Antheraea assamensis and Samia cynthia ric

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Int. J. Indust. Entomol. 30(1) 6-16 (2015) ISSN 1598-3579, http://dx.doi.org/10.7852/ijie.2015.30.1.6

Genetic characterization of microsporidians infecting Indian non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) by using PCR based ISSR and RAPD markers assay. Wazid Hassan* and B. Surendra Nath

Molecular Pathology Division, Seribiotech Research Laboratory, Central Silk Board, CSB Campus, Kodathi, Carmelram Post, Bangalore - 560035, Karnataka, India

Abstract This study established the genetic characterisation of 10 microsporidian isolates infecting non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) collected from biogeographical forest locations in the State of Assam, India, using PCR-based markers assays: inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD). A Nosema type species (NIK-1s_mys) was used as control for comparison. The shape of mature microsporidian spores were observed oval to elongated, measuring 3.80 to 4.90 µm in length and 2.60 to 3.05 µm in width. Fourteen ISSR primers generated reproducible profiles and yielded 178 fragments, of which 175 were polymorphic (98%), while 16 RAPD primers generated reproducible profiles with 198 amplified fragments displaying 95% of polymorphism. Estimation of genetic distance coefficients based on dice coefficients method and clustering with un-weighted pair group method using arithmetic average (UPGMA) analysis was done to unravel the genetic diversity of microsporidians infecting Indian muga and eri silkworm. The similarity coefficients varied from 0.385 to 0.941 in ISSR and 0.083 to 0.938 in RAPD data. UPGMA analysis generated dendrograms with two microsporidian groups, which appear to be different from each other. Based on Euclidean distance matrix method, 2-dimensional distribution also revealed considerable variability among different identified microsporidians. Clustering of these microsporidian isolates was in accordance with their host and biogeographic origin. Both techniques represent a useful and efficient tool for taxonomical grouping as well as for phylogenetic classification of different microsporidians in general and genotyping of these pathogens in particular. © 2015 The Korean Society of Sericultural Sciences Int. J. Indust. Entomol. 30(1), 6-16 (2015)

Introduction

Received : 9 Jan 2015 Revised : 26 Mar 2015 Accepted : 27 Mar 2015 Keywords: Microsporidians, Antheraea assamensis, Samia cynthia ricini, RAPD, ISSR, genetic characterisation

amenable for domestication and exhibits multivoltine nature with polyphagous feeding habit. Eri silk mostly confined to

Among the non-mulberry (vanya) silkworms, eri silkworm,

the Brahmaputra valley of Assam, India in the tribal inhabited

[Samia cynthia ricini, Donovan (Lepidoptera: Saturniidae)] is

districts and other North Eastern states of the country, significantly

*Corresponding author. Wazid Hassan Molecular Pathology Division, Seribiotech Research Laboratory, CSB Campus, Carmelram Post, Kodathi, Bangalore – 560035, India. Tel: +0091 080 28440651 / FAX: +0091 080 28439597 E-mail: [email protected]  © 2015 The Korean Society of Sericultural Sciences 6

Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 6-16 (2015)

contributes to Indian commercial silk production. Like mulberry

inter simple sequence repeats or ISSRs) are very useful for detecting

silkworm, eri silkworms are prone to several virulent and infectious

genetic polymorphisms in microsatellite and intermicrosatellite

diseases and pests. However, they are comparatively more resistant

loci and have been found to be a novel technique for fingerprinting

to the diseases and pests than other silkworm types.

and thus differentiating, closely related individuals (Zietkiewicz et

Muga silkworm [Antheraea assamensis, Helfer (Lepidoptera:

al. 1994). The usefulness of ISSR genetic markers has been well

Saturniidae)] is an economically important insect in India. A. assamensis

established by various researchers (Nagaoka and Oghihara 1997,

is an endemic species prevalent in the Brahmaputra valley. Muga culture

Fang et al. 1997, Raina et al. 2001, Reddy et al. 1999, Vijayan 2004,

for the people of Assam is part of their culture, tradition and customs,

Rao et al. 2005). The ISSR-PCR strategy is especially attractive

rather than a profitable professional. Presently, about 30,000 families

because it does not need sequence information for primer synthesis,

in Assam are directly associated with muga culture. Muga silkworm

enjoying the advantage of random markers.

is very prone to several virulent and infectious diseases and pest. The

In Random Amplified Polymorphic DNA-PCR assay a set of

germs and symptoms of disease are more or less similar to those other

primers of arbitrary nucleotide sequences been used (Welsh and

silkworm species diseases. All the major pathogenic microbes cause

McClelland 1990, Williams et al. 1990), which have described

disease in muga silkworm and the most common among them are

as potential molecular marker system for the analysis of genetic

Pebrine, Grassarie, Flacherie, and Muscardine. Pebrine is caused by

diversity and phylogeny in a wide variety of organisms (Hadrys et

Nosema sp. In pebrine disease, silkworm stops feeding resulting in

al. 1992, Lu and Rank 1996).

unequal size larvae, they become it sluggish and die. The dead larvae turn black due to secondary bacterial infection.

