posterior to the ventral sucker,near the border with the hind body. Gonadsare located in the hind body as opposed tothose of other trematodes arranged in tandemjust behind the ventral sucker. Differences inbody morphology therefore, are key factorsin separating trematode groups.

The diplostomid body form is quite distinctfrom that of a dorsoventrally flattenedcrustacean (Argulus and Dolops species), thedorsum of which is covered by a carapaceand the ventral side with paired appendageswith hair-like projections for swimming(Mwita 2006). The close genetic relationshipobserved for these two groups is highlyunexpected. The two groups however,separated independently in the MP tree.

The families Diplostomidae andClinostomidae analyzed in the present studycomprise a group of allogenic parasites thatutilise the clariid fishes as secondaryintermediate hosts before maturing in fisheating birds (Paperna, 1980; Williams andJones 1994). The other group ofmonophyletic trematodes analysed areautogenic parasites of the Clariidae and otherfish families (Williams and Jones 1994).Whether these life forms (allogenic orautogenic) are partially or wholly controlledby the 18S rDNA gene, such that atrematode is adapted to life in a secondaryfish host and later identifies a fish eatingbird as a final host, requires furtherinvestigations. Similar results were observedby Campos et al. (1998) where theSchistosomatidae, Diplostomidae andFellodistomidae were separated as amonophyletic clade.


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Trees estimated in the present study stronglysupported monophyletic relations among thenematode families infecting the clariid fishspecies of Lake Victoria. In the NJ tree,Contracaecum sp. was in the most basalposition among the nematodes analysed.However, this was not the case for the MPtree in which Contracaecum sp. was locatedbasal to the rest of the groups includingDiplostomidae and crustaceans. Poorresolution with respect to the phylogeneticrelationship of Contracaecum sp. among thenematodes has been reported by (Nadler andHudspeth, 1998, Szostakowska andFagerholm 2007).

The MP tree divided the nematodes into twogroups; the Camallanidae / Heterocheillidae(Anisakidae) and the Rhabdochonidae /Quimperidae. Like the trematodes, thenematodes differ in certain aspects of theirmorphology, the Camallanidae, for instance,have an elongate oesophagus divided intoanterior muscular and posterior glandular.The buccal capsule is lined with chitinousplates. The second group of Rhabdochonidae/ Quimperidae however have few structuresin common and this is supported by poorbootstrap values based on geneticrelationships.

In a similar manner to the trematode clade,Contracaecum sp. is an allogenic parasiteand all the other nematodes analysed areautogenic parasites of the clariids in LakeVictoria and elsewhere. Whether the samereason(s) as for the trematodes above applyin the division of nematodes into groupsobserved is not immediately apparent,therefore needs further investigation.However, the fact that the allogenicnematodes and trematodes share the samesecondary intermediate host (clariid fish) andthe final bird hosts such as cormorantsherons, pelicans, egrets and darters (Barsonand Marshall, 2004), renders support thatthese parasites have a common gene thatenables the two groups to utilise the sameecological environment as observed in thepresent study. So the evolutionary

relationship is rather tied to the feedingecology and life history traits, which evolveover a long period, rather than merelygenetics.

AKNOWLEDGEMENTThe authors thank the Lake VictoriaEnvironment Management Project,University of Dar Es Salaam subcomponentwho financed this study. Tanzania FisheriesResearch Institute (TAFIRI), Mwanza centrefor provision of laboratory space and Prof.Steven M. Carr of Memorial University,Newfoundland in whose’ laboratory themolecular work was conducted.

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PHYLOGENETIC RELATIONSHIPS OF THE METAZOAN …