Genetic Relatedness of Diplostomum Species (Digenea: Diplostomidae) Infesting Nile Tilapia (Oreochromis Niloticus L.) in Western Kenya

doi:10.4236/ojapps.2013.38055

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Open Journal of Applied Sciences, 2013, 3, 441-448

Published Online December 2013 (http://www.scirp.org/journal/ojapps)

http://dx.doi.org/10.4236/ojapps.2013.38055

Open Access OJAppS

Genetic Relatedness of Diplostomum Species (Digenea:

Diplostomidae) Infesting Nile Tilapia (Oreochromis

Niloticus L.) in Western Kenya

Violet M. Ndeda*, Dickson O. Owiti, Ben O. Aketch, David M. Onyango

Department of Zoology, Maseno University, Kisumu City, Kenya

Email: *[email protected]

Received May 24, 2013; revised June 26, 2013; accepted July 3, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-

ABSTRACT

Diplostomum species metacercariae are trematode parasites that pose serious economic threats to aquaculture practice

globally. Identification of Diplostomum at metacercariae stage has remained equivocal, hence lack of elucidation of the

actual role of these species in fish population. 21 Diplostomoid metacercariae obtained from eyes of Nile tilapia were

characterized using 18S and ITS rDNA (ITS1-5.8S-ITS2) genes. Phylogenetic analyses of ITS rDNA gene dataset in

the metacercariae revealed close relationship to Diplostomum mashonense and D. baeri. Molecular identification using

18S rDNA sequences revealed close relationship to D. compactum, D. phoxini and D. spathaceum. Overall, genetic

analyses in this study depicted a significant unrecognized genetic diversity among Diplostomum species. Successful

differentiation of Diplostomum genera in this study using ribosomal markers suggested that 18S and ITS rDNA genes

are effective genetic markers for inter-species phylogenetic analysis and should be employed in future for identification

of diplostomoidea.

Keywords: Diplostomum; 18S rDNA; Internal Transcribed Spacer Region

1. Introduction

Diplostomum metacercariae are trematode parasites with

complex three host lifecycle that are considered major

pathogens of fish. They mature in the small intestine of

piscivorous birds and are passed on to snails as first in-

termediate host and fish as second intermediate host dur-

ing their life cycle [1]. Once attached to the fish, Dip-

lostomum metacercariae moves to the lens, retina and

aqueous humour of fish eyes as well as the brain, spinal

cord and nasal spaces thereby resulting into substantial

losses of wild and farmed fish [1,2].

Diplostomum parasites have been encountered in fish

farms in the northern hemisphere (Europe and North

America) and as such received wide theoretical and em-

pirical attention due to their pathogenic consequences to

fingerlings [3-6]. The eye fluke infection has been asso-

ciated with decreased growth of fish in rearing conditions

[7,8] and increased mortality in fish as a result of reduced

vision caused by cataracts which impairs the fish’s feed-

ing efficiency [9] and makes the fish more vulnerable to

avian predation [10]. Taxonomic studies on the diplos-

tomid digeneans from the African continent are scarce

and limited to few Diplostomum species descriptions

published between 1930s-1960s, and virtually little is

known of their natural history [e.g., 11,12]. This is re-

lated to low sampling efforts in tropical countries due to

lack of expertise in the field of fish parasitology. In addi-

tion, identification of these parasites is problematic due

to 1) the presence of morphologically similar species; 2)

the phenotypic plasticity of the adults and metacercariae;

3) the simple larval morphology; and 4) the difficulties in

linking life-cycle stages [13].

These problems represent a major impediment for the

assessment of the distribution and the actual role of dip-

lostomids in fish populations. Furthermore, most of the

published African diplostomid studies have focused on

the catfish Clarias gariepinus [14-17]. Recently, reports

have indicated presence of Diplostomiasis infection

*Corresponding author.

V. M. NDEDA ET AL.

442

among Oreochromis niloticus in Kenya [18,19] and from

another African freshwater fish host, Synodontis nigrita

[17] in Nigeria. This is an indication that Diplostomum

parasites affect a number of fish species and therefore

more research should be undertaken to elucidate the di-

versity of these parasites in other fish populations. As

such, basing taxonomic identification of African Dip-

lostomum species to a few ancient descriptions is mis-

leading. In addition, relying on results from one fish spe-

cies (Clarias gariepinus) makes it unreliable to extrapo-

late results to other fish species because of differences in

feeding and resting behaviours among fish species. Inap-

propriate naming of Diplostomum species in tropical

countries has led to misidentification of the species and

thus inaccurate estimates of species diversity. For exam-

ple, a study by [16] assigned the name D. mashonen se to

a parasite species recovered from the brain of clarid fish

in Tanzania, however, a recent study on the same by [17],

elicited a controversy in the naming of the parasites

thereby reallocating D. mashonense to Tylodelphys mash-

onense using ITS1-5.8S-ITS2 molecular markers. This

controversy depicts a complicated taxonomic situation in

Africa coupled with poor taxonomic resources and ex-

pertise. Therefore, misidentification of species is a fact

that cannot be neglected because, concurrent infections

with the eye-infecting diplostomids appear to be frequent

and widespread geographically being reported in Tanza-

nia, South Africa and Kenya [16,19,20].

This fact, coupled with the morphological similarity of

the metacercariae makes the practical species diagnosis

based on morphology very difficult. Use of rapid and

accurate molecular identifications using standardized

tools and barcoding approaches appear most suited [21]

for unravelling diplostomid taxonomic diversity in dif-

ferent regions worldwide.

To date, a total of eight named species of Diplosto-

mum using ITS rDNA sequences are now available.

These include: complete sequences of the ITS1-5.8S-

ITS2 gene cluster for Diplostomum huronense (La Rue,

1927), Diplostomum indistinctum (Guberlet, 1923) and

Diplostomum baeri (Dubois, 1937) from fish and/or gulls

collected in Canada [22-24] and partial ITS1 sequences

for Diplostomum baeri, Diplostomum mergi (Dubois,

1932), Diplostomum paracaudum (Iles, 1959), Diplosto-

mum parviventosum (Dubois, 1932), Diplostomum pseu-

dospathaceum (Niewiadomska, 1984) and Diplostomum

spathaceum (Rudolphi, 1819) from larval stages col-

lected in Poland [25]. Furthermore, ITS1-5.8S-ITS2 se-

quences for nine additional presumed species (uniden-

tified isolates labelled as Diplostomum spp. 1-9) have

been generated recently from fish metacercariae in Can-

ada [23,24,26]. These studies only shed light on previ-

ous studies conducted in the St. Lawrence River in Can-

ada which is geographically limited to the northern he-

misphere (Europe and North America). Therefore more

research should be prioritized on Diplostomum parasites

affecting fish in order to update the taxonomic database

in Africa. The principal goal of this study was to estab-

lish the genetic relatedness of Diplostomum spp. parasi-

tizing populations of Oreochromis niloticus L. within

Kisumu municipality, in western Kenya using ribosomal

molecular markers.

2. Materials and Methods

The study was conducted in three locations within Ki-

sumu municipality (Figure 1) bordering Lake Victoria in

western Kenya, after every 3 weeks from December,

2011 to February, 2012. Kisumu region was preferred for

the study because of the Economic Stimulus Program

(ESP) initiated by the government in 2009 that targeted

fish farmers in the region and led to the construction of

over 300 fish ponds in the municipality. In addition, Ki-

sumu region experiences four distinct seasons, i.e. two

rainy seasons and two dry seasons. The rainy seasons are

further sub-divided into the long rainy season and the

short rainy season [27]. Likewise, the dry season is also

subdivided into a long dry season, and a short dry season.

The long rainy season usually begins in March through to

May. This is normally followed by a long dry spell,

which starts in June and ends in August. The short rainy

season starts in October and lasts for two months until

November, followed by the long dry spell which starts in

December through to February [28]. Period of sampling

for this study (December 2011-February 2012) was pre-

ferred based on previous literature by [19] in Kenya that

reported diplostomid transmission patterns to be higher

during dry seasons compared to wet seasons. Maximum

temperatures in Kisumu occur in the long dry spell with

an annual maximum temperature range of about 27˚C to

about 32˚C [29]. Minimum temperature ranges from

14˚C to 18˚C, with the peak minimum temperature re-

corded in August through September [29]. At least three

farms were selected per location based on their proximity

to one hatchery centre in the municipality which serves

as source of the much required quality fingerlings for

supply to prospective farmers. These locations border

each other hence ease of accessibility.

2.1. Fish Sampling Procedure and

Transportation

Sample size used in this study was estimated according

to the formula by [30]. Sampling was done in three main

settlement areas of Kisumu after every three weeks for a

period of three months (December 2011-February 2012).

Sixty four (64) Nile tilapia fish were randomly sampled

per pond, for every three ponds per farm using a seine

et of 1.5 m diameter and 6mm mesh. Therefore a total n

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V. M. NDEDA ET AL.

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443

Figure 1. Map showing Kisumu Municipality and the study area (marked in box).

18S300R [5’TCAGGCTCCCTCTCCGG-3’ (400 nt)];

of 27 fish ponds were sampled in Kisumu municipality.

Sampled fish were then transported in an iced cool box at

8˚C to the department of Zoology laboratory, Maseno

University, for further analysis.

