Morphological and Genetic Variations of the

Biochem Genet DOI 10.1007/s10528-014-9647-8

Morphological and Genetic Variations of the Freshwater Leech, Hirudinaria spp., in Peninsular Malaysia L. K. Chong • Alan H. K. Ong • S. G. Tan • K. A. S. Taranjeet • M. M. Peris • A. M. M. A. Sana H. R. Hassan

•

Received: 25 March 2013 / Accepted: 20 November 2013 Ó Springer Science+Business Media New York 2014

Abstract In this study the genetic diversity of local freshwater leeches (Hirudinaria spp.) was inferred using mtDNA COI gene analysis and compared with the gross external variations of 26 freshwater leech specimens obtained from the wild and leech farms. Based on a neighbor-joining tree generated from 516 COI base sequences, four distinct clades of Hirudinaria were seen with interspecific genetic divergence in the range of 7.6–14.5%. The external morphological variations based on the presence of stripes, location of gonopores, and anus separated the samples into four morphologically distinct groups matching the four clades obtained from the molecular data. Two black stripes at the ventral region were observed only in specimens found clustered with clades that contained the GenBank-reported H. manillensis, whereas the brown or dark green coloration without stripes on the ventral region was seen in samples that clustered with H. javanica and H. bpling clades.

L. K. Chong A. M. M. A. Sana Malaysian University of Science and Technology, Petaling Jaya, Selangor, Malaysia A. H. K. Ong (&) Department of Pre-Clinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Lot PT 21144, Jalan Sungai Long, Bandar Sungai Long, Cheras, 43000 Kajang, Selangor, Malaysia e-mail: [email protected] S. G. Tan Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia K. A. S. Taranjeet M. M. Peris Universiti Tunku Abdul Rahman, Kuala Lumpur, Wilayah Persekutuan, Malaysia H. R. Hassan Fisheries Research Institute, Batu Maung, Penang, Malaysia

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Keywords Asian freshwater leech COI analysis Coloration patterns Hirudinaria Genetic distance

Introduction Freshwater leeches (Annelida, Hirudinea) have a wide global distribution, with a substantial number of genera and their corresponding species found in Asia (Sket and Trontelj 2008). As with Hirudo medicinalis, Hirudinaria manillensis (commonly known as the buffalo leech) (Kutschera and Roth 2006) is a sanguivorous (hemophagic), freshwater leech with three jaws, belonging to the family Hirudinidae (Phillips and Siddall 2009). Hirudinaria manillensis, however, is mainly found in Southeast Asian countries, such as the Philippines, Thailand, Vietnam, and Malaysia, as well as in China (Zhang et al. 2008) and Taiwan (Lai and Chen 2010). In Malaysia, freshwater leeches with H. manillensis-like morphology are commonly cultured in farms (Elliott and Kutschera 2011; Teh et al. 2011) for their use as a component in traditional medicine (Tan 2008), in leech therapy for blood-related problems (Bickel et al. 1994; Zhang et al. 2008), and as sources of medicinally bioactive ingredients (Electricwala et al. 1991; Zaidi et al. 2011). Molecular classification places Hirudinaria as a sister taxon to Goddardobdella (Phillips and Siddall 2009). Nevertheless, the morphological features currently used for species identification and selection in local farming activities remain ambiguous, as farmers depend primarily on the external patterns, which have been documented for only a small number of the wild freshwater medicinal leeches within this region (Kutschera and Roth 2006; Lai and Chen 2010; Moore 1938; Nesemann and Sharma 2001; Phillips and Siddall 2009). Contrary to the more established genetic studies on the Hirudo genus (Phillips and Siddall 2009; Siddall et al. 2007; Trontelj and Utevsky 2005), the genetic diversity of the Asian buffalo leech (H. manillensis) and its genetic variants have also not been properly investigated. Most local farmers also believe that the variations seen in their collections are due to the habitats from which they were collected. As such, the present study focused on the use of COI sequencing data and external coloration patterns and morphology of wild and farmed freshwater leeches to compare and establish the genetic diversity of Hirudinaria spp. in Peninsular Malaysia.

