Assessment of antiviral medicinal plant extracts against grasserie disease and their impact on biochemical and hematological parameters in silkworm (Bombyx mori L.)

Introduction

The mulberry silkworm, Bombyx mori L., plays a pivotal role in the global silk industry and serves as a vital source of income and foreign exchange for several silk-producing countries (Krishnaswami et al., 1973; Isaiarasu et al., 2011) [7,3] . In tropical regions where silkworm rearing is practiced throughout the year, the insects are frequently exposed to a range of pathogenic infections, making disease management one of the major challenges in sericulture (Samson et al., 1998; Rajeswari and Isaiarasu, 2004; Isaiarasu et al., 2011) [18,16,3] . These diseases collectively account for considerable economic losses, often reducing cocoon yields by 30-40% (Chandrasekharan et al., 2006) [25] . Among them, Grasserie, a viral disease caused by the Bombyx mori nucleopolyhedrovirus (BmNPV), remains the most destructive, persisting year-round under tropical climatic conditions (Sivaprakasam, 1999; Latha et al., 2011; Manjunath et al., 2020) [20,8,11] . Medicinal and aromatic plants, known for their abundance of bioactive phytochemicals, have long been utilized in traditional medicine for their therapeutic properties. These plants produce a wide array of secondary metabolites, which, though not directly linked to primary metabolism, play key roles in ecological interactions and serve as natural defense agents against pathogens (Jain et al., 2004; Isaiarasu et al., 2011) [5,3] . The antimicrobial and antiviral nature of these compounds offers promising applications in silkworm health management, particularly in mitigating viral infections such as BmNPV.

Apart from inhibiting viral replication, these botanicals are also reported to enhance growth, immunity, and overall vitality in silkworms (Latha et al., 2011) [8] . Several earlier studies have confirmed the antiviral and antimicrobial efficacy of various plant-derived substances, underscoring their potential for sustainable disease control in sericulture (Gouda, 1991; Sridevi, 2003; Murthy, 2004; Shubha, 2005; Manimegalai and Chandramohan, 2006) [1,21,14,19,10] . The growth and physiological development of insects are largely governed by biochemical constituents such as carbohydrates, proteins, and lipids (Ito and Horie, 1959; Wyatt and Kalf, 1956; Latha et al., 2011; Ananda Kumar and Michael, 2012) [4,22,8,23] . In particular, haemolymph proteins are crucial for transport processes, enzymatic functions, and immune responses, with their synthesis influenced by both genetic and hormonal regulation (Hurliman and Chen, 1974; Latha et al., 2011) [2,8] . The haemolymph, an extracellular fluid analogous to blood in vertebrates, performs multiple physiological roles including nutrient transport, excretion, moulting, metamorphosis, and defense mechanisms such as phagocytosis and encapsulation (Gad and Alzahofi, 2010; Nazar et al., 2020) [26,15] . The present study was undertaken to evaluate the antiviral potential of three medicinal plant extracts-Aloe vera, Phyllanthus niruri, and Andrographis paniculata-against Grasserie disease in B. mori. These plants were selected based on their previously reported antiviral properties, easy availability, and suitability for eco-friendly disease management. Furthermore, the study assessed the influence of these plant extracts on protein activity and haemolymph parameters in BmNPV-infected silkworms. The experimental work was conducted at Sri Krishnadevaraya University, Anantapur, with the overarching objective of identifying effective, plant-based antiviral agents for sustainable disease management in sericulture.

Materials and Methods

Preparation of Plant Extracts

Extracts from three medicinal plants-Aloe vera, Phyllanthus niruri, and Andrographis paniculata-were prepared with minor procedural modifications. Fresh plant materials were initially shade-dried to retain their bioactive constituents and subsequently ground into a fine powder using an electric blender. All extraction procedures were carried out at Sri Krishnadevaraya University, Anantapur. For each plant species, 10 g of the powdered material was soaked in 100 ml of distilled water for six hours to facilitate the release of active compounds. The mixture was then subjected to continuous stirring for one hour on a magnetic stirrer to ensure complete extraction.

After stirring, the resulting mixture was filtered through Whatman filter paper to separate the solid residues. The filtrate was stored at 4 °C for subsequent experimental use, following the method described by Manjunath et al. (2020) [11] . To obtain the working solution, each extract was diluted to a 5% concentration using double-distilled water, ensuring consistency across all treatments. These prepared plant extracts were later employed to evaluate their efficacy in managing Grasserie disease in Bombyx mori.

