IJIE
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)
References
The small subunit ribosomal RNA gene sequence of Pleistophora anguillarum and the use of PCR primers for diagnostic detection of
Baker MD, Vossbrinck CR, Didier ES, Maddox JV, Shadduck JA (1995) Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. J Eukaryot Microbiol 42, 564 -570. Bretagne S, Costa JM, Besmond C, Carsique R, Calderone R (1997) Microsatellite polymorphism in the promoter sequence of the elongation factor 3 gene of Candida albicans as the basis for a typing system. J Clin Microbiol 35, 1777-1780. Brooks WM, Cranford JD (1972) Microsporidoses of the hymenopterous parasites, Campoletis sonorensis and Cardiochiles nigriceps, larval parasites of Heliothis species. J Invertebr Pathol 20, 77-94. Canning EU, Curry A, Cheney SA, Lafranchi-Tristem NJ, Kawakami Y, Hatakeyama Y, Iwano H, Ishihara R (1999) Nosema tyriae n. sp. and Nosema sp. microsporidian parasites of cinnabar moth Tyria jacobaeae. J Invertebr Pathol 74, 29-38. Dice L R (1945) Measures of the amount of ecological association between species. Ecology 26, 297-302. Fang DQ, Roose ML, Krueger RR, Federici CT (1997) Fingerprinting
the parasite. J Eukaryot Microbiol 45, 556-560. Keeling P (2009) Five Questions about Microsporidia. PLoS Pathoglogy 5(9), e1000489. Doi,10.1371/ journal.ppat.1000489. Keeling PJ (2003) Congruent evidence from α-tubulin and β-tubulin gene phylogenies for a zygomycete origin of microsporidia. Funga Genet Biol 38, 298-309. Kumar AR, Sathish V, Nair GB, Nagaraju J (2007) Genetic characterization of Vibrio cholerae strains by inter simple sequence repeat-PCR. FEMS. Microbiol Lett 1-8. DOI:10.1111/j.15746968.2007.00762.x Lu R, Rank GH (1996) Use of RAPD analyses to estimate population genetic parameters in the alfalfa leafcutting bee, Megachile rotundata. Genome 39, 655-663. Maddox JV, Sprenkel RK (1978) Some enigmatic microsporidia of the genus Nosema. Misc. Pub Entomol Soc Am 11, 65-84. Malone LA, Wigley PJ (1981) The morphology and development of Nosema carpocapsae, a microsporidian pathogen of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae) in New Zealand. J Invertebr Pathol 38, 315-329.
trifoliate orange germ plasm accessions with isozymes, RFLPs and
Mathis A, Michel M, Kuster H, Muller C, Weber R, Deplazes P (1997)
inter-simple sequence repeat markers. Theor Appl Genet 95, 211-219.
Two Encephalitozoon cuniculi strains of human origin are infectious
Gur-Arie R, Cohen CJ, Eitan Y, Shelef L, Hallerman EM, Kashi Y (2000) Simple sequence repeats in Escherichia coli: abundance, distribution, composition and polymorphism. Genome Res 10, 62-71. Hadrys H, Balick M, Schierwater B (1992) Applications of random
to rabbits. Parasitology 114, 29-35. Medeiros J, Tavares J, Simoes N, Solter LF (2004) A new isolate of the microsporidium Vairimorpha necatrix (Microsporidia: Burenellidae) recorded in the azores. J Invertebr Pathol 85, 58-60.
15
Wazid Hassan and B. Surendra Nath Genetic characterization of microsporidians infecting Indian non-mulberry silkworms
Mercer CF, Wigley PJ (1987) A microsporidian pathogen of the
Tsai SJ, Lo CF, Soichi Y, Wang CH (2003) The characterization of
poroporo stem borer, Sceliodes cordalis (Dbld) (Lepidoptera:
microsporidian isolates (Nosematidae: Nosema) from five important
Pyralidae). J Invertbr Pathol 49, 93-101. Nagaoka T, Oghihara Y (1997) Applicability of inter-simple sequence repeat polymorphism in wheat for use as DNA markers in comparison to RFLP and RAPD markers. Theor Appl Genet 94, 597-602. Nath BS, Hassan W, Rao SN, Prakash NBV, Gupta SK, Mohanan NM,
lepidopteran pests in Taiwan. J Invertbr Pathol 83, 51-59. Undeen AH, Alger NE (1971) A density gradient method for fractionating microsporidian spores. J Invertbr Pathol 18, 419-420. Undeen AH, Cockburn AF (1989) The extraction of DNA from microsporidia spores. J Invertbr Pathol 54, 132-133.