Materials and Methods Sample Collection and Storage A total of 26 specimens (Table 1) sampled from various states of Peninsular Malaysia (Fig. 1) were identified according to their dorsal, ventral, and lateral coloration patterns (Lai and Chen 2010; Nesemann and Sharma 2001; Whitman 1886). Subsequently, the leeches were also viewed with a dissecting microscope (Leica Zoom 2000, Model No. Z45V, Germany), under 109 magnification for the locations of the anus and gonopores. All samples were either frozen at -20°C or fixed and stored in 95% ethanol and kept at -20°C prior to DNA extraction.

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Dark green

Brown

Brown

Brown

Brown

Brown with twin stripes




AF2

BF1

BF2

BP1

BP2

CF1

CF2

CS1

CR1


Dark green

AF1

CR2

Color and pattern (ventral)

Lab code

C

C

C

C

C

B

B

B

B

A

A

Type

Table 1 Freshwater leech samples used in this study

H. manillensis

H. manillensis

H. manillensis

H. manillensis

H. manillensis

H. bpling

H. bpling

H. bpling

H. bpling

H. bpling

Hirudinaria bpling

Closest species match (based on position of anus and gonopores)

River

River

Swamp

Farm

Farm

Paddy Field

Paddy Field

Farm

Farm

Farm

Farm

Source

100°180 52.5200 E

5°190 4.0700 N

100°180 52.5200 E

5°190 4.0700 N

100°250 50.400 E

5°240 30.900 N

100°180 52.300 E

5°190 00.200 N

100°180 52.300 E

5°190 00.200 N

100°330 15.3500 E

5°320 34.0400 N

100°330 15.3500 E

5°320 34.0400 N

100°180 52.300 E

5°190 00.200 N

100°180 52.300 E

5°190 00.200 N

100°180 52.300 E

5°190 00.200 N

Penang

Penang

Penang

Penang

Penang

Kedah

Kedah

Penang

Penang

Penang

Penang

5°190 00.200 N 100°180 52.300 E

Geographic origin (state)