Treatment Details T1: Aloe vera + BmNPV of 1×10 5 POB ml -1 T2: Phyllanthus niruri + BmNPV of 1×10 5 POB ml -1 T3: Andrographis paniculata + BmNPV of 1x10 5 POB ml -1 T4: BmNPV of 1x10 5 POB ml -1 T5: Distilled water control

Schedule of Treatment

To evaluate the antiviral efficacy of the selected medicinal plant extracts, five treatment groups (T 1 -T 5 ) were established. The extracts were applied to fresh mulberry leaves, which were subsequently fed to Bombyx mori larvae on the first day of the fourth and fifth instar stages. The experimental larvae belonged to the FC₁×FC₂ bivoltine double hybrid.

Observations were made to assess the impact of the treatments on cocoon characteristics, protein concentration, and total haemocyte count (THC). For reliability and reproducibility, the experiment was conducted in three replications, each consisting of 100 larvae, following the standardized method described by Shubha (2005) [19] . This experimental design provided a strong statistical basis for evaluating the antiviral potential of the medicinal plant extracts and their influence on the physiological and biochemical responses of B. mori larvae infected with Grasserie disease.

Haemolymph Collection

Haemolymph samples were collected from silkworms belonging to all treatment groups (T 1 -T 5 ) in pre-chilled test tubes to preserve sample stability and prevent coagulation. The collected haemolymph was immediately centrifuged at 1500 rpm for 10 minutes, and the resulting supernatant was carefully transferred into sterile microtubes. The samples were stored at-20 °C until further biochemical analysis, following the procedure outlined by Isaiarasu et al. (2011) [3] .

Estimation of Total Soluble Proteins

The total soluble protein content in the haemolymph was quantified using the Lowry method (Lowry et al., 1951) [9] , employing bovine serum albumin (BSA) as the standard reference. The estimation procedure followed the protocol described by Ananda Kumar and Michael (2012) [23] to ensure accuracy and consistency across all samples.

Counting the Total Haemocytes (THC)

For the estimation of total haemocyte count (THC), 1 ml of phosphate-buffered saline (PBS) was added to each haemolymph sample as an anticoagulant. A 10 µl aliquot of the diluted haemolymph was then loaded onto a Neubauer hemocytometer, and haemocytes were counted under a stereo microscope at 40× magnification. The method employed was based on the standard procedure reported by Nazar et al. (2020) [15] .

Ten cocoons were randomly selected from each replication on the sixth day after spinning. Each cocoon was weighed individually, and the average cocoon weight was calculated. This procedure was performed according to the method described by Ananda Kumar and Michael (2012) [23] and Kapoor et al. (2022) [6] .

For determining shell weight, ten cocoons from each replication were randomly chosen. The pupa was carefully removed, and the remaining cocoon shells were weighed. The mean shell weight was calculated as per the procedures outlined by Ananda Kumar and Michael (2012) [23] and Kapoor et al. (2022) [6] .

The shell ratio was computed using the ratio of shell weight to total cocoon weight, expressed as a percentage, following Kapoor et al. (2022) [6] .

Shell weight Shell Ratio =

x 100 Cocoon weight Pupal Weight (g) For the determination of pupal weight, ten cocoons were randomly selected from each replication. The pupa was separated from the cocoon, and its weight was measured individually. The average pupal weight was then calculated in accordance with the method described by Kapoor et al. (2022) [6] .

Throughout the rearing period, the numbers of healthy and diseased larvae were recorded for each treatment. The disease incidence was determined using the following formula, as suggested by Kapoor et al. (2022) [6] .

