Research article · 2025 · Full text
International Journal of Biology SciencesIdentification and infectivity of different microsporidian isolates collected from sericulture (Bombyx mori L) areas
Volume 7(1) · Pages 36–40 · DOI: 10.33545/26649926.2025.v7.i1a.269 ↗
Research areasSilkworms and Sericulture ResearchViral Infectious Diseases and Gene Expression in InsectsAgricultural Systems and Practices
Abstract
Author-writtenPebrine is the most destructive disease of silkworm, Bombyx mori L., as it is transmitted transovarially to the next generation and causes crop loss. Pebrine disease is caused by microsporidian strains whose pathogenicity, rate of spread, and mode of transmission differ. This has recently become a serious concern for seed producers in India. The threat has been increasing, and disease incidence has increased in various locations in Karnataka and Tamil Nadu. A disease survey was conducted at various locations in these two states to record disease incidence rates. Ten microsporidian isolates were collected and inoculated for bioassay studies into healthy larvae to determine their infectivity, multiplication, and transovarial transmission. Rearing was conducted for two successive generations, and the mortality rate in the second generation varied for all the microsporidian isolates, with pupal survival ranging from 21-65%. All ten isolates of microsporidia were found to exhibit transovarial transmission. The fecundity significantly decreased and the hatching percentage decreased from 5% to 22% in the second generation of infected batches. The presence of microsporidia was confirmed through PCR amplification of the SSU rRNA gene of Microsporidia. Five microsporidian isolates were selected for gene sequence analysis on the basis of their collection at different locations and identified as being homologous to Nosema plutellae, Nosema fumiferenae and Nosema species. This study helps in understanding the infectivity levels of different microsporidia prevalent in the sericulture field.
Summary
Source-checkedPlain-language reading of the full paper, prepared with AI and checked against the text. Each point links to the authors' own words.
Why it matters
- Pebrine is considered the most destructive disease affecting the silkworm Bombyx mori L. because it causes crop loss and is passed from parents to offspring. View evidence ↗
- The complexity of pebrine disease has increased due to the emergence of various microsporidian strains that differ in how they spread, multiply, and cause disease. View evidence ↗
- Because microsporidian spores vary significantly in size and shape, simple microscopic observation is insufficient for accurate identification. View evidence ↗
The question
- The study aims to identify microsporidia found in sericulture areas and evaluate their infectivity across successive generations. View evidence ↗
What they did
- Researchers conducted a disease survey in silkworm seed areas across Karnataka and Tamil Nadu to assess the prevalence of pebrine. View evidence ↗
- To test infectivity, ten microsporidian isolates were inoculated into third instar Sarupat silkworm larvae, using three replications of 200 larvae each. View evidence ↗
- Five of the ten isolates were sequenced and analyzed using BLAST to determine their homology to known species. View evidence ↗
What they found
- Ten microsporidian isolates were identified in the field, showing spore intensities between 3 and 25 per microscopic view. View evidence ↗
- Infected silkworms showed high mortality during the spinning stage, with survival rates between 57% and 83%. View evidence ↗
- In the first generation, pupal survival rates varied by strain, ranging from 41% to 78%. View evidence ↗
- The number of eggs produced (fecundity) decreased significantly in the second generation, ranging from 206 to 350 eggs across isolates. View evidence ↗
- Hatching rates for infected eggs in the second generation dropped to between 5% and 22%. View evidence ↗
- Pupation rates in the second generation ranged from 21% to 65%, with the NIT-de1 isolate identified as the most virulent. View evidence ↗
- All ten microsporidian isolates were confirmed to be transmitted transovarially, as evidenced by the presence of spores in eggs on the 7th day of development. View evidence ↗
- Molecular analysis showed that isolate NIT-de2 was over 90% similar to Nosema fumiferenae, while NIK-hd2 was related to Nosema plutellae. View evidence ↗
What it means
- The authors conclude that field-identified Nosema species can cross-infect silkworms, which poses a risk of economic loss to the sericulture industry. View evidence ↗
Visual summary
3 visualsIllustrations explain the paper; charts are replotted from the paper's own table values.