So far, no research works are reported on the molecular characterization of microsporidian isolates infecting the Indian muga

Microsporidia are a diverse group of obligate intracellular

(A. mylitta) and eri (S. c. ricini) silkworms, with special reference

eukaryotic parasites with 1300 described species in 160 genera

to vast bio-geographical forest areas in different districts of Assam

approximately (Wittner and Weiss 1999, Keeling 2009).

State, India. The present study was undertaken to establish genetic

Microsporidia are distinctive eukaryotes, which do not have

characterization of 10 different microsporidian isolates infecting

centrioles and mitochondrial apparatus, although, nuclei are present

Indian non-mulberry silkworm, A. assamensis and S. c. ricini using

in distinct number (Vossbrinck and Woese 1986, Vossbrinck et

ISSR and RAPD-PCR markers assay.

al. 1987). Earlier microsporidia were classified in the kingdom Protista. However, recent molecular phylogenetic analysis using various genes viz., α-tubulin, β-tubulin and Hsp-70, suggest that

Materials and Methods

microsporidia are more closely related to fungi (Hirt et al. 1997, Keeling 2003). Microsporidians infect a wide range of invertebrates

Origin of microsporidian spores and purification

and vertebrates including insects, fishes, mammals and protists (Wittner and Weiss 1999, Wasson and Peper 2000, Weiss 2001).

Ten microsporidians were originally collected from the diseased

Classification of microsporidians based on ultrastructural

individual A. assamensis and S. c. ricini silkmoths during 2010 to 2013

differences have been replaced by molecular phylogenetic analysis

in nine locations belonging to six geographic regions covering different

based on DNA marker profiles (Baker et al. 1995, Hartskeerl et

traditional muga and eri culture reserved forest areas in the districts of

al. 1995, Mathis et al. 1997, Hung et al. 1998). The Inter simple

Jorhat, Dhemaji, Darrang, kamrup, Korbi Anglong, and Lakhimpur

sequence repeats (ISSR), (Zeitkiewicz et al. 1994) and Random

in Assam, India (Fig. 1). The microsporidian spores were isolated

amplified polymorphic DNA (RAPD), (Williams et al. 1990)

from infected muga and eri silkmoths by maceration and suspended

were identified as potential molecular marker systems. The ISSR

them in 0.85% NaCl followed by filtration through cheese cloth and

and RAPD-PCR markers are successfully been used to generate

centrifugation at 3500 r/min for 10 min. The spore pellet was purified

molecular markers, to create genetic diversity and phylogenetic

by Percoll gradient centrifugation (Undeen and Alger 1971). Each of

relationship among different microsporidians identified from

the purified microsporidian isolates were maintained in vivo in isolation,

different silkworms (Tsai et al. 2003, Rao et al. 2005, 2007, Nath et

through per oral inoculation and designated as MIA-7mJr, MIA-8mDm,

al. 2011, Hassan and Nath 2014 ).

MIA-9mMd, MIA-10mKp, MIA-1eBr ,MIA-2eBr, MIA-3eDj, MIA-

ISSR primers that amplify regions between SSRs (referred as

4eLr, MIA-5eDu, MIA-6eTr and the type species is designated as NIK-

7

Wazid Hassan and B. Surendra Nath Genetic characterization of microsporidians infecting Indian non-mulberry silkworms

1s_mys. The details of microsporidian isolates, places of collection,

Measurement of spore length and width

host, shape and size are presented in Table 1. The morphology of purified microsporidian spores was observed using phase contrast microscope. The length and width of microsporidian spores were measured according to the method of Undeen and Vavra (1997). The fresh mature spores were spread in water agar on glass micro-slides and measured using an ocular micrometer under phase contrast microscope and all the measurements are presented in micrometers as mean values of 12 individual observations (Table 1).

DNA extraction and purification Genomic DNA was extracted from the sporoplasms discharged Fig. 1. Map of Assam showing the distribution of non-mulberry silkworm Muga (A. assamensis) and Eri (S. c. ricini) in six biogeographical areas.

from spores using the glass bead method (Undeen and Cockburn 1989). DNA concentration and quality was determined by spectrophotometry at 260 and 280 nm and on 0.8% agarose gel

Table 1. Details of ten microsporidian isolates and type species: their place of collection, host and morphology Sl. No.

Name of microsporidian isolates

1

MIA-7mJr

2

MIA-8mDm

Antheraea assamensis

Dhemaji forest area, District: Dhemaji, Assam, India 27˚4'81.11N / 94˚5’57.28E/70.0

Elongated 4.40±0.12 2.66±0.08 oval

3

MIA-9mMd

Antheraea assamensis

Mangaldoi forest area, District: Darrang, Assam, India 26˚4'33.00N / 92˚0’33.00E/70.0

Elongated 4.10±0.21 2.90±0.04 oval

4

MIA-10mKp

Antheraea assamensis

Kamrup forest area, District: Kamrup, Assam, India 26˚3'16.08N / 91˚5’98.39E/68.0

Elongated 3.90±0.08 2.80±0.06 oval

5

MIA-1eBr

Samia. c. ricini

Barpathar forest area, District: Korbi Anglong, Assam, India Elongated 3.80±0.08 2.60±0.01 26˚2’76.31N / 93˚8’97.38E/90.0 oval

6

MIA-2eBr

Samia. c. ricini

Barpathar forest area, District: Korbi Anglong, Assam, India Elongated 3.80±0.05 2.70±0.12 26˚2’76.31N / 93˚8’97.38E/90.0 oval

7

MIA-3eDj

Samia. c. ricini

Dhemaji forest area, District: Dhemaji, Assam, India 27˚4’81.11N / 94˚5’57.28E/70.0

Elongated 3.90±0.20 2.95±0.10 oval

8

MIA-4eLr

Samia. c. ricini

Lakhimpur forest area, District: Lakhimpur, Assam, India 27˚2’06.35N / 94˚1’51.37E/78.0

Elongated 4.05±0.08 3.05±0.01 oval

9

MIA-5eDu

Samia. c. ricini

Diphu forest area, District: Korbi Anglong, Assam, India 25˚8’46.52N / 93˚4’29.87E/74.0

Elongated 4.00±0.07 2.70±0.01 oval

10

MIA-6eTr

Samia. c. ricini

11

Host

Place of collection (Forest area/Village), District, Latitude/Longitude/Elevation

Spore shape

Length

Width

Antheraea Jorhat forest area, District: Jorhat, Assam, India. 26˚7'56.02N Elongated 4.90±0.23 2.90±0.01 assamensis / 94˚2’09.45E/92.0 oval

NIK-1s­_mys Bombyx mori

Titabar forest area, District: Jorhat, Assam, India 26˚5’88.08N Elongated 3.85±0.18 2.80±0.01 / 94˚1’87.21E/68.0 oval CSTRI, Mysore, District: Mysore, Karnataka, India. 12˚17’44.9154N/76˚38’21.7716E/770.10

Oval

Note: MIA, Microsporidia India Assam; NIK, Nosema India Karnataka; CSR&TI, Central Sericulture Research and Training Institute

8

Spore size (mµ)

3.80±0.08 2.60±0.01

Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 6-16 (2015)

visualization, using a known quantity of λDNA (10 ng/μL) as a

DNA contamination was checked using insect mitochondrial

standard before use in subsequent PCRs. The possibility of host

primers.

Table 2 The nucleotide sequences of the primers, number of amplified fragments, fragment size, and number of polymorphic fragments scored using ISSR and RAPD profiles of eleven microsporidians DNA in PCR. Sl. No.

Primers identification

Nuceotide sequence (5'→3')

No of fragments amplified

Fragments Size range (bp)

No of polymorphic fragments

ISSR primers 1

812

GAGAGAGAGAGAGAGAA

13

300-3000

13

2

816

CACACACACACACACAT

13

400-3600

13

3

817

CACACACACACACACAA

12

400-3500

12

4

818

CACACACACACACACAG

13

500-3500

13

5

825

ACACACACACACACACT

11

450-3500

11

6

826

ACACACACACACACACC

12

450-3800

12

7

827

ACACACACACACACACG

12

700-3000

11

8

834

AGAGAGAGAGAGAGAGYT

13

400-3500

12

9

842

GAGAGAGAGAGAGAGAYG

12

450-3350

12

10

855

ACACACACACACACACYT

13

500-2500

13

11

856

ACACACACACACACACYA

13

400-2500

13

12

862

AGCAGCAGCAGCAGCAGC

13

550-3000

13

13

864

ATGATGATGATGATGATG

14

500-4000

13

14

881

GGGTGGGGTGGGGTG

14

600-3800

14

Total

178

175

RAPD primers 1

OPW-1

CTCAGTGTCC

13

500-2700

12

2

OPW-2

ACCCCGCCAA

13

500-2200

12

3

OPW-4

CAGAAGCGGA

11

300-2200

10

4

OPW-5

GGCGGATAAG

12

500-3200

12

5

OPW-6

AGGCCCGATG

14

350-2900

13

6

OPW-7

CTGGACGTCA

14

350-2800

12

7

OPW-8

GACTGCCTCT

12

350-3000

12

8

OPW-9

GTGACCGAGT

12

300-2700

12

9

OPW-10

TCGCATCCCT

13

500-2700

13

10

OPW-11

CTGATGCGTG

12

350-1800

12

11

OPW-12

TGGGCAGAAG

14

400-2200

13

12

OPW-16

CAGCCTACCA

12

350-2800

12

13

OPW-17

GTCCTGGGTT

12

300-3000

12

14

OPW-18

TTCAGGGCAC

08

500-2550

08

15

OPW-19

CAAAGCGCTC

11

300-2000

11

16

OPW-20

TGTGGCAGCA

13

350-2600

12

Total

196

188

9

Wazid Hassan and B. Surendra Nath Genetic characterization of microsporidians infecting Indian non-mulberry silkworms

PCR amplification of the DNA with ISSR primers

with bromophenol blue gel loading dye and were size fractionated by electrophoresis on 1.5% agarose gel. RAPD amplified fragments

The protocol of Zietkiewicz et al. (1994) was followed with minor

similarly processed further like ISSR.

modifications. 20 ISSR primers from primer set 9 (Biotechnology Laboratory, University of British Columbia, Vancouver, B.C.) were

Molecular data analysis

tested for PCR amplification, of which 14 primers (11 di, 2 tri, and 1 penta-, nucleotides) which were high polymorphic and reproducible

Analysis of the patterns was based on the presence or absence of

observations were used for PCR amplification. These primers were

unambiguously reproducible amplified bands and their size. ISSR

mostly 15 to 18 mers (Table 2). The PCR amplification was carried

and RAPD markers were scored according to the presence (1) or

out in 20 µL of reaction volume, containing 1 x PCR buffer, 30 ng

absence (0) of a band across ten isolates of microsporidians; each

of template DNA, 200 µM of each dNTP’s, 2.5 mM MgCl2, 100 pM

primer was scored separately. The banding scoring were repeated

of a single primer and 1 U of Taq DNA polymerase. Samples were

three times and only the reproducible conspicuous bands were

amplified on a DNA thermal cycler (MJ Research Inc., Watertown,

included in the analysis. The total number of fragments amplified,

Mass.). After initial denaturation at 94°C for 2 min, 35 cycles of

the number of polymorphic fragments scored and the percentage

30 s denaturation at 94°C, 30 s annealing at 50°C and a 2 min

of polymorphic bands were documented. The NTSYS-pc version

extension at 72°C were performed before a final extension of 10

2.11T computer program (Applied Biostatistics, Setauket, NY) was

min at 72°C and subsequent cooling at 4°C. The ISSR amplification

used for genetic distance analysis. The ISSR and RAPD data were

PCR products were mixed with bromophenol blue gel loading dye

analyzed using SIMQUAL (similarity for qualitative data) method

and were size fractionated by electrophoresis on 2.0% agarose gel

to generate genetic distance values among different microsporidian

(Promega Corporation, Madison, USA ) in 1 x Tris-borate-EDTA

isolates using Dice coefficients (Dice, 1945) (S = 2Nab/(2Nab+Na+Nb),

buffer (89 mM Tris, 89 mM Boric acid, 2 mM EDTA, pH 8.0)

where Nab is the number of bands common to lanes a and b, Na is

and gels were stained with ethidium bromide (0.5 µg/mL) for 30

the total number of bands present in a and Nb is the total number of

min (Sambrook et al. 1989). A standard molecular weight marker

bands in lane b) (Nei and Li 1979). The Dice similarity coefficients

(Thermo Scientific, USA) was used in each electrophoretic run

were then used to generate dendrograms using the un-weighted

and the UV-transilluminated gels were photographed by using

pair group method with arithmetic averages (UPGMA) employing

Gel Documentation System (Syngene Corporation, Cambridge,

the SAHN (sequential, agglomerative, hierarchical and nested

UK). Three replicate experiments were carried out to verify the

clustering) module. To check the robustness of the obtained UPGMA

reproducibility of the markers on different occasions.

based dendrograms and their confidence limits, bootstrapping with 1000 replications was performed using the WINBOOT software