18S637R [5’TACGCTATTGGAGCTGGAGTTACCG-

3’ (600 nt)] were used [33]. Amplification of the portion

of the rDNA that included the complete ITS1-5.8S-ITS2

region was done via the polymerase chain reaction (PCR)

in MJ Gradient thermocycler (Gene amp. PCR system

9700, Applied Biosystems USA) according to [22] pro-

tocol.

2.2. Laboratory Procedures

2.2.1. Examina tion of Fish Specimens f or

Diplostomum Parasites

Primer sequences of:

Fish eyes were dissected and then examined for meta-

cercariae with a stereoscopic microscope using proce-

dures described by [31]. The metacercariae extracted

from each eye were counted as separate lots, and placed

in a petri dish containing saline solution before storing in

95% ethanol. Isolated metacercariae were stored in mi-

crocentrifuge tubes at 4˚C and labeled according to their

collection sites.

D1 [5’-AGGAATTCCTGGTAAGTGCAAG-3’];

D2 [5’-CGTTACTGAGGGAATCCTGGT-3’] were em-

ployed [33]. The products were run through electropho-

resis using a 1% agarose gel containing ethidium bro-

mide (0.5 μg/mL) alongside 0.5 μg/μl; Fermentas: Gen-

eRuler™ 1kbp DNA Ladder (for ITS) and 100 bp (for

18S) and visualized under Ultra Violet light.

PCR products were then purified using the Gene-Jet

PCR purification kit (Fermentas, No. K 0701) following

the manufacturer’s protocol.

2.2.2. Ge netic Cha ra cterization of Diplostomum

Species

DNA was extracted from individual diplostomoids fol-

lowing the method of [32]. DNA amplification of the

18S rDNA sequence was performed according to proto-

col described by [33] using MJ Gradient thermocycler

(Gene amp. PCR system 9700, Applied Biosystems USA).

2.3. DNA Sequencing

Sequencing was performed at the Inqaba laboratories in

South Africa. ITS1-5.8S-ITS2 region was sequenced

using primers BD1 (5’-GTC GTA ACA AGG TTT CCG

TA-3’) and BD2 (5’-TAT GCT TAA ATT CAG CGG

Primer sequences of:

18S9F [5’TGATCCTGCCAGTAGCATATGCTTG-3’];

V. M. NDEDA ET AL.

444

GT-3’) of [34] which were used as the forward and re-

verse primers, respectively. Sequencing for 18S rDNA

gene was performed using the forward PCR primer only.

2.4. Data Analysis

2.4.1. Diplostomum Species Identification and

Delineation

Contiguous sequences of the small subunit region and

internal transcribed spacer regions of ribosomal DNA

from 21 specimens were created from forward and re-

verse chromatograms and edited using DNABaser ver-

sion 2.7. Multiple alignments of the contigs was con-

ducted using Muscle 3.8.31 multiple alignment software.

The nucleotide sequence data of ITS rDNA and 18S

rDNA sequences were then submitted to Basic Local

Alignment Search Tool (BLAST,

www.ncbi.nlm.nih.gov/blast) for similarity searches in

Gen Bank. MEGA version 4.0.2 [35] was used to cluster

potential species using the Neighbour-Joining pheno-

grams. The reliability of internal branches in the Neigh-

bour-Joining trees was assessed using bootstrap analysis

with 1000 replicates. The resulting networks were rooted

with the out-group taxa.

2.4.2. Neighbour-Joining Analyses of ITS rDNA and

18S rDNA Sequences from Diplostomum

Specimens Collected from Fish in Kisumu

Municipality

Phylogenetic analyses were conducted based on the

alignment of partial and complete sequences of ITS

rDNA and 18S rDNA using the NJ method. The resultant

tree as shown in Figures 2 and 3 presented bootstrap

consensus values of >50% for almost all branches con-

firming that the samples were indeed members of the

Diplostomum genus and were closely related to Dip-

lostomum phoxini, Diplostomum compactum, Diplosto-

mum spathaceum, Diplostomum mashonense and Dip-

lostomum baeri.

The NJ analyses for 18S rDNA sequences alone re-

vealed presence of single species of Ichthyocotylurus,

Strigidae and Bolbophorus, two species of Apharyngos-

trigea and Posthodiplostomum, one species of Alaria and

at least three species of Diplostomids (Diplostomum

phoxini, Diplostomum compactum and Diplostomum spa-

thaceum) that were closely related to the sample speci-

mens (Figure 2). The NJ analyses for ITS rDNA (Figure

3) revealed genetic relationship of the sample specimens

to two Diplostomum species (Diplostomum mashonense

and Diplostomum baeri). The resultant tree presented

bootstrap consensus values of >50% for almost all

branches. The bootstrap (Felsenstein, 1988) consensus

tree was inferred from 100 replicates (Figure 3) and

1000 replicates (Figure 2) and taken to represent the

relationship of the taxa analyzed. The trees are drawn to

scale, with branch lengths in the same units as those of

the evolutionary distances used to infer the phylogenetic

tree.

