To disinfect breeding farms, some chemicals are currently used, such as hydrated lime at 0.5%, formaldehyde at 30% and formaldehyde at 2%, applied in the breeding shed to prevent any contamination by viral pathogens during the cultivation of silkworms (Suman et al., 2021). These products can cause risks to the health of the producer and damage the production of cocoons due to their use for long periods, which has led to the selection of resistant pathogens (Brancalhão et al. 2013). Therefore, mechanisms used to prevent infection and the spread of the BmNPV virus in the environment can help to reduce the damage caused in the field of sericulture.
The first signs of BmNPV infection observed in larvae were similar to those previously described (Brancalhão et al. 2009) for a virulent strain of Bombyx mori nucleopolyhedrovirus (BmNPV), present in the northwest and south regions of Paraná state (Brazil), whose genome was already sequenced (Ardisson-Araújo et al. 2014).
Silkworms were experimentally infected with the virus that did not survive and showed liquefaction and subsequent rupture of the integument, regardless of treatment with Bm5. These results were also described previously (Bueno et al. 2019), and despite that having used Bm5, there was no effect on larval liquefaction. It was previously described that the liquefaction effect cannot be attributed only to viral cathepsin (V-Cath) but also to the activity of another enzyme, such as viral chitinase (chiA), which increases the spread of virions within the host's vital organs, causing terminal tissue liquefaction of the insect and ultimately causing its death, thus facilitating the process of horizontal transmission of the virus (Rohrmann 2019). These enzymes perform a synergistic effect during virus replication within infected cells, which determines the dependence of these enzymes to promote the liquefaction of the host's tissues (Hodgson et al. 2011). Studies in Autographa californica multiple nucleopolyhedrovirus indicated that the action of cathepsin protease is necessary for the release of chitinase, cathepsin, and OB, thus causing terminal lysis of the host and dissemination of OB in the skies of infected insects (Hodgson et al, 2021). However, the Bm5, an inhibitor candidate for BmNPV-Cath, is not sufficient to prevent the total liquefaction of larvae and prevent the spread of the virus to the environment but minimizes this disruption, making a broad-spectrum inhibitor necessary for both viral hydrolytic enzymes.
Previous studies using treatment with bacterial prodigiosin have shown inhibition of BmNPV at different stages of viral replication in 24.72% of cells infected with cell lysis (Zhou et al., 2016). Our results were superior using a single dose of Bm5 applied immediately after inoculation of the virus to promote the significant survival of larvae and pupae, which can contribute to the field of sericulture in the prevention of the onset of grasserie disease caused by BmNPV.
According to our results, the cocoon quantity (CQ) variable of the treatment i3(Vir + Bm5) was similar to that of the control groups, demonstrating that even the infected caterpillars were able to build the cocoon, similar to the results reported by Bueno et al., 2019. The larvae that were infected with BmNPV and treated with Bm5 built cocoons of good quality, presenting an average weight of 1.9 mg. Our results were also superior to those previously reported (Bhaskar et al. 2011), who used different plant extracts to treat larvae infected with BmNPV and obtained average cocoon weights of 1.65 g. Higher values were also reported (Porto et al. 2005), who worked on the performance of four breeds of silkworms and reported an average weight between 2.03 and 2.26 g. In Brazil, the BRATAC silk industry considers first-class cocoons, those with an average weight of 1.9 g, which have a better selling price in the market (Sabbag et al. 2013). Therefore, according to this statement, the cocoon weight (CWe) results from the Bm5 treatment presented here are considered first-class cocoons.
All of the control and the infected larvae that receive Bm5 reported here show first-class cocoons, and the cocoon from the infected and treated with Bm5 larva shows a good cocoon shell weight (CSW) parameter, as reported for control larvae treated only with potable water. On the other hand, the results obtained in this work indicate that although the silk content (SC) remains practically unchanged among the groups, regardless of the treatment with Bm5, the total number of cocoons is decisive for production. The treatment with Bm5 shows differences in silk production when considering the number of cocoons obtained but not regarding the silk content. In addition, it was possible to prove that the Pluronic F-127 used as a diluent of Bm5 did not cause toxic or harmful depressive effects on silkworm biology, regardless of the infected insects or not, as well as on the production of cocoons and the productive parameters; the same was detected by (Bueno et al. 2019).
Insect defenses against antioxidant compounds include esterase, which can be classified into two groups. The α-esterase group includes carboxylesterases, cholinesterase, and acetylesterase, and the β-esterase group includes only carboxylesterases and cholinesterase (Oakeshott et al. 1993). These enzymes play a role in metabolic detoxification, a mechanism by which compounds such as insecticides, for example, are modified to form less toxic to the insect or eliminated quickly, preventing its action at the target site (Wu et al., 2004; Beckel et al., 2006). As shown, in this work, 11 esterase bands in the midgut and 6 in the fat body of B. mori hybrid larvae were detected. Six bands in the midgut and eight in the fat body of B. mori were described previously (Eguchi and Sugimoto, 1965). Several other authors have determined a different number of bands in the midgut of different breeds and hybrids of B. mori (Jingade et al. 2011). These results indicate that specific esterase expression can occur according to the larval instar, related to a variation in the number and intensity of enzyme fractions because in the early and later stages of development, fewer bands can be detected. Additionally, in this study, no inhibition or higher expression of any bands other than those detected in the electrophoretic pattern was observed; therefore, viral infection by BmNPV or even treatment with Bm5 compound and the diluent Pluronic F-127 did not alter the enzymatic profile. These data indicate that this detoxification system involved in the metabolism of xenobiotic compounds was not activated. The analysis of antioxidant systems in B. mori showed some changes in the enzymes catalase and superoxide dismutase, and the cholinergic system revealed changes only in the beginning of BmNPV infection (Vessaro-Silva et al. 2019).
In this sense, the results from Bm5 on the infected larvae suggest that this could be utilized to prevent total losses from cocoon production on breeding silkworm farms during BmNPV infection. The compound Bm5 has been shown to be effective in combating viral infection and can be used in combination with other control measures. Further studies are needed in the search for new compounds that can inhibit the action of the two enzymes involved in BmNPV infection to prevent the spread of this virus in silkworm cultivation.