PCR amplification of the DNA with RAPD primers

(Yap and Nelson 1996). In addition, the genetic variability further tested by multidimensional scaling of the ISSR and RAPD data

RAPD-PCR reactions were performed according to the protocols

was carried out using the ALSCAL algorithm (SPSS Inc. Chicago

of Welsh and McClelland (1990) and Williams et al. (1990). Twenty

USA). The dissimilarity matrixes were created using Euclidean

different RAPD primers (Operon Technologies, Inc, Alameda, CA)

distance and the same was used for the classical Young-Householder

were used for PCR amplification. The PCR amplifications were

multidimensional scaling procedure in this method (Young et al.

carried out in MJ Research Thermal Cycler PTC-200 (MJ Research

1984, Young and Harris 1990).

Inc. Watertown, MA.) in 20 µL reaction mixture containing 1 x PCR buffer, 200 µM each dNTP’s, 2.5 mM MgCl2, 0.2 µM of a single primer, 30 ng template DNA and 1 U of Taq DNA polymerase (Thermo

RESULTS

Scientific). Amplification reactions were carried out for 35 cycles after an initial denaturation for 3 min at 93°C. Each PCR cycle comprised

Spore morphology

three steps: denaturation at 93°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 2 min with a final extension of 10

The different microsporidian isolates identified from diseased

min at 72°C. The RAPD amplification PCR products were mixed

A. mylitta and S. c. ricini silkworm, (Table 1) were characterized

10

Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 6-16 (2015)

using spore morphology. The shape of mature spore of four muga

used to generate ISSR-PCR amplification patterns. Twenty ISSR

microsporidians found elongated oval from measuring 3.90 to

primers were tested for PCR amplification with all 10 newly identified

4.90 μm in length and 2.66 to 2.90 μm in width. Similarly, six eri

microsporidian isolates along with a type species. Out of 20 primers,

microsporidians also found elongated oval from measuring 3.80

fourteen primers produced good amplification products. They were

to 4.05 μm in length and 2.60 to 3..05 μm in width (Table 1). The

used for ISSR markers analysis. A majority of 11 primers annealed

shape of NIK-1s_mys, a type species is oval measuring 3.80 μm in

to dinucleotide repeats, 2 annealed to tri- and 1 to penta nucleotide

length and 2.60 μm in width (Table 1).

repeats. Very high degree of polymorphism was detected with all 14 ISSR primers. Total 178 fragments were scored from the fourteen

Genetic variability revealed by the ISSR markers

ISSR primers. Out of 178 fragments, 175 (98%) were polymorphic (Table 2). The ISSR-PCR amplified fragments profile generated

Genomic DNA from 11 different microsporidian isolates were

with ISSR-827 primer shown in Fig. 2A clearly shows the extent of polymorphism among the different microsporidian isolates. The total number of ISSR markers varied in different isolates with different primers. Some of the bands were common to all isolates and the rest were present only in specific isolates. Almost all 11 microsporidian isolates had different ISSR profiles. The size of the amplified fragments ranged from 700 to 3000 bp (Table 2). The ISSR-PCR fingerprinting patterns of 11 microsporidian isolates with various ISSR primers were used to calculate genetic distance values with Dice similarity coefficient method (Dice, 1945). The relationship among the 11 microsporidian isolates, as revealed by genetic similarity calculated from ISSR data, varied from 0.385 to 0.941 (Table 3A), suggesting significant variability among the microsporidians. The lowest value

Fig. 2A. Inter simple sequence repeat (ISSR) banding profiles obtained on 2% agarose gel for the eleven microsporidian isolates with the primer ISSR-827. The lane marked M shows the molecular size marker.

of Dice similarity coefficient (0.385) was found between the MIA6eTr and MIA-7mJr isolates. The highest similarity coefficient (0.941) was found between MIA-1eBr and MIA-2eBr (Table 3A). The

Table 3. A Dice genetic similarity distance matrix values based on ISSR data among eleven microsporidian isolates MIA-7mJr