3. Discussion

The principal findings of this study indicated that mul-

tiple species infections of Diplostomum were common in

the fish community. These findings were revealed using

ITS and 18S ribosomal DNA. Five species of lens-infect-

ing Diplostomum were found to be closely related to the

specimens analysed in this study based on the distance

matrix method that considered the phenotypic similarities

of the species. The use of outgroup taxa (Tylodelphys sp.)

that was closely related to Diplostomum and the mono-

phyly of the cryptic species revealed herein, suggested

that each species complex originated from a common

ancestor. According to this study, ITS sequence data of

specimen D32 was closely related to Diplostomum mash-

onense (Beverley-Burton, 1963). Similarity of specimen

D32 to D. mashonense was associated with the resultant

tree that presented a bootstrap consensus value of 100%

for the branch (Figure 3). This is in agreement with the

observation by [16] who pointed out striking similarity

between D. mashonense (Beverley-Burton, 1963) and

Tylodelphys spp. 1 and 2, and later discriminated D.

mashonense (FJ 470402) from Tylodephys spp. using

morphometric variability analysis. Similarity between the

two Diplostomum species (D. mashonense and D32) sug-

gest a strong association between Diplostomum sp. stud-

ied in Tanzania and Diplostomum in Kisumu, Kenya.

Thus, similarity in parasite distribution in the different

fish host species might be as a consequence of co-evolu-

tionary interactions associated with geographical diver-

gence of the species.

Phylogenetic analysis of ITS rDNA sequence data

from adult forms of Diplostomum by [22] lends support

to 1 sequence from this study which demonstrated highly

similar consensus sequences to D. baeri (JQ 665460)

classified as American species (Figure 3). Specimens

closely related to D. baeri included D42, D52, D35, D57,

D44, D26, and D46 which were equally assessed by ITS

rDNA and strongly supported by a high bootstrap value

(>99%). 18S rDNA sequence data was closely related to

D. compactum, D. spathaceum and D. phoxini classified

as American or European species. Specimen M7 and

M10 were closely related to D. compactum and D. spa-

thaceum, whereas specimens M5-M20 were closely re-

lated to D. phoxini. Similarity of the specimens to re-

ported Diplostomum sp. was associated with the resultant

tree that presented bootstrap consensus values of >50%

for almost all branches (Figures 2 and 3). The present

study therefore provides a preliminary confirmation of

diplostomoid species residing in both continents with a

possibility of recent divergence or hybridization. Spatial

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V. M. NDEDA ET AL.

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445

Figure 2. Neighbour joining tree depicting genetic relationship between sample specimens labelled “M” and other reported

Diplostomum sp. as inferred from 18S rDNA sequences. Numbers at the nodes represent bootstrap values. Bolbophorus sp.

was set as out group.

Figure 3. Neighbour-joining tree of sequences constructed in this study in comparison with representatives in the Dip-

lostomum sp. as inferred from ITS rDNA (Specimen labelled D’s) and 18S rDNA (Specimen labelled M’s) sequences.

Numbers at the nodes represe nt bootstr a p value s. Tylodelphys spp. was set as an out group.

V. M. NDEDA ET AL.

446

istribution of Diplostomum sp. at the three fish locations of Fish, Samara Publi

in Kisumu municipality demonstrated five Diplostomum

communities among the fish population. This is an indi-

cation of increased gene flow among the parasites mak-

ing it impossible for only one parasite to adapt to the

environment. Although our study was not designed to

investigate how definitive hosts influence gene flow in

parasites, we gave speculations based on the biological

basis of the parasite life cycle. Host mobility has been

proposed to be the main determinant of gene flow in

parasites since they are commonly dependent on their

host for dispersal [36]. According to [37], parasites with

complex life cycles such as Diplostomum include multi-

ple host species within their lifecycle and therefore gene

flow is expected to be determined by the host with the

highest dispersal rate. In this study, it was likely that high

mobility in the bird species commonly seen around the

farms (the great Egret, Cormorants and Pied-billed Grebe)

was sufficient to cause high levels of gene flow among

the spatially isolated Diplostomum parasites in the dif-

ferent farms when taking into account that each defini-

tive host can harbour dozens or hundreds of adult para-

sites.

4. Conclusion

Internal transcribed spacer (ITS) and 18S rDNA

dge the help from Maseno

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ITS: Internal Transcribed Spacer

rDNA: Ribosomal DNA