No. of diseased larvae Disease incidence (%) =

Results and Discussion

Influence of Plant Extracts on Cocoon Parameters

The application of medicinal plant extracts significantly influenced cocoon-related traits such as cocoon weight, shell weight, and shell ratio. Among the different treatments, the Aloe vera + BmNPV group (T 1 ) recorded the highest cocoon weight (1.85 g), followed by the distilled water control (T 5 ) at 1.81 g, and Phyllanthus niruri + BmNPV (T 2 ) at 1.75 g. The lowest cocoon weight (1.25 g) was observed in the BmNPVonly treatment (T 4 ) (Table 1). Similar trends were observed for shell weight and shell ratio, where T 1 exhibited superior performance, followed by T 5 , T 2 , and T 3 , while T 4 consistently showed the lowest values. These results highlight the protective and growth-promoting potential of Aloe vera, which enhanced cocoon production even under viral infection stress. The findings of this study are in line with earlier research. Manoharan (1996) [12] reported an increase in cocoon weight from 1.50 g to 1.68 g and in shell weight from 0.28 g to 0.312 g when aqueous extracts of Psoralea corylifolia, Tribulus terrestris, Acanthospermum hispidum, Caesalpinia coriaria, and Bougainvillea antiviral protein were administered to the PM × NB4D2 hybrid, compared with the control values (1.45 g and 0.280 g, respectively). This enhancement was attributed to the dual role of these botanicals in suppressing BmNPV infection and improving silk yield and quality. Similarly, Sivaprakasam (1999) [20] demonstrated that Bougainvillea antiviral protein purified from B. spectabilis conferred strong protection against BmNPV while simultaneously enhancing silk production in PM × NB4D2 hybrids. Supporting these observations, Latha et al. (2011) [8] also reported improved cocoon quality following the administration of plant-based antiviral formulations.

Collectively, these results reinforce the efficacy of plantderived bioactive compounds, particularly Aloe vera, in promoting silkworm health and productivity by mitigating viral infection effects and enhancing cocoon parameters.

The administration of medicinal plant extracts significantly reduced the incidence of Grasserie disease in the FC₁×FC₂ bivoltine silkworm double hybrid. All treatment groups showed a marked decline in disease incidence compared to the untreated control, indicating the antiviral potential of the tested botanicals. The lowest disease incidence was observed in the distilled water control (T 5 ), followed closely by the Aloe vera + BmNPV treatment (T 1 ), which recorded 23.6% incidence. Moderate disease levels were observed in the Phyllanthus niruri + BmNPV group (T 2 ) with 56.9%, while the Andrographis paniculata + BmNPV group (T 3 ) exhibited a higher incidence of 75.2%. The highest infection rate (78.4%) occurred in the BmNPV-only treatment (T 4 ) (Table 1; Figure 1). These observations confirm that Aloe vera is particularly effective in suppressing Grasserie disease, exhibiting stronger antiviral activity compared to the other two plant extracts and the virus-only control. The variation in antiviral responses among the treatments suggests that the bioactive constituents of these plants may differ in their ability to inhibit viral replication or boost host immunity. Such results emphasize the potential application of botanical formulations as part of an integrated disease management strategy in sericulture (Ram, 2000) [17] , with Aloe vera emerging as a highly promising candidate for mitigating viral infections and enhancing silkworm health.

Earlier investigations support these findings. Sridevi (2003) [21] and Manjunath et al. (2020) [11] reported a substantial reduction in the incidence of diseases such as Grasserie and Flacherie in silkworm hybrids (CSR2×CSR4 and PM×CSR2) when supplemented with medicinal plant extracts, compared to untreated controls. These studies, in agreement with the present results, demonstrate that plant-based antiviral treatments can effectively reduce disease prevalence and improve both health and productivity in silkworms across different hybrids. The consistent antiviral performance of medicinal plants underscores their potential as eco-friendly, sustainable alternatives for disease management in modern sericulture practices.

Influence of Plant Extracts on Total Haemocyte Count (THC)

The administration of medicinal plant extracts had a significant effect on the total haemocyte count (THC) in silkworms infected with BmNPV. In the BmNPV-only treatment (T 4 ), THC initially increased from 3,194 ml⁻¹ on day 1 to 5,923 ml⁻¹ by day 3 post-inoculation. However, from day 4 onward, a sharp decline was observed, with THC dropping to 194 ml⁻¹ by day 6. In contrast, the control group (T 5 ) exhibited a gradual rise in THC from 3,154 ml⁻¹ on day 1 to 6,058 ml⁻¹ by day 5, followed by a slight decrease to 4,320 ml⁻¹ during the spinning stage (Table 2; Figure 2).