MIA8mDm

MIA9mMd

MIA10mKp

MIA-1eBr MIA-2eBr MIA-3eDj MIA-4eLr

MIA5eDu

MIA-6eTr

MIA-7mJr

1.000

MIA-8mDm

0.636

1.000

MIA-9mMd

0.737

0.762

1.000

MIA-10mKp

0.609

0.800

0.818

1.000

MIA-1eBr

0.462

0.500

0.480

0.552

1.000

MIA-2eBr

0.417

0.462

0.522

0.593

0.941

1.000

MIA-3eDj

0.429

0.667

0.519

0.710

0.529

0.500

1.000

MIA-4eLr

0.545

0.571

0.563

0.611

0.718

0.649

0.585

1.000

MIA-5eDu

0.400

0.593

0.583

0.643

0.645

0.690

0.788

0.632

1.000

MIA-6eTr

0.385

0.643

0.560

0.759

0.500

0.533

0.867

0.564

0.839

1.000

NIK-1s_mys

0.480

0.519

0.500

0.571

0.774

0.759

0.727

0.632

0.800

0.710

NIK-1s_ mys

1.000

Note: Values are calculated from 14 ISSR primers 11

Wazid Hassan and B. Surendra Nath Genetic characterization of microsporidians infecting Indian non-mulberry silkworms

genetic similarity values were used for constructing the dendrogram

Genetic variability revealed by the RAPD markers

using the un-weighted pair group method with arithmetic averages (UPGMA) method (Fig. 3A). The obtained dendrogram grouped 11

Twenty RAPD primers were screened for fingerprinting of the

microsporidian isolates in to two major (A and B) clusters. Cluster

10 newly identified microsporidian isolates along with type species

A included four isolates: MIA-7mJr, MIA-8mDm, MIA-9mMd, and

out of which sixteen primers that yielded good amplification were

MIA-10mKp, isolated from muga silkworm and collected from four

utilized. The amplified products obtained with primer OPW-6 are

district i.e., Jorhat, Dhemaji, Darang and Kamrup respectively. Cluster

depicted at Fig. 2B. The size of amplified products with different

B, consisted seven isolates viz., MIA-1eBr, MIA-2eBr, MIA-3eDj,

primers ranged from 350 to 2900 bp (Table 2). Total 196 RAPD

MIA-4eLr, MIA-5eDu, and MIA-6eTr isolated from eri silkworm and

fragments were generated with 16 primers of which 95% were

collected from Korbi Anglong, Lakhimpur, Jorhat, and Dhemaji and

polymorphic (Table 2). Values of genetic distance obtained from

type species NIK-1s_mys.

each pair wise comparison of RAPD fragments are shown at

Fig. 3A. Dendrogram constructed from ISSR data showing genetic relationships among the eleven microsporidian isolates using UPGMA method. Numbers on each node indicate bootstrap values.

Fig. 2B. Random amplified polymorphic DNA (RAPD) banding profiles obtained on 1.5% agarose gel for the eleven microsporidian isolates with the primer OPW-6. The lane marked M shows the molecular size marker.

Table 3. B Dice genetic similarity distance matrix values based on RAPD data among eleven microsporidian isolates MIA7mJr

MIA8mDm

MIA9mMd

MIA1mKp

MIA-1eBr MIA-2eBr MIA-3eDj MIA-4eLr

MIA5eDu

MIA-6eTr NIK-1s_Mys

MIA-7mJr

1.000

MIA-8mDm

0.229

1.000

MIA-9mMd

0.158

0.452

1.000

MIA-1mKp

0.356

0.316

0.537

1.000

MIA-1eBr

0.270

0.267

0.303

0.350

1.000

MIA-2eBr

0.270

0.200

0.242

0.300

0.938

1.000

MIA-3eDj

0.217

0.256

0.333

0.245

0.390

0.439

1.000

MIA-4eLr

0.143

0.095

0.083

0.194

0.261

0.261

0.188

1.000

MIA-5eDu

0.242

0.154

0.207

0.278

0.429

0.429

0.324

0.632

1.000

MIA-6eTr

0.182

0.216

0.300

0.213

0.308

0.308

0.500

0.400

0.457

1.000

NIK-1s_Mys

0.195

0.235

0.270

0.227

0.278

0.222

0.311

0.222

0.188

0.419

Note: Values are calculated from 16 RAPD primers. 12

1.000

Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 6-16 (2015)

Fig. 3B. Dendrogram constructed from RAPD data showing genetic relationships among the eleven microsporidian isolates using UPGMA method. Numbers on each node indicate bootstrap values.

Fig. 4A. Spatial distribution of eleven different microsporidians based on the ALSCAL multidimensional scaling method using Euclidean distance matrix with ISSR data. Details of microsporidians isolates are e1=MIA-1eBr, e2=MIA-1eBr, e3= MIA-3eDj, e4= MIA-4eLr, e5=MIA-5eDu, e6=MIA-1eTr, m1=MIA-7mJr, m2=MIA-8mDm, m3=MIA-9mMd, m4=MIA10mKp and s1=NIK-1s_mys.

Table 3B. The relationship between 10 isolates and type species as revealed by genetic distance from dice similarity matrix RAPD data varied from 0.083 to 0.938 (Table 3B). The genetic distance similarity matrix was least (0.083) between MIA-6eTr and MIA7mJr while it was highest (0.938) between MIA-1eBr and MIA2eBr (Table 3B). UPGMA based dendrogram utilizing the genetic distance values of RAPD data is presented in Fig. 3B. The dendrogram indicated clustering of the different microsporidians into two groups (A and B). Group A contained four isolates i.e., MIA7mJr, MIA-8mDm, MIA-9mMd, and MIA-10mKp. All isolates infect muga silkworm and collected from four different districts i.e.,

isolated from Mysore, Karnataka, India (Fig. 3B).

Fig. 4B. Spatial distribution of eleven different microsporidians based on the ALSCAL multidimensional scaling method using Euclidean distance matrix with RAPD data. Details of microsporidians isolates are e1=MIA-1eBr, e2=MIA-1eBr, e3= MIA-3eDj, e4= MIA-4eLr, e5=MIA-5eDu, e6=MIA-1eTr, m1=MIA-7mJr, m2=MIA-8mDm, m3=MIA-9mMd, m4=MIA10mKp and s1=NIK-1s_mys.

Two dimensional distribution of microsporidians as revealed by ALSCAL method

and the muga isolates which differed from type species, were considered

Jorhat, Dhemaji, Darang and Kamrup. The B group contained seven isolates viz., MIA-1eBr, MIA-2eBr, MIA-3eDj, MIA-4eLr, MIA5eDu, and MIA-6eTr isolated from eri silkworm and collected from four different districts in Assam, India and type species NIK-1s_mys

to be different variants. The grouping of different microsporidians based The two-dimensional scaling of the ISSR and RAPD data, using ALSCAL algorithm based on Euclidean distance matrix, has clearly

on Euclidean distance matrix is very similar as like in the UPGMA based dendrogram (Fig. 4A and 4B).

delineated each of the newly identified microsporidian from the muga and eri silkworms as well as from the type species, NIK-1s_mys (Fig. 4A and 4B). Of the 10 microsporidian isolates, two eri microsporidians

Discussion

MIA-5eDu and MIA-6eTr are found to be little closer to NIK-1s_mys, indicating that eri microsoridians are slightly similar to the type species

The identified microsporidian isolates were characterized using

13

Wazid Hassan and B. Surendra Nath Genetic characterization of microsporidians infecting Indian non-mulberry silkworms

spore morphology and PCR based ISSR and RAPD fingerprinting.

scaling has not only supported the information generated by the

The spore of type species, NIK-1s_mys is oval in shape and size

UPGMA dendrogram, but it has made the Euclidean distances

measuring 3.80 (length), 2.60 (width) (Table 1). NIK-1s_mys is

among different microsporidians more clear. In both illustrations

similar to the type species N. bombycis maintained at Sericultural

the grouping were almost similar and it is important to note that

Experimentation Station, Tokyo, Japan with GenBank accession

in the UPGMA the genetic distance values was used to construct

number D85503. Canning et al. (1999) suggested the determination

dendrogram using the method of Nei and Li (1979), whereas in

of the status of new microsporidian isolates should be made against

ALSCAL- multidimensional scaling the Euclidean method (Young

D85503. Hence, NIK-1s_mys is included as reference species in the

et al. 1984, Young and Harris 1990) was used to obtain similarity

this study for comparing new microsporidian isolates identified form

matrix. The results from both methods gave almost similar patterns

A. assamensis and S. c. ricini. Earlier, researchers had used the small

of grouping; in both methods microsporidians isolated from A.

differences in microsporidian spore size, as an indication of genus.

assamensis and S. c. ricini were discriminated from each other

However, small differences in spore size could not be considered as

and from type species as well (Figs. 3A, 3B, 4A, and 4B). The

a taxonomic character. The spore size for a given species may vary

grouping pattern of newly identified microsporidians supported their

with the host size (Brooks and Cranford 1972) and is affected by

sympatric speciation origin in the bio-geographical sericulture area

temperature (Maddox and Sprenkel 1978, Medeiros et al. 2004) age

of Assam, India.

of the host and the medium in which they are measured (Malone

The ISSR fingerprinting results of Rao et al. (2005) showed that,

and Wigley 1981, Mercer and Wigley 1987). In this study, we found

the dinucleotide repeats (AG)n, (GT)n; trinuclotide repeats (ATG)

muga microsporidians were slightly bigger than eri microsporidians

n

(Table 1) which support Brooks and Cranford (1972) finding.