GPS coordinate

FJ610333

FJ610332

FJ610331

FJ610330

FJ610329

FJ610334

FJ610328

FJ610327

FJ610326

FJ610325

FJ610324

GenBank Acc No

H4

H4

H4

H4

H4

H2

H2

H3

H2

H1

H1

Haplotype

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Dark green





Dark green

Brown



Dark green


Lab code

T

T13

T14

T23

T25

T31

T32

P13

P23

P25

M7

Table 1 continued

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C

A

C

C

B

A

C

C

C

C

A

Type

P. granulosa

H. javanica

P. granulosa

Poecilabdella granulosa

H. bpling

H. bpling

H. manillensis

H. manillensis

H. manillensis

H. manillensis

H. bpling


Paddy Field

River

River

River

Swamp

Swamp

Swamp

Swamp

Swamp

Swamp

Zoo pond

Source

102°130 8.400 E

2°140 56.400 N

101°440 23.900 E

3°30 10.800 N

101°350 34.800 E

3°00 28.800 N

101°350 38.400 E

3°00 28.800 N

101°50 2.400 E

4°430 26.400 N

101°50 2.400 E

4°430 26.400 N

101°50 2.400 E

4°430 26.400 N

101°50 2.400 E

4°430 26.400 N

101°50 2.400 E

4°430 26.400 N

101°50 2.400 E

4°430 26.400 N

Melaka

Selangor

Selangor

Selangor

Terengganu

Terengganu

Terengganu

Terengganu

Terengganu

Terengganu

Perak

4°510 200 N 100°450 3.600 E


GPS coordinate

JQ738395

JQ738408

JQ738401

JQ738400

JQ738406

JQ738405

JQ738404

JQ738403

JQ738402

JQ738407

FJ610335

GenBank Acc No

H7

H8

H7

H6

H5

H5

H4

H4

H4

H4

H2

Haplotype

Biochem Genet






Lab code

M12

M15

M18

M21

Table 1 continued

C

C

C

C

Type

P. granulosa

P. granulosa

P. granulosa

P. granulosa


Paddy Field

Paddy Field

Paddy Field

Paddy Field

Source

102°130 8.400 E

2°140 56.400 N

102°130 8.400 E

2°140 56.400 N

102°130 8.400 E

2°140 56.400 N

Melaka

Melaka

Melaka

Melaka

2°140 56.400 N 102°130 8.400 E


GPS coordinate

JQ738399

JQ738398

JQ738397

JQ738396

GenBank Acc No

H7

H7

H6

H7

Haplotype

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Fig. 1 Leech sampling locations within the states of Peninsular Malaysia

DNA Extraction, Amplification, and Sequencing DNA was extracted from the caudal sucker region following Borda and Siddall (2004) or from skin tissues of the ventral region following Trontelj and Utevsky (2005) using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). PCR amplification was done using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994) for the mitochondrial cytochrome oxidase I gene (mtDNA COI). The total PCR volume for mtDNA COI was 25 ll, containing 19 Taq buffer, 0.4 lM each primer, 2 mM MgCl2, 0.8 mM dNTP, 2.5 U Chromo Taq (Vivantis), and 50 ng DNA. The amplification conditions and cycles were identical to Borda et al. (2008), except for an adjustment of the annealing temperature from 48 to 46°C. All PCR products were purified with a GeneAll PCR SV kit (General Biosystems Korea) and sequenced in both directions using the ABI 3730XL DNA analyzer. These sequences have been deposited in GenBank (accession nos. FJ610324-FJ610335 and JQ738395-JQ738408; Table 1).

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Sequence Alignment and Phylogenetic Analyses Multiple sequence alignment and nucleotide composition for the COI sequences were done using ClustalW with default parameters in Mega version 4.0 (Tamura et al. 2007). In order to make better inferences of the sequences obtained from the various leech types in our collection, our COI sequences were trimmed from 532 to 516 bp to include sequences of Hirudinaria spp., Hirudo spp., a Goddardobdella species, and an Aliolimnatis species from GenBank (Table 2) and used for sequence comparisons as well as phylogenetic analyses. The jModeltest software (Posada 2008) was used to search for the best-fit model for the data using only Hirudinaria so as to maximize the accuracy of the model. The likelihood settings were set at default while the Akaike Information criterion (AIC) was set to calculate parameter importance and model averaging. The genetic distances were calculated using the TIM3?G model with a gamma value of 0.0640 generated for our samples by jModeltest in PAUP version 4b (Swofford 2002). The phylogenetic tree was generated using PAUP for distance (neighbor-joining) using the model selected by jModeltest with 1,000 bootstrap replicates.

Results and Discussion From a total of 532 bases generated, eight haplotypes characterized by 106 polymorphic sites (92 parsimony informative sites and 14 singletons) were revealed. Basically, the distance-based tree generated in this study (Fig. 2) indicated the presence of four clades. One consisted of haplotypes that clustered with H. manillensis from Vietnam (GQ868748 and GQ868747); another had representatives Table 2 COI sequences of Hirudo spp. and Hirudinaria spp. used in sequence comparisons and phylogenetic analyses Species

Geographic origin

GenBank Acc No

Reference

Hirudinaria manillensis

Puerto Rico

AY425449

Borda and Siddall (2004)


Vietnam

GQ368748

Phillips and Siddall (2009)


Vietnam

GQ368747



Thailand

GQ368746


Hirudinaria javanica

Vietnam

GQ368745


Hirudinaria bpling

Thailand

JQ846012

Phillips (2012)

