Modulazione circadiana delle risposte immunitarie innate in Bombyx mori

The circadian clock is an internal timekeeping mechanism that enables organisms to physiologically anticipate and synchronise to daily changes in the environment such as shifting day light hours. The molecular mechanism of the clock is endogenously driven and rhythmically promotes and represses gene expression with a periodicity of around 24-hours. The clock regulates many biological functions and has been shown to modulate innate immune defences throughout the 24-hour day. In Drosophila, the immune response to several bacterial infections is regulated by the clock, and flies are more susceptible to systemic bacterial infections occurring in the day compared to at night. Oral ingestion is a more typical route of pathogenesis, though it is less well studied. We aimed to determine whether the immune response to an oral infection with Enterococcus mundtii in germ-free 1st day 5th instar (L5D1) Bombyx mori is regulated by the circadian clock or is modulated by light. We performed oral infections in B. mori reared in different photoperiods, including constant darkness. We found that larvae reared in 12:12 LD exhibited a time-of-day dependent variation in their immune responses, with larvae infected in the day (ZT3) more sensitive to the pathogen compared to night infected larvae (ZT15). When infected in DD, there was no difference in the survival response depending on the time of infection, indicating the immune response is not regulated by the circadian clock. We then characterised the 24-hour expression profiles of core clock and immune genes, circulating hemocytes and the structure of the midgut in germ-free L5D1 B. mori. The results indicate L5D1 B. mori lack a mature circadian oscillator, though several individual genes, including Clock (Clk), were cycling in 12:12 LD. Circulating hemocyte numbers vary in a cyclic fashion and are increased in the day but do not cycle in DD. We did not observe any daily variation in the midgut structure. A transcriptomic analysis of the midgut following oral infection identified distinct differential gene expressions following morning and night infections. Progenitors of hydrogen peroxide, juvenile hormone regulation, including ecdysone, and pathogen recognition receptors were rapidly upregulated 3 hours post infection at night. Moreover, antimicrobial peptides were activated 6 hours post night infection and a gene set enrichment analysis showed the Toll and imd pathway was activated 9 hours post a night infection. These immune modulators were not upregulated following a day-time infection. Although L5D1 B. mori lack a mature circadian clock, the Clk gene was rhythmically expressed in the brain and only in 12:12 LD. In a light-dependent manner, Clk regulates prothoracic hormone, that regulates ecdysone, that is an upstream activator Peptidoglycan Recognition Receptor-LC, a membrane bound pathogen sensor that activates the imd immune pathway. Therefore, even in the absence of a mature circadian clock the rhythmic expression of Clk in 12:12 LD and not DD, may be causing light driven modulation of innate immune responses that are not regulated by the circadian clock. We generated a period gene KO mutant and characterised the line for silk productivity, gene expression, egg hatching rhythm, daily hemocyte profile, daily variations in the midgut structure and performed an oral infection with E. mundtii in 12:12 LD. There was no difference in silk production compared to the wildtype, mutants lost hatching rhythmicity and per expression was considerably downregulated. Hemocytes fluctuated in the day but not in a circadian fashion. The per null line was more sensitive to oral infection with E. mundtii compared to the wildtype although the variation in survival depending on the time of infection was maintained with day-time infected larvae being more susceptible to the pathogen. The gut structure was unchanged thought the day but the peritrophic matrix was considerably compacted in the mutant.