Functional characterisation of Fe (II) and 2OG-dependent dioxygenase TcALKBH4 in the red flour beetle, Tribolium castaneum

Alpha-ketoglutarate-dependent dioxygenase ALKB homologue 4 (ALKBH4) is a member of the Fe (II) and 2-oxoglutarate-dependent ALKB homologue family that plays important roles in epigenetic regulation by alkyl lesions removal in mammals. However, the roles of ALKBH4 in insects are not clear. Here, TcALKBH4 was cloned and functionally characterised in Tribolium castaneum. Temporal expression revealed that TcALKBH4 was highly expressed in early embryos and early pupae. Spatial expression showed that TcALKBH4 was highly expressed in the adult testis, and followed by the ovary. RNA interference targeting TcALKBH4 at different developmental stages in T. castaneum led to apparent phenotypes including the failure of development in larvae, the reduction of food intake and the deficiency of fertility in adult. However, further dot blot analyses showed that TcALKBH4 RNAi does not seem to influence 6 mA levels in vivo. qRT-PCR was used to further explore the underlying molecular mechanisms; the result showed that TcALKBH4 mediates the development of larvae possibly through 20E signalling pathway, and the fertility of female and male adult might be regulated by the expression of vitellogenesis and JH signalling pathway, respectively. Altogether, these findings will provide new insights into the potential function of ALKBH4 in insects.

Automated summary

ALKBH4 plays a crucial role in epigenetic regulation in mammals by repairing alkyl lesions, but its function in insects is not well understood. In this study, the TcALKBH4 gene was cloned and characterized in Tribolium castaneum. The expression pattern of TcALKBH4 showed high expression in early embryos and early pupae, and in the adult testis and ovary. RNA interference targeting TcALKBH4 at different developmental stages resulted in distinct phenotypes, including developmental failure in larvae, reduced food intake, and fertility deficiency in adults. However, TcALKBH4 RNAi did not affect 6mA levels in vivo. qRT-PCR analysis suggested that TcALKBH4 may mediate larval development through the 20E signaling pathway, and the fertility of female and male adults may be regulated by vitellogenesis and JