Diapause is a developmental transition in insects based on seasonal adaptation to adversity; it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.
Bombyx mori silk is a super-long natural protein fiber with a unique structure and excellent performance. Innovative silk structures with high performance are in great demand, thus resulting in an industrial bottleneck. Herein, the outer layer sericin SER3 is ectopically expressed in the posterior silk gland (PSG) in silkworms via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a fiber with sericin microsomes dispersed in fibroin fibrils. The water-soluble SER3 protein secreted by PSG causes P25’s detachment from the fibroin unit of the Fib-H/Fib-L/P25 polymer, and accumulation between the fibroin layer and the sericin layer. Consequently, the water solubility and stability of the fibroin-colloid in the silk glandular cavity, and the crystallinity increase, and the mechanical properties of cocoon fibers, moisture absorption and moisture liberation of the silk also improve. Meanwhile, the mutant overcomes the problems of low survival and abnormal silk gland development, thus enabling higher production efficiency of cocoon silk. In summary, we describe a silk gland transgenic target protein selection strategy to alter the silk fiber structure and to innovate its properties. This work provides an efficient and green method to produce silk fibers with new functions.
Metabolic disorders of the circulatory system of animals (e.g., hyperglycemia and hyperlipidemia) can significantly affect immune function; however, since there is currently no reliable animal model for hyperproteinemia, its effects on immunity remain unclear. In this study, we established an animal model for hyperproteinemia in an invertebrate silkworm model, with a controllable plasma protein concentration (PPC) and no primary disease effects. We evaluated the influence of hyperproteinemia on innate immunity. The results showed that high PPC enhanced hemolymph phagocytosis via inducing a rapid increase in granulocytes. Moreover, while oenocytoids increased, the plasmacytes quickly dwindled. High PPC inhibited hemolymph melanization due to decreased phenoloxidase (PO) activity in the hemolymph via inhibiting the expression of the prophenoloxidase-encoding genes, PPO1 and PPO2. High PPC upregulated the gene expression of antimicrobial peptides via differential activation of the Toll and Imd signaling pathways associated with NF-κB signaling, followed by an induction of inconsistent antibacterial activity towards Gram-positive and Gram-negative bacteria in an animal model of high PPC. Therefore, high PPC has multiple significant effects on the innate immune function of the silkworm circulatory system.
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.
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