Gene expression analysis of transcriptional co activators and pro apoptotic markers in the development of Bombyx mori L under thermal stress

Abstract

The silkworm Bombyx mori L. is a commercially advantageous insect with significant agricultural value, which exhibits enormous thermal sensitivity. Global warming and climate change pose a focal threat to their growth and reproduction. High temperature negatively impacts nearly all biological processes, including biochemical and physiological processes. While the optimum temperature range for normal silkworm growth is 20°C to 28°C and maximum productivity occurs between 23°C to 28°C. When temperature exceeds 30°C that directly harms its health. High temperatures pose a significant threat to all organisms, potentially leading to its death. Exposure to thermal stress compels cells to generate an overabundance of reactive oxygen species and free radicals, which subsequently culminates in oxidative stress. This oxidative burden inflicts significant damage at both the physiological and structural levels within the cell. These ROS known to play an important role in the initiation of apoptosis, a form of programmed cell death, mediated through both mitochondria-dependent and independent pathways. Apoptosis integral to insect development, notably occurring during the metamorphosis of the silkworm. Despite this established role, the specific mechanism by which thermal stress induced oxidative stress and subsequent ROS mediated signaling trigger apoptosis in Bombyx mori remains poorly understood. The current study addresses this gap by performing gene expression analysis to investigate the responses of B. mori to thermal stress induced apoptosis. Specifically we assessed the differential expression of genes encoding transcriptional co-activators, pro-apoptotic markers and inflammatory response genes across varidus developmental stages following exposure to thermal stress. This investigation aims to delineate the intricate signaling cascade linking environmental stress, oxidative damage. and programmed cell death in this economically important insect species. For the purpose of evaluating the impact of thermal stress on the gene expression pattern we used the quantitative RT PCR method. Heat stress induced oxidative stress also leads to an imbalance in the antioxidant defense system and it also affects the growth and development of silkworms. In an effort to determine the impact of thermal stress, morphometric parameters of larvae. pupae and adults were studied and to determine the antioxidant activity various biochemical assays were also done. To evaluate the changes in the histological architecture of midgut tissue were also done. The quantitative and qualitative parameters of Mg cocoons in response to thermal stress were also evaluated. Following the fourth moult, fifth instar larvae were divided into two groups, control (C) group and thermal stress exposed (T) group. The group 1 was treated with normal rearing temperature about 25+2°C and the group 2 larvae were treated with 40+2°C temperature in 1 hr per day during the fifth instar stage. The treatments were also continued during the pupal and adult stage. In our observations, the high temperature exerts a negative impact on the morphometric parameters of larva, pupa and adult. The length of fifth instar larva, weight of larva, pupa and adult were decreased in the thermal stress exposed group compared to control. The mortality rate was increased in the heat shock exposed group in comparison with control. Moreover there was a significant increase in the lipid peroxidation level in different tissues of different developmental stages of silkworm. This indicates the level of oxidative stress induced by thermal stress. In addition to that there was noted a significant increase and decrease in the antioxidant defense system in the heat shock exposed group of different tissues at different developmental stages of silkworm in contrast to control. Thermal stress exposed groups showed an alternation in the activity of antioxidant enzymes such as SOD, CAT, POX, GST, GPX, and GR. Furthermore an alteration in the gene expression of BmApaf-1, BmICE-2, Caspase3. BmYKki, MBF1. TNFSFS5 and IL6 in different developmental stages of silkworm. Thermal stress exerts an upregulated expression of apoptotic related genes such as BmApaf-1, BmICE-2 and Caspase3 gene in different tissues of different developmental stages of silkworm. This indicates the _ activation of apoptotic pathways in silkworm Bombyx mori in response to thermal stress. Upregulated expression of transcriptional co-activator genes (BmYki and MBFI1) in the experimental group may protect the organism from thermal stress. Besides that the increased expression of IL6 and TNFSF5 implicated the incidence of inflammation in thermal stress exposed group in comparison with control. Moreover the heat shock protein genes play a critical role in the defense against thermal stress. The upregulated expression of Hsp70 gene in all the developmental stages indicates the protection against thermal stress. The present study also analyzed the impact of thermal stress on the various cocoon parameters like length, weight. shell weight, shell ratio, filament length, filament weight and reelability percentage. It is obvious that thermal stress negatively affects the quality and quantity of cocoons produced. So the result revealed that thermal stress induced oxidative stress exerts a negative impact on the overall growth and productivity of cocoons. Moreover the thermal stress induced ROS initiates the activation of apoptotic pathways and also activates the stress response genes to withstand the thermal stress. This study gives an idea about the overall biochemical and molecular aspects of stress response in silkworm on exposure to thermal stress.