Cycle of Transovarial Transmission
AI-generated conceptual illustration (Bombyx mori silkworm moths, eggs, and larvae). It explains the idea and does not show study data.
View source evidence ↗
Pupal survival (%) (Mean± SD): NIK-ku1 vs NIK-ku2 vs NIK-ra1 vs NIK-ra2 vs NIK-hd1
Replotted from Table 1 (page 3) of the original paper. Every bar is the paper's own table value.
View source evidence ↗
Fecundity (2nd Gen.) (Mean± SD): NIK-ku1 vs NIK-ku2 vs NIK-ra1 vs NIK-ra2 vs NIK-hd1
Replotted from Table 1 (page 3) of the original paper. Every bar is the paper's own table value.
View source evidence ↗Figures & tables
From the original paper
Table 1
Figure 1
Figure 2
Figure 3Introduction
Pebrine disease caused by the intracellular parasite Nosema bombycis is a highly destructive disease that affects the silkworm Bombyx mori L. This disease is transmitted to the next generation and leads to significant losses in silk cocoon production. The disease has become more complex in recent years because of the identification of various strains and species infecting silkworms. These strains vary in their pathogenicity, spread rate, transmission method, and multiplication behavior. This poses a significant challenge for silkworm seed (egg) producers in India, as identifying and eliminating infected lots requires substantial effort due to the high multiplication rate of the disease, which can severely impact the silk industry. The rapid spread of this disease can cause extensive damage to silkworm populations and result in complete crop failure in severe cases. The complexity of the disease has increased due to the continuous emergence of different types of microsporidian infections in silkworms (Sharma et al., 2003) [13] . Some of these infections are caused by genera such as Vairimorpha, Pleistophora, and Thelohania, each with unique infection patterns (Samson, 2000) [12] . Nosema bombycis was initially identified as the primary cause of microsporidiosis in silkworms by the end of the 19th century, and multiple microsporidia from different genera are now responsible for the disease. These microsporidia vary in spore shape, size, and pathogenicity, with some showing low multiplication rates in silkworms (Bhat & Nataraju, 2006) [4] . While some microsporidia coexist harmlessly in the gut, others, such as NIK-4m can be highly pathogenic, causing acute disease through cyst formation on the gut surface (Ananthlashmi et al., 1994) [2] .
The occurrence of microsporidians has been reported in sericulture areas, and infected lots are often discarded without determining their impact on crop loss. Understanding the infectivity and transovarial transmission of microsporidia is crucial for clarifying the extent of crop damage. Microsporidian spores exhibit a diverse range of shapes and sizes when viewed under a microscope, necessitating more advanced identification techniques beyond morphology-based methods. Vossbrinck et al. (1987) [15] demonstrated the utility of PCR analysis of SSU-rRNA gene sequences for the molecular identification of microsporidians. Various methods for the amplification of SSU-rRNA gene fragments have subsequently been utilized for diagnosing and identifying insect microsporidia (Kawakami et al., 2001) [8] . Limited information is available on the characteristics of spores in silkworms, the pathology of pebrine disease, and fluctuations in its incidence. Therefore, this study aims to investigate the fundamental aspects of microsporidia in sericulture, including their identification and infectivity in subsequent generations.
Materials and methods
Materials and methods
Sample collection and multiplication
A disease survey was carried out in silkworm seed areas in various locations in Karnataka and Tamil Nadu states to determine the prevalence of pebrine disease. Microsporidia collected from the fields were cultured by inoculating them into healthy larvae. The spores of the microsporidia were extracted from infected larvae, pupae, and moths by homogenizing them in a 0.6% potassium carbonate solution and then centrifuging the suspension at 3,000 × g for 3 minutes. The resulting pellets were washed with water 3 to 5 times until a clear suspension was achieved, and the spores were quantified at a concentration of 1x10 6 spores/ml for bioassay studies.
Infectivity of microsporidian isolates
The infectivity of the microsporidian isolates was tested by inoculating them into third instar multivoltine silkworm (Sarupat) larvae on the first day. Three replications with 200 larvae each were used for the bioassay studies, and a control group of larvae fed mulberry leaves with distilled water was also maintained separately until spinning. Dead larvae were examined daily for the presence of microsporidial spores. Cocooning activities were carried out for 10 microsporidia, and five batches of eggs were prepared from the infected moths of each microsporidia and incubated at 25 °C until hatching for the second generation. Two hundred larvae were randomly selected after the second molt, and rearing was performed following standard procedures. Mother moths were tested for the presence of spores, and positive batches of eggs were incubated at 25 °C for embryonic development, with eggs tested for vertical transmission. Data on larval mortality, pupal mortality, fecundity, hatching percentage, and transovarial transmission rate were recorded.
PCR amplification and sequencing
Genomic DNA was extracted from the microsporidian isolates, and the Nosema-specific small subunit (SSU) rRNA gene was amplified from all ten microsporidian isolates via the forward primer 5'-CACCAGGTTGATTCTGCC-3' and reverse primer 5'-TTATGATCCTGCTAATGGTTC-3' designed by Baker et al. (1995) [3] . The PCR master mix was prepared with 1 µl (10 pg/µl) of each of the forward and reverse primers along with 5 µl of the emerald master mix (TAKARA Bio, USA). The volume was made up to 10 µl with water, including 2 µl of template. The reaction parameters were as follows: 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 35 s. The PCR samples were run on a 1.2% agarose gel with ethidium bromide at 100 V for 40 mins. The amplified products were sequenced via an ABI PRISM automated sequencer.
Phylogenetic analysis
A phylogenetic tree was constructed with nucleotide sequences through molecular evolutionary genetic analysis via MEGA 11 (Tamura et al., 2021) [9] . A neighbor-joining tree was constructed by the Kimura 2-parameter with 1000 bootstrap replications and a default random seed. Five microsporidia were selected from the ten isolates from the same locations and sequenced. The SSU-rRNA genes of the five microsporidian isolates were subjected to homology searches via BLAST (Alschul et al., 1990) [1] . Similar gene sequences were downloaded from NCBI, and multiple sequence alignment was performed with the Clustal W program.
Results
Results
The infectivity of microsporidians
Ten microsporidian isolates were found in the sericulture field, with spore intensities ranging from 3 to 25 per microscopic view (Fig. 1). The isolates were cultured and optimized for spore concentration for inoculation. Different strains showed varying infectivity to silkworms, with larval mortality starting 8 days post inoculation. Dead larvae were confirmed through microscopic examination, which revealed oval/elongated spores. Mortality rates are high during spinning, with survival rates ranging from 57-83%. Cocoons were harvested on the 6 th day after spinning, and pupal survival rates varied among strains ranging from 41-78% in the first generation. Dead pupae were confirmed by the presence of microsporidia spores through microscopic examination. Fecundity significantly decreased in all the isolates, ranging from 206 to 350 eggs in the second generation. The hatching percentage of the infected eggs decreased from 5-22% in the second generation. Transovarial transmission of the microsporidians was confirmed by examination of eggs on the 7 th day of embryonic development, which revealed transovarial transmission in all the isolates. Pupation rates varied among the ten isolates, ranging from 21-65%, with NIT-de1 being the most virulent. Mortality was observed in the chawki stage, with mortality rates dependent on spore dose in the embryonic stage (Table 1).
| Micro sporidia isolates | Larval Survival (%) (Mean± SD) | Pupal survival (%) (Mean± SD) | Fecundity (2nd Gen.) (Mean± SD) | Hatching % (2nd Gen.) (Mean± SD) | Pupation rate % (2nd Gen.) (Mean± SD) |
|---|---|---|---|---|---|
| NIK-ku1 | 64±3.606 | 41±4.20 | 232±21.07 | 87±1.528 | 25±3.055 |
| NIK-ku2 | 68±4.583 | 64±4.041 | 272±53.61 | 89±3.618 | 36±3.512 |
| NIK-ra1 | 70±3.215 | 62±2.000 | 267±18.68 | 85±2.351 | 34±4.000 |
| NIK-ra2 | 66±5.508 | 50±4.041 | 299±41.99 | 84±1.670 | 25±4.000 |
| NIK-hd1 | 73±5.132 | 65±5.000 | 342±11.31 | 80±1.929 | 37±4.583 |
| NIK-hd2 | 57±3.606 | 50±2.517 | 289±51.31 | 79±1.593 | 24±3.215 |
| NIK-kk1 | 71±6.807 | 61±4.163 | 292±25.16 | 82±2.351 | 46±2.000 |
| NIK-kk2 | 83±3.055 | 78±2.517 | 350±11.32 | 90±1.929 | 65±3.512 |
| NIT-de1 | 74±4.041 | 55±6.110 | 320±17.32 | 87±2.517 | 32±5.033 |
| NIT-de2 | 62±2.517 | 53±5.508 | 206±9.02 | 73±4.509 | 21±2.646 |
| Control | 94±1.000 | 91±0.577 | 422±12.01 | 95±1.103 | 92±1.00 |
| CD (5%) | 6.489 | 5.949 | 32.4 | 3.18 | 3.95 |
Table text extracted from the original PDF; superscript letters are shown inline. The original table image is below.
View original table image
Identification of microsporidia
The SSU rRNA gene of all ten microsporidia was amplified, producing products ranging in size from 1150 to 1175 bp, with an expected product size of 1230 bp in the Nosema genus (Fig. 2). The amplified products were purified and validated through sequencing, and the gene sequences were compared among the isolates and submitted to GenBank.
Molecular phylogeny
The SSU rRNA gene sequences of the five microsporidia isolates with the highest similarity according to BLAST
Discussion
All ten isolates were found to be infective to silkworms, and various strains were prevalent in the field. Ananthalakshmi et al. (1994) [2] isolated three different strains of microsporidians from Karnataka, whereas Samson et al.
(1999) [11] isolated two microsporidians from lepidopterans that were infective to silkworms. Advanced moth techniques have been used for an appropriate diagnosis of pebrine disease, along with improved inspection methods for different developmental stages of silkworms. Many advancements have been made in conventional diagnostic techniques, and the development of molecular techniques has provided new tools for taxonomic and phylogenetic studies. Nageswararao et al. (2004) [10] studied the pathogenicity, mode of transmission, tissue specificity of infection, and small subunit rRNA (SSU-rRNA) gene sequences of three new microsporidian isolates from the silkworm B. mori. They identified NIK-2r as the type species N. bombycis, whereas NIK-3h and NIK-4m were identified as Vairimorpha sp. Surendranath et al. (2012) [14] identified five new microsporidian isolates from silkworms and confirmed them as Nosema species through SSU-rRNA sequence-based phylogenetic analysis. Bhuvaneswari & Surendranath (2015) [5] extracted seven microsporidian isolates from infected lepidopteran pests in the sericulture zones of Karnataka, India, and conducted PCR-based analysis to determine the phylogenetic relationships among the key isolates. The phylogenetic analysis, genetic distance, and NCBI BLAST analysis indicated that the identified microsporidians are closely related to the typical Nosema subgroup. The identified microsporidian isolates belong to the Nosema genus, with NIK-hd2 showing homology with Nosema plutellae, which infects the Diamondback moth (Gautam et al., 2018) [7] and silkworms. NIT-de2 shows homology with Nosema fumiferanae, which infects larvae of the spruce budworm Choristoneura fumiferana (Frankenhuyzen et al., 2007) [6] . The nucleotide differences in the SSU-rRNA gene clearly distinguished all five microsporidians from the standard type species.
Conclusion
All ten microsporidian isolates were found to be infective to Bombyx mori and transmitted via transovarial transmission. This study revealed that microsporidians identified from the field as Nosema species can cross-infect silkworms and potentially cause economic losses in the sericulture industry.
Acknowledgement
AcknowledgmentThe authors are thankful to the Central Silk Board for supporting the work and thankful to K. R. Rathnamma, Senior Technical Assistant of Seed pathology section for assistance in experiments, Silkworm rearing, and data collection.
Figures and tables from the paper
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