Hirudinaria bpling

Thailand

JQ846013

Phillips (2012)

Hirudo medicinalis

Slovenia

AY763149

Trontelj and Utevsky (2005)

Hirudo verbana

Slovenia

EF446696

Siddall et al. (2007)

Hirudo troctina

Morocco

GQ368751


Hirudo orientalis

Azerbaijan

GQ368750


Goddardobdella elegans

Australia

GQ368743



Australia

GQ368744



Australia

GQ368742


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Fig. 2 Clustering of leech haplotypes based on a 516 bp sequence of the COI region. Neighbor-joining tree generated using all 26 samples and other genera, as listed in Table 2, and rooted using Goddardobdella elegans species from Genbank

that clustered with H. manillensis from Thailand (GQ868746) and Puerto Rico (AY425449). The remaining two clades are the H. javanica (GQ368745) and the H. bpling clades (JQ846012 and JQ846013). Our results showed three levels of genetic distance ranging from values of less than 1%, values of 1–5%, and values of more than 7% (Table 3). The low genetic

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H2

H3

H4

H5

H6

H7

H8

Hirudinaria javanica GQ368745

H. bpling JQ846012

H. bpling JQ846013

H. manillensis

2

3

4

5

6

7

8

9

10

11

12

16

15

14

13

H1

1

AY763149

Hirudo medicinalis

GQ368747

H. manillensis

GQ368746

H. manillensis

GQ368748

H. manillensis

AY425449

Haplotype

No.

0.204

0.136

0.145

0.136

0.134

0.012

0.012

0.129

0.130

0.136

0.136

0.047

0.139

0.006

0.004

–

1

0.204

0.139

0.145

0.138

0.136

0.012

0.012

0.129

0.130

0.136

0.136

0.047

0.141

0.000

2

0.204

0.139

0.145

0.138

0.136

0.012

0.012

0.129

0.130

0.136

0.136

0.047

0.141

3

0.185

0.004

0.089

0.004

0.086

0.138

0.138

0.112

0.114

0.082

0.082

0.132

4

0.202

0.130

0.127

0.130

0.116

0.051

0.051

0.115

0.117

0.119

0.119

5

0.177

0.076

0.008

0.076

0.031

0.133

0.133

0.095

0.096

0.004

6

0.182

0.076

0.008

0.076

0.029

0.133

0.133

0.095

0.096

7

0.194

0.107

0.100

0.107

0.102

0.129

0.129

0.002

8

0.192

0.105

0.098

0.104

0.101

0.126

0.126

9

0.204

0.133

0.143

0.133

0.138

0.000

10

Table 3 Genetic distances between haplotypes of freshwater leeches isolated from Peninsular Malaysia

0.204

0.133

0.143

0.133

0.138

11

0.194

0.081

0.037

0.081

12

0.180

0.000

0.085

13

0.183

0.085

14

0.181

15

16

17

18

Biochem Genet

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123

GQ368750

Hirudo orientalis

GQ368751

0.230

0.228

0.220

1

0.230

0.228

0.222

2

0.230

0.228

0.222

3

0.190

0.200

0.200

4

0.222

0.236

0.244

5

0.192

0.200

0.204

6

0.197

0.205

0.209

7

0.217

0.207

0.224

8

0.214

0.204

0.222

9

0.234

0.231

0.230

10

Hirudinaria manillensis, H. javanica, H. bpling, and Hirudo species included for comparison purposes

19

Hirudo troctina

EF446696

Hirudo verbana

17

18

Haplotype

No.

