Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(4): 676-679 (2017)

PCR-based detection of microsporidia in silkworms

using non-conventional RNA polymerase primers

Gourab Roy, Kalidas Mandal and G. Ravikumar*

Seri-Biotech Research Laboratory, Central Silk Board, Carmelaram Post, Kodathi, Bangalore, 560 035, India.

ABSTRACT

Microsporidia are obligate intracellular, spore-forming parasites that infect both invertebrates and vertebrates. They

infect silkworms causing the deadly pebrine disease leading to heavy crop loss in sericulture. Because of the horizon-

tal and vertical transmittance, outbreaks should be detected at an early stage and persistent infections should also

be identi ed to prevent further transmittance. So far, microscopic examination method remains the conventional

detection method for screening of microsporidia in sericulture. Molecular diagnosis tools have an advantage over

microscopic detection as they are more speci c, sensitive and aid in early detection. Microsporidia detection by PCR

method using primers designed from SSU-rRNA is widely used. In this study, we developed a PCR assay for the detec-

tion of microsporidia using primers designed from the conserved regions of RNA polymerase gene. Under optimized

PCR conditions, the assay yielded a ~650 bp DNA fragment from microsporidia infected silkworms, Bombyx mori

and Antheraea mylitta. Sequence analysis of the ampli ed products has shown homology to various microsporidia

including Nosema bombycis and N. antheraea. No non-speci c products were observed. This method could help in

early detection of microsporidia infection at any developmental stage of the silkworm and thereby reducing the crop

loss.

KEY WORDS: DETECTION, MICROSPORIDIA, PCR

676

ARTICLE INFORMATION:

*Corresponding Author: ravikumarpillai@gmail.com

Received 21

Oct, 2017

Accepted after revision 18

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/10

INTRODUCTION

Silkworm, Bombyx mori is one of the most important

domesticated insects, which produces luxuriant silk

thread in the form of cocoon by consuming mulberry

leaves during larval period. In India the bulk of the

commercial silk produced is mulberry silk whereas, Eri,

Tasar & Muga silk contribute to a lesser extent. These

silkworms are susceptible to various diseases resulting

in substantial crop loss which is estimated to be 40%

in India (Singh et al., 2012). The common pathogens

infecting them are microsporidians including Nosema

Gourab Roy, Kalidas Mandal and G. Ravikumar

bombycis, nucleopolyhedrovirus (NPV) and densovirus

(mainly DNV1&2), infectious  acherie virus (IFV), cyto-

plasmic polyhedrovirus (CPV) and bacteria. The micro-

sporidians cause pebrine disease; NPV causes grasserie;

and DNVs, IFV and bacterial pathogens cause  ach-

erie diseases. Among all, the microsporidians disease

is responsible for the signi cant economic loss in the

sericulture industry. Microsporidiasis remained a threat

to silk industry since time immemorial, because of its

unique and recurrent occurrence and is the only dis-

ease transmitted both horizontally and vertically (Bhat

et al., 2009). Several species and strains of microsporidia

have been isolated from infected silkworms among

which pebrine caused by Nosema bombycis is the most

prevalent. Other microsporidian species (Vairimorpha,

Pleistophora, Thelohania etc.) which differ in their spore

morphology, sites of infection and virulence, have also

been isolated from silkworms (Kawarabata, 2003, Gupta

et al., 2017).

Since the control of disease is often met with lim-

ited success, early detection of pathogens is essential to

control of emerging, reemerging, and in preventing the

spread of infectious diseases. Microsporidian are easily

detected by light microscopy when infections are heavy

and spores are present. However, early infections without

spores, or light infections with low numbers of spores are

easily missed. This limitation has made it dif cult to con-

duct investigations into microsporidian prevalence and

transmission. To overcome these dif culties, PCR- based

techniques have been developed to detect the major path-

ogens of silkworms with great speci city and sensitivity

(Hatakeyama and Hayasaka 2003, Hamiduzzaman et al.,

2010, Ravikumar et al., 2011, Fu et al., 2016).

Due to the availability of sequence information and

the presence of conserved and variable sequence regions

within the SSU rRNA genes, PCR-based methods have

typically used primers of this gene for the detection of

microsporidians (Franzen and Muller, 1999). Herein, we

report that primers designed from the RNA polymerase

of microsporidians can also be used to detect micro-

sporidians from silkworms. To our knowledge, this is the

 rst report on the detection of microspordians using its

RNA polymerase primers from silkworms.

MATERIALS AND METHODS

SILKWORM AND MICROSPORIDIAN INFECTION

The silkworm rearing and microsporidian infection were

essentially performed as reported by us (Ravikumar

et al, 2011). Silkworms, B. mori (Pure Mysore) were fed

on mulberry leaves. For Microsporidian infection, 3rd

instar day 1 larvae were orally fed with 2000 spores/

larva and periodical observations were taken. Control

larvae did not receive microsporidian infection. On 4

and 8

day post infection (p.i.), larval mid gut tissues

were dissected out and used for DNA extraction, fol-

lowed by PCR. DNA was also extracted from pupa, adult

and eggs of infected and normal silkworms.

DNA EXTRACTION

DNA extracted from the mid gut of infected and control

using Hi-Pure DNA extraction Kit (Himedia) according to

manufacturer’s protocol. DNA from mulberry leaves and

pebrine infected A. mylitta DNA were used as reported

earlier (Ravikumar et al., 2011). The DNA was analyzed

in 1% agarose gel electrophoresis and quanti ed using a

Nanodrop (Thermo Corporation) spectrophotometer.

PCR AND CLONING

A set of primers were designed from the conserved region of

available microsporidian RNA polymerase sequences from

NCBI database. The primers used were: Sense: 5’-CCICAY-

TTYCCIAARGARGAYTA-3’ and antisense: 5’-AARGAY-

ITIGARGGIACIAAYGA-3’. (I: deoxyinosine; R: A, G; Y: T,

C). PCR reactions were carried out using 1X Taq buffer,

2.5 mM dNTPs, 25 mM MgCl2, 0.5U Taq DNA polymerase

(Fermentas) and 100 ng of DNA. The DNA from control

silkworms and mulberry DNA were employed as negative

controls. PCR reactions were carried out (Eppendorf) using

the following cycles: 94°C for 2 min, 30 cycles of 94°C for

40 s, 48°C for 30 s, and 72°C for 30s and 1 cycle of 72° C

for 5 min. PCR products were analyzed in 1% agarose gel

electrophoresis, stained in Sybergreen (HiMedia) and visu-

alized under UV transillumination. The PCR products were

cloned in pJET blunt end cloning vector (Fermentas) and

positive clones were con rmed by colony PCR. Puri ed

plasmids were sequenced at Euro ns, Bangalore, followed

by BLAST analysis (NCBI).

RESULTS AND DISCUSSION

Results are presented in Figure 1. PCR ampli cations

have resulted in discrete and desired product. DNA

extracted from the microsporidian infected silkworm

yielded speci c ampli cation products of ~ 650 bp (Lane

1-4) using RNA polymerase primers. No non- speci c

products were observed. The negative controls; DNA

from normal silkworm and the plant DNA from mul-

berry showed no PCR products, indicating the speci c-

ity of the PCR. The banding intensity on day 8th was

higher to that of on 4th day showing the proliferation

of pathogen at an advanced stage of infection. Fur-

ther con rmation of the PCR products was done by

sequencing and BLAST analysis. BLAST showed 92- 99

% homology to RNA polymerases of various isolates of

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PCR-BASED DETECTION OF MICROSPORIDIA IN SILKWORMS 677

Gourab Roy, Kalidas Mandal and G. Ravikumar

FIGURE 1. PCR ampli cation of microsporidia DNA (~650bp). M: Molecular weight marker, Lane 1&2: DNA

from microsporidia infected B. mori larvae, day 4 and 8 p.i., respectively; Lane 3&4: DNA from micro-

sporidia infected A. mylitta larvae day 4 and 8 p.i. Lane 5&6: DNA from uninfected B. mori and mulberry

leaves, respectively, were used as controls.

N. bombycis, Nosema Sp, N. antheraea, N. ceranae, N.

pernyi and other microsporidia. The same results were

obtained from other developmental stages, pupa, adult

and eggs (data not shown) of the silkworm. In addi-

tion, the same primer sets could detect microsporidian

of tasar silkworm A. mylitta showing the ef cacy of the

RNA polymerase-based primers in detecting microspo-

ridian of other silkworm species than B. mori. The con-

served regions of RNA polymerase gene was effectively

utilized for the detection of microsporidia in the present

work and it can be used for detection of microsporidia

of other insects/organisms also. Highly conserved SSU-r

RNA gene primers were successfully used for the detec-

tion and classi cation of microsporidia across organ-

isms with high speci city and sensitivity (Weiss and

Vossbrinck, 1999; Jehle et al., 2006, Ravikumar et al.,

2011). PCR diagnosis of N. pernyi using SSU-rRNA prim-

ers provided increased speci city and sensitivity when

compared with light microscopy in Antheraea pernyi

(Jiang et al., 2011). For the effective control of pebrine

disease, outbreaks should be detected at an early stage

and persistent infections should also be identi ed to

prevent further transmittance of the disease.

In our study, microsporidia were detected by PCR at

4th day of p.i, whereas the spores were visible under

microscope only on day 8 and afterwards. Hence, this

method can be useful in the early detection of micro-

sporidia which is critical in reducing crop loss in seri-

culture. Further, real-time quantitative PCR assay can

be used with RNA polymerase primers for increased

sensitivity. The results of this study suggest that RNA

polymerase primers from microsporidia can be used

for pebrine detection in sericulture. To the best of our

knowledge, this is the  rst study in which PCR was used

for the successful detection of microsporidia using RNA

polymerase primers in silkworms.

ACKNOWLEDGEMENTS

Authors are thankful to Messrs. S. N. Gundurao and N.

Pillapa for rearing silkworms. Financial support was

provided by Central Silk Board (CSB), Bangalore, India.

Mr. Gourab Roy is thankful to CSB for a Junior Research

Fellowship.

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Gourab Roy, Kalidas Mandal and G. Ravikumar

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PCR-BASED DETECTION OF MICROSPORIDIA IN SILKWORMS 679

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