A feasibility study towards cryopreservation of silkworm eggs: response of non-diapause silkworm eggs to low temperature.

Insect embryos are very sensitive to chilling temperatures and vary with species and their developmental stages. Insect eggs are small and can supercool to temperatures ranging from -5 degrees C to -40 degrees C. In general, insects rely on a variety of ecological and physiological adaptations to survive low temperatures, making cryopreservation technique significantly complex. Mulberry silkworm (Bombyx mori L., Lepidoptera: Bombycidae) eggs are cleidoic with chorion (approximately 20-25 µm thick). Preservation of non-diapause eggs to a limited period is practiced usually to delay hatching. The advantage of early embryonic periods having resistance to low temperature is utilized for chilling of eggs and preservation for long periods. However, technique for cryopreservation of silkworm eggs is not yet developed and the identification of precise embryonic stage and chill sensitivity is necessary for effective silkworm cryopreservation. The paper reports the chill-sensitivity and tolerance of non-diapause silkworm embryos of mulberry silkworm at various embryonic stages. Silkworm embryo of 48h-age are relatively chill-sensitive as compared to other embryonic ages. This is vital information for the development of cryopreservation protocol for silkworm eggs.

Upregulation of Atg5 and AIF gene expression in synchronization with programmed cellular death events in integumental epithelium of Bombyx mori induced by a dipteran parasitoid infection.

Infection of the commercially important silkworm, Bombyx mori by a tachnid parasitoid, Exorista bombycis induced activation of genes and cellular responses associated with apoptosis in integumental epithelial cells. Composite cellular profile showed initial autophagy, intermediate endoplasmic reticulum degranulation and deformed nucleus as well as later DNA fragmentation indicating apoptosis. Two cell death-associated proteins, autophagy 5-like (Atg5L) and apoptosis-inducing factor (AIF), in addition to caspase, are identified from the infected integumental epithelium through mass spectrometric analysis. Genes encoding these proteins showed age-dependent activation after the infection as revealed by quantitative expression analysis. Atg5 showed early upregulation in association with signs of autophagy whereas AIF showed late upregulation in association with DNA condensation and fragmentation. Expression of AIF showed negative correlation with that of Atg5 after the infection. On the other hand, in control, caspase expression showed positive correlation with AIF expression indicative of regulated expression in normal larval epithelium, which was absent after infection. Activation of Atg5, AIF and caspase genes in close association with different cell death events revealed the synchronized differential expression of apoptosis-associated genes in response to the macroparasitism. Enhanced expression of Atg5, AIF and caspase genes coupled with the appearance of cell death symptoms indicate parasitism-induced activation of genetic machinery to modulate cell death events in the epithelium, which was hither to unknown in invertebrate systems.

Protein A linked latex antisera test for detection of infectious flacherie of silkworm, Bombyx mori L. caused by B. mori infectious flacherie virus.

Protein A linked latex antisera (PALLAS) test was used for detection of B. mori infectious flacherie virus (BmIFV) in silkworms. The test was found superior to direct latex agglutination (DLA) test for detection of BmIFV in infected larval and faecal matter extracts and purified preparations. Latex beads sensitized with anti-BmIFV IgG were able to detect up to 75 ng/ml BmIFV in PALLAS test and 300 ng/ml in DLA test. BmIFV was detected in diluted extracts of infected larvae (up to 1:200) and their faecal matter (up to 1:500). BmIFV progressive infection was detected at 12 and 24 hr of post infection in PALLAS and DLA tests, respectively in both larval and faecal matter extracts. PALLAS tests provides a simple, specific and highly sensitive test for detection of infectious flacherie virus.