The Aloe vera + BmNPV treatment (T 1 ) displayed a pattern similar to the control, with THC increasing from 3,205 ml⁻¹ on day 1 to 5,924 ml⁻¹ by day 5, before declining to 4,256 ml⁻¹ on day 6, closely approximating the control values. This indicates that Aloe vera helped maintain haemocyte levels in infected larvae, likely due to its antiviral and immunomodulatory properties. Treatments T 2 (Phyllanthus niruri + BmNPV) and T 3 (Andrographis paniculata + BmNPV) exhibited similar trends, though they ranked third and fourth, respectively, in maintaining THC levels. These observations are consistent with previous reports. Ananda Kumar and Michael (2011) [23] and Nazar et al. (2020) [15] noted that silkworm larvae infected with pathogens such as Bacillus thuringiensis showed marked reductions in THC, with Flacherie-infected larvae experiencing a 15.3% decline compared to healthy controls. Such decreases in haemocyte counts are indicative of physiological stress and weakened immunity, often resulting from reduced feeding and metabolic activity in infected larvae (Balavenkatasubbalah and Sivaprasad, 2014; Nazar et al., 2020) [24,15] . Overall, these results underscore the potential of medicinal plant extracts in enhancing immune function and sustaining haemocyte levels, thereby improving resistance to viral infections and supporting healthier growth in B. mori. Treatments 1 st day (THC ml -1 ) 2 nd day (THC ml -1 ) 3 rd day (THC ml -1 ) 4 th day (THC ml -1 ) 5 th day (THC ml -1 ) 6 th day (THC ml -1 ) T1: Aloe vera + BmNPV of 1×10

Estimation of Total Soluble Proteins in the Haemolymph

The total soluble protein content in the haemolymph of bivoltine hybrid silkworms exhibited a steady increase from the first day through to the spinning stage across all treatment groups. This age-dependent rise in protein levels is consistent with previous findings reported by Ananda Kumar and Michael (2012) [23] , reflecting the progressive accumulation of haemolymph proteins during larval development.

Among the treatments, the Aloe vera + BmNPV group (T 1 ) recorded the highest protein concentration on day six, reaching 92.6 mg ml⁻¹, closely followed by the healthy control (T 5 ) at 91.3 mg ml⁻¹. Treatments T 2 (Phyllanthus niruri + BmNPV)

and T 3 (Andrographis paniculata + BmNPV) also exhibited appreciable protein levels, though these were lower than T1 and T 5 . The BmNPV-only treatment (T 4 ) showed the lowest protein concentration, with a markedly reduced value of 12.3 mg ml⁻¹ (Table 3; Figure 3).

These results indicate that Aloe vera extract effectively maintained higher haemolymph protein levels even in the presence of BmNPV infection, closely mirroring those of healthy larvae. The pronounced decline in protein concentration in the virus-only group underscores the detrimental effect of BmNPV on silkworm physiology. Conversely, the ability of Aloe vera and other medicinal plant extracts to support protein synthesis and bolster immune function highlights their potential as protective agents in silkworm rearing.

Conclusion

Among the three medicinal plants tested-Aloe vera, Phyllanthus niruri, and Andrographis paniculata-Aloe vera demonstrated the highest antiviral efficacy against Grasserie disease in Bombyx mori, while also significantly enhancing key cocoon parameters. All tested plant extracts positively influenced total haemocyte count (THC) and haemolymph protein levels, contributing to improved silkworm health.

Based on these findings, Aloe vera shows promise as a natural additive for bed disinfectants and can be effectively incorporated into integrated strategies for the management of viral diseases in silkworm rearing.

