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Abstract:
Endogenous biological clocks synchronize physiological processes to the exogenous geophysical cycles. The development and reproduction of Clunio marinus is regulated by a circalunar clock, which is entrained by moonlight, tidal cycles of mechanical agitation and temperature fluctuations. Interestingly, not all Clunio populations are sensitive to the three zeitgebers. Previous analysis revealed
the population derived from Helgoland (He-2SL) to be insensitive to moonlight, and that this loss is genetically determined. Further results suggested that duplicated NinaB genes could be responsible for the loss of sensitivity to moonlight in He-2SL. NinaB codes for an enzyme, combining an oxygenase activity with an isomerase-activity, and takes part in the production of a visual chromophore by
catalysing the conversion from carotenoids into retinoids. Clunio uniquely possesses 3 copies of the gene. To investigate, if duplicated NinaB genes are functional, we cloned and sequenced the full-length
NinaB, NinaB2 and NinaB3 transcripts from moonlight-sensitive (Por-2SL) and moonlight-insensitive populations (He-2SL) in at least three technical replicates. The majority of the full-length transcripts could be read through, suggesting that duplicated genes are indeed functional. The major difference between the two populations was a deletion of 155 base pairs within the NinaB2 gene in He-2SL likely
leading to a truncated protein. The presence of this mutation was genotyped in 41 Por-2SL and 82 field-caught males. The mutation was absent in Por-2SL and present only in He-2SL, although it was not fixed. Finally, to investigate the expression of NinaB 1-3 genes, we established a new in situ hybridization protocol. Using this, we showed the expression or rOpsin1 takes place in the eyes of the Clunio marinus larvae in the developmental stage L3.