microsporidian species isolated from the mulberry silkworm,

, (CTC)n, (GTT)n were most abundant in the genome of different

The genomic DNA from ten microsporidian isolates was used

Bombyx mori. Based on the di-, tri- nucleotide amplification

to generate ISSR and RAPD-PCR amplification patterns. The

results, it is clear that the different microsporidian isolates identified

constructed both dendrograms clearly revealed that clustering of

from the A. assamensis and S. c. ricini, are different from the

11 microsporidian isolates based on their host silkworm viz., four

microsporidians identified from the mulberry silkworms (B.mori).

muga microsporidians [MIA-7mJr, MIA-8mDm, MIA-9mMd,

ISSR and RAPD profiles in the present study clearly delineated

and MIA-10mKp] separated in a separate group while six eri

the 10 microsporidian isolates with good bootstrap confidence

microsporidians [MIA-1eBr, MIA-2eBr, MIA-3eDj, MIA-4eLr,

values from type species, NIK-1s_mys and confirming the

MIA-5eDu, and MIA-6eTr] were separated in other grouped along

capability of ISSR and RAPD markers to discriminate the different

with a type species. This indicates all the eri microsporidians had

microsporidian isolates (Fig. 3a, b). Rivera et al. (1995), Mathis et

close phylogenetic relationship with type species NIK-1s_mys. The

al. (1997), Bretagne et al. (1997), Gur-Arie et al. (2000), Shivaji et

dendrograms showed clear separation of all 10 novel microsporidian

al. (2000), Sreenu et al. (2003), Tsai et al. (2003), Rao et al. (2005,

isolates from each other with a good bootstrap value. In order to

2007), Kumar et al. (2007), Nath et al., (2011), Hassan et al.(2014)

distinguished the micirosporidian isolates from each other, further

used ISSR and RAPD markers for genetic characterization and

analysis of dendrograms suggest that two eri microsporidian isolates

identification of various bacteria, microsporidia and fungi. Thus, the

[MIA-5eDu and MIA-6eTr] look slightly similar with type species,

present study forms the first report on molecular characterization

NIK-1s_mys.

of microsporidian isolates from muga and eri silkworm based

Even the multidimensional scaling of the ISSR and RAPD data,

on ISSR and RAPD-PCR markers assay. The ISSR and RAPD-

using the ALSCAL algorithm on Euclidean distance matrix has

PCR are one of the simplest and quickest marker systems with

clearly separated the microsporidians from each other and type

high reproducibility, including the virtue of its unique efficiency in

species, NIK-1s_mys (Fig. 4A and 4B). The multidimensional

distinguishing even closely related organisms and is important for

scaling method is one of the multivariate approaches of grouping

proper identification of different microsporidian isolates. The study

based on the Euclidean distance matrix (Young et al. 1984, Young

confirms that molecular tools including ISSR and RAPD markers

and Harris 1990). It anticipates being more informative about

analysis are alternative and facilitate more useful genetic diversity

differentiating distinct and closely related isolates. The use of

studies of different microsporidians infecting various organisms,

pictorial representation of data using ALSCAL- multidimensional

since this technique requires only a small amount of genomic DNA

14

Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 6-16 (2015)

and can produce high levels of polymorphism. The study inferred that the ten newly identified microsporidians

amplified polymorphic DNA (RAPD) in molecular ecology. Mol Ecol 1, 55-63.

from muga and eri silkworms differed in their spore morphology

Hartskeerl RA, Van Gool T, Schuitema AR, Didier ES, Terpstra

and the ISSR and RAPD PCR profiles indicating genetic variability

WJ (1995) Genetic and immunological characterization of the

among them. The high level of polymorphism realized from this

microsporidian Septata intestinalis Cali, Kotter and Orenstein, 1993:

study further proves the efficacy of ISSR and RAPD markers assay.

reclassification to Encephalitozoon intestinalis. Parasitology 110, 277-285. Hassan W, Nath BS, (2014) Genetic diversity and phylogenetic

Acknowledgements

relationships among microsporidian isolates from the Indian tasar silkworm, Antheraea mylitta,as revealed by RAPD fingerprinting

The authors are grateful to Department of Biotechnology (DBT), Government of India for the financial support. Wazid Hassan is a

technique. Int J Indust Entomol 29(2), 169-178. Doi.org/10.7852/ ijie.2014.29.2.169

recipient of Research Fellowship by DBT and PhD Scholarship

Hirt RP, Healy B, Vossbrinck CR, Canning EU, Embley TM (1997)

by University Grants Commission (Maulana Azad National

Amitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular

Fellowship).

evidence that microsporidia once contained mitochondria. Curr Biol 7, 995-998. Hung HW, Lo CF, Tseng CC, Peng SE, Chou CM, Kou GH (1998)

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