Table 3 continued

0.234

0.231

0.230

11

0.207

0.215

0.214

12

0.190

0.198

0.195

13

0.197

0.205

0.207

14

0.191

0.198

0.196

15

0.087

0.098

0.101

16

0.094

0.101

17

0.096

18

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Biochem Genet

distance of less than 1% was typically observed between individuals with haplotypes H1, H2, and H3 (0.2–0.6%) in clade 1; between haplotype H8 and H. javanica in clade 2 (0.2%); between haplotypes H6, H7, and H. manillensis from Thailand (GQ368746) (0.4–0.8%) in clade 3; as well as between members of haplotype H4 with H. manillensis from Vietnam (GQ368747 and GQ368748) within clade 4 (0.4%) (Table 3). An intermediate genetic distance was seen between H. manillensis from Puerto Rico (GQ368749) with H6, H7, and H. manillensis from Thailand (2.9–3.7%), as well as between the subclades in Clade 1 (1.2–5.1%). The highest genetic divergence range was seen between the four main clades of Hirudinaria (7.6–14.5%). Although the intraspecific divergence observed in this study was consistent with that of Hirudo medicinalis (\2.0%; Siddall et al. 2007), the divergence range between the clade consisting of H. manillensis from Vietnam and H. manillensis originating from Thailand and Puerto Rico (reportedly a species introduced from Thailand; Phillips and Siddall 2009) was as high as the divergence values seen between Hirudo species (7.6–14.5%) and those reported in Siddall et al. (2007). The morphological variations observed on the ventral region (presence of stripes, location of gonopores, and location of anus) were able to differentiate the leech specimens in this study into four groups: those that were described for H. javanica (seven annuli between gonopores) by Phillips (2012) and Whitman (1886) and the rest with five annuli between the gonopores. The specimens with five annuli between the gonopores can be further separated into two groups: those without stripes (type A and type B) and those with stripes (type C) at the ventral region (Fig. 3; Table 1). The specimens without stripes had the anus located at the 102nd annuli and fit the description of H. bpling by Phillips (2012). Type C specimens were samples that clustered with the COI sequences of H. manillensis previously reported in GeneBank; they consisted of two groups that differed in the position of the anus. In one group which clustered with H. manillensis from Vietnam (Clade 4), the anus was located at the 102nd annuli as described for H. manillensis in Lai and Chen (2010) and Phillips (2012). In the other which, clustered with H. manillensis from Puerto Rico and Thailand (Clade 3), the anus was between the 100th and 101st annuli. Sister clades 3 and 4 of H. manillensis showed high genetic distance and gross external morphological difference based on the position of the anus. Such morphological difference has not yet been reported within any leech species. However, because only very limited genetic data and morphological descriptions are available for comparison of the identified species for this clade (Puerto Rico and Thailand specimens), we are not able to confirm if our specimens clustered into Clade 3 are indeed H. manillensis. In conclusion, the COI sequence analysis showed leeches from the wild and from the farm clustered into H. javanica, H. bpling, and two sister clades of H. manillensis. Based on the external morphological variations, specimens could be separated into the corresponding four groups of leeches. In particular, the presence of two black stripes along the ventral region was observed only in specimens that clustered with H. manillensis previously reported in GenBank. This study also showed that the farmed specimens were not just H. manillensis; H. bpling

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(i) Type A

(ii)

(iii) Type B Ventral twin stripes

Type C Dorsal view Ventral view Fig. 3 Leech specimens collected from the wild. Dorsal view The overall external dorsal morphological features of Hirudinaria include (i) segmentally interrupted median line, (ii) two rows of box-like patterns on each side of the midline, and (iii) lateral patterns consisting of an orange yellowish marginal stripe with lateral black lines evenly spaced in each segment. Ventral view Variations observed in ventral colors and patterns of types A, B, and C

specimens were also identified in our collection from the same farm. Moreover, since the farmed stocks were collected from the wild, it is very likely that the farms were cultivating various Hirudinaria spp. Acknowledgments This study was supported by the Fundamental Research Grant Scheme (FRGS/1/10/ NSNH/MUST/02/1) under the Ministry of Higher Education (MOHE). The authors would also like to express their gratitude to Mr. Francis King and Mr. Ahmad Izham for providing the samples used in this study.

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