Bajpai AK (2011) Genetic diversity among microsporidian isolates
Undeen AH, Vavra J (1997) Research methods for entomopathogenic
from the silkworm, Bombyx mori, as revealed by randomly amplified
protozoa. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect
polymorphic DNA (RAPD) markers. Acta Parasitologica 56(4), 333-338.
Pathology, Academic Press, San Diego, USA, pp. 117-151.
Nei M, Li WH (1979) Mathematical model for studying genetic
Vijayan K (2004) Genetic relationships of Japanese and Indian mulberry
variation in terms of restriction endonucleases. Proc Natl Acad Sci
(Morus spp.) genotypes revealed by DNA fingerprinting. Plant Syst
USA 76, 5269-5273.
Evol 243, 221-232.
Raina SN, Rani V, Kojima T, Oghihara Y, Singh KP, Devarumath RM
Vossbrinck CR, Maddox JV, Friedman S, Debrunner-Vossbrinck BA,
(2001) RAPD and ISSR fingerprints as useful genetic markers for
Woese CR (1987) Ribosomal RNA sequence suggests microsporidia
analysis of genetic diversity, varietal identification and phylogenetic
are extremely ancient eukaryotes. Nature 326, 411-414.
relationships in parent (Arachis hypogea) cultivars and wild species. Genome 44, 763-772. Rao SN, Nath BS, Saratchandra B (2005) Characterization and phylogenetic relationships among microsporidia infectingsilkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis. Genome 48, 355-366. Rao SN, Nath BS, Bhuvaneswari G, Raje US (2007) Genetic diversity and phylogenetic relationships among microsporidia infecting the
Vossbrinck CR, Woese CR (1986) Eukaryotic ribosomes that lack a 5.8s RNA. Nature 320, 287-288. Wasson K, Peper RL (2000) Mammalian microsporidiosis. Vet Pathol 37, 113-128. Weiss LM (2001) Microsporidia 2001: Cincinnati. J Eukaryot Microbiol(Suppl.), 47S-49S. Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213-7218.
silkworm, Bombyx mori, using random amplification of polymorphic
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990)
DNA: Morphological and ultrastructural characterization. J Invertbr
DNA polymorphisms amplified by arbitrary primers are useful as
Pathol 96, 193-204. Reddy KD, Nagaraju J, Abraham EG (1999) Genetic characterization of the silkworm, Bombyx mori by simple sequence repeat (SSR)anchored PCR. Heredity 83, 681-687.
genetic markers. Nucleic Acids Research 18, 6531-6535. Wittner M, Weiss LM (1999) The microsporidia and microsporidiosis. ASM, Washington, (D.C), Pp.1-553. Yap IV, Nelson RJ (1996) WINBOOT: A program for performing
Rivera IG, Chowdhury MA, Huq A, Jacobs D, Martins MT, Colwell
bootstrap analysis of binary data to determine the confidence limits
RR (1995) Enterobacterial repetitive intergenic consensus sequences
of UPGMA-based dendrograms. IRRI discussion paper series No.14,
and the PCR to generate fingerprints of genomic DNAs from Vibrio
International Rice research Institute, Manila, Philippines.
Cholerae O1, O139, and non-O1 strains. Appl Environ Microbiol 61, 2898–2904.
Young FW, Easterling DV, Forsyth BN (1984) The general Euclidean model for scaling three mode dissimilarities: Theory and allocation.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
In: Law, H.G., Snyder, G.W. Jr., Hattie, J., McDonald, R.P. (Eds.).
laboratory manual. second ed. Cold Spring Harbor Laboratory, Cold
Research methods for multi-node data analysis in the behavioural
Spring Harbor, New York.
sciences. Praeger, New York, USA.
Shivaji S, Bhanu NV, Aggarwal RK (2000) Identification of Yersinia
Young FW, Harris DF (1990) Multidimensional scaling: procedure
pestis as the causative organism of plague in India as determined by
ALSCAL. In: Norusis M. (Ed.) SPSS base system: user’s guide,
16S rDNA sequencing and RAPD-based genomic fingerprinting.
Chicago, USA, pp. 397-461.
FEMS Microbiol Lett 189, 247-252. Sreenu VB, Alevoor V, Nagaraju J, Nagarajaram HA (2003) MICdb: database of prokaryotic microsatellites. Nucleic Acids Res 31, 106-108.
16
Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple-sequence repeat (SSR)- anchored polymerase chain reaction amplification. Genomics 20, 176-183.