Temporal transcriptome reveals that circadian clock is involved in the dynamic regulation of immune response to bacterial infection in Bombyx mori

Abstract

The circadian clock plays a critical role in the regulation of host immune defense. However, the mechanistic basis for this regulation is largely unknown. Herein, the core clock gene cryptochrome1 (cry1) knockout line in Bombyx mori, an invertebrate animal model, was constructed to obtain the silkworm with dysfunctional molecular clock, and the dynamic regulation of the circadian clock on the immune responsiveness within 24 h of Staphylococcus aureus infection was analyzed. We found that deletion of cry1 decreased viability of silkworms and significantly reduced resistance of larvae to S. aureus. Time series RNA-seq analysis identified thousands of rhythmically expressed genes, including immune response genes, in the larval immune tissue, fat bodies. Uninfected cry1 knockout silkworms exhibited expression patterns of rhythmically expressed genes similar to wild-type (WT) silkworms infected with S. aureus. However, cry1 knockout silkworms exhibited a seriously weakened response to S. aureus infection. The immune response peaked at 6 and 24 h after infection, during which "transcription storms" occurred, and the expression levels of the immune response genes, PGRP and antimicrobial peptides (AMPs), were significantly upregulated in WT. In contrast, cry1 knockout did not effectively activate Toll, Imd, or NF-κB signaling pathways during the immune adjustment period from 12 to 18 h after infection, resulting in failure to initiate the immune responsiveness peak at 24 h after infection. This may be related to inhibited silkworm fat body energy metabolism. These results demonstrated the dynamic regulation of circadian clock on silkworm immune response to bacterial infection and provided important insights into host antimicrobial defense mechanisms.

Inhibition of Expression of the Circadian Clock Gene Cryptochrome 1 Causes Abnormal Glucometabolic and Cell Growth in Bombyx mori Cells

“…Under the light cycle of LD 12:12, the mRNA levels of BmCry2 , BmTim , and BmClk in Cry1-KD cells remained low, while the mRNA levels of BmPer and BmCyc recovered, and the amplitudes of clock genes increased ( Figure S4 ). More importantly, the expression of BmCry2 , BmPer , BmTim , and BmClk in Cry1-KD cells resumed oscillatory rhythm due to photoentrainment, which is consistent with the observations of several instances of clock gene knockout in silkworms [ 47 , 48 , 49 , 50 , 51 ]. We speculate that there are two possible reasons for this: (1) there is still a small amount of remaining BmCRY1 protein in Cry1-KD cells, which responds to photo signals and resets circadian rhythms; (2) other clock genes, such as BmCRY2, have the function of receiving photo signals.…”

The clock gene Cryptochrome 1 is involved in the photoresponse of embryonic hatching behavior in Bombyx mori

“…Studies have shown that the egg hatching and adult emergence behavior rhythms of silkworms are disrupted by the deletion of the clock gene Per or Tim (Ikeda et al, 2019, 2021; Nartey et al, 2021). Our study has also shown that the hatching behavioral rhythm of Cry1 knockout silkworm is also inhibited (Qiu et al, 2023b). In addition, the absence of other clock genes in insects can lead to irregular egg hatching or adult emergence (Mark et al, 2021; Markert et al, 2016; Merlin et al, 2013; Wang et al, 2023).…”

“…The WT and Cry1 mutant (Cry1 −/− ) larvae were raised in the laboratory with mulberry leaves, under a 12 h light:12 h dark (light-dark [LD] 12:12) cycle at a constant temperature of 25.0 ± 0.5°C with 70%-80% humidity. TALENs were used as described previously to knockout the clock gene Cry1 in the DaZao strain (Qiu et al, 2023b). The knockout target site was located at +83 bp from the transcription start site, and the left and right binding sites for TALENs were 5′-CGGCGTTGGAAGAAA-3′ and 5′-ATTTTCATTTTTG-3′, respectively.…”

Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori

“…TALEN-mediated gene targeting knockout has been used to obtain the Cry1 mutant (Cry1 À/À ). The mutant failed to produce complete mRNA and proteins (Qiu et al, 2023). Briefly, the second exon of the Cry1 gene, a 20 bp length DNA fragment located at the transcription start site +83 bp, was used as the knockout target.…”

“…Quantitative reverse transcription PCR (qRT-PCR) was performed as previously described (Qiu et al, 2023). Brain, PG and fat body (FB) were dissected at ZT0 and washed three times with PBS.…”