References

1. Gouda RR. Studies on methods to increase silk yield of Bombyx mori L. [Ph.D. Thesis]. Tamil Nadu Agricultural University, Coimbatore, India; 1991. p. 242.
2. Hurliman RF, Chen PS. Ontogenetische Veranderungen des enzymmusters in der haemolymphe von Phormia rigina. Revue suisse de zoologie. 1974;81:648-654. DOI: https://doi.org/10.5962/bhl.part.76028.
3. Isaiarasu L, Sakthivel N, Ravikumar J, Samuthiravelu P. Effect of herbal extracts on the microbial pathogens causing flacherie and muscardine diseases in the mulberry silkworm, Bombyx mori L. J Biopestic. 2011;4(2):150155.
4. Ito T, Horie Y. Carbohydrate metabolism of the midgut of the silkworm, Bombyx mori L. Arch Biochem Biophys. 1959;80:174-176.
5. Jain R, Nagpal S, Jain S, Jain SC. Chemical and biochemical evaluation of Bauhinia species. J Med Arom Plant Sci. 2004;26(1):48-50.
6. Kapoor S, Bhat MA, Sudan D. A comparison of different bed disinfectants on the rearing performance of the double hybrid silkworm FC1×FC2. Pharma Innov J. 2022;11(10):522-527.
7. Krishnaswami S, Narashimanna MN, Suyananrayana SK, Kumararaj S. Sericulture Manual 2: Silkworm Rearing. Vol. 15. FAO Agricultural Services Bulletin. FAO, Rome; 1973. p. 131.
8. Latha S, Bhaskar RN, Pallavi, Chikkalingaiah. Effect of botanical treatment to BmNPV polyhedral bodies on cocoon parameters of silkworm Bombyx mori (PM×CSR2). Int J Adv Biotechnol Res. 2011;1(1):45-51.
9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin-phenol reagent. J Biol Chem. 1951;193(1):265-275. DOI: https://doi.org/10.1016/S0021-9258(19)52451-6.
10. Manimegalai S, Chandramohan N. Efficacy of botanicals against grasserie disease of silkworm, Bombyx mori L. Sericologia. 2006;46(1):15-23.
11. Manjunath GC, Doreswamy C, Bhaskar RN. Evaluation of certain medicinal plant extracts for the management of late larval flacherie disease of silkworm, Bombyx mori L. J Entomol Zool Stud. 2020;8(4):260-264.
12. Manoharan S. Evaluation of certain botanicals for the management of grasserie of Bombyx mori L. [M.Sc. (Agri.) Thesis]. Tamil Nadu Agricultural University, Coimbatore, India; 1996. p. 78.
13. Murugan K, Jeyabalan D, Kumar NS, Nathan SS, Sivaprakasam N. Growth promoting effects of plant products on silkworm-a biotechnological approach. J Sci Ind Res. 1998;57(10-11):740-745.
14. UAS, Bangalore, India; 2004. p. 78.
15. Nazar MZ, Umer A, Sarwar ZM, Faiz FU, Hussain S. Haemolymph assay and morphological changes of silkworm, Bombyx mori L. (Bombycidae: Lepidoptera) infected with Bacillus thuringiensis var. sotto. Pak J Agric Res. 2020;33(2):212-216. DOI: International Journal of Advanced Biochemistry Research https://www.biochemjournal.com https://doi.org/10.17582/journal.pjar/2020/33.2.212.216.
16. Rajeswari K, Isaiarasu L. Influence of the leaf, flower and pod extracts of Moringa oleifera on the growth and reproductive parameters of Bombyx mori L. Entomon. 2004;29(4):331-338.
17. Ram R. Appropriate silkworm rearing technology. In: Agrawal HO, Seth MK, editors. Sericulture in India. Bishen Singh Mahendra Pal Singh Press, Dehradun, India; 2000. p. 289-302.
18. Samson MV, Singh RN, Sasidharan TO. Resham Jyothi-a wide spectrum bed disinfectant. Indian Silk. 1998;37(3):9-10.
19. UAS, Bangalore, India; 2005. p. 84.
20. Sivaprakasam N. Botanical formulation for management of grasserie disease of silkworm, Bombyx mori L. In: Proceedings of the National Seminar on Tropical Sericulture, Bangalore; 1999. p. 335-337.
21. Sridevi G. Effect of mulberry leaves fortified with medicinal botanicals on the performance of mulberry silkworm, Bombyx mori L. [M.Sc. (Seri.) Thesis]. UAS, Bangalore, India; 2003. p. 116.
22. Wyatt GR, Kalf GF. Trehalase in insects. Fed Proc. 1956;15:188.
23. Ananda Kumar MD, Michael AS. Changes in the activities of proteins and carbohydrates in haemolymph of silkworm Bombyx mori treated with greenleaf. Int J Environ Sci. 2012;3(1):119-125. DOI: https://doi.org/10.6088/ijes.2012030131013.
24. Balavenkatasubbaiah M, Sivaprasad V. Flacherie disease and its management in silkworm rearing. Central Sericultural Research and Training Institute, Mysore; 2014. Available from: https://csrtimys.res.in/sites/default/files/menufiles/forewar ning-kn-flacherie.pdf.
25. Chandrasekharan K, Nataraju B, Balavenkatasubbaiah M, Sharma SD, Selvakumar T, Dandin SB. Grasserie and post cocoon mortality in silkworm. Indian Silk. 2006;45(4):12-13.
26. Gad AA, Alzahofi NN. Changes in the haemocytes of Bombyx mori larvae (Lepidoptera: Bombycidae) in relation to Escherichia coli and Bacillus thuringiensis infection. Egypt Sci Mag. 2010;6(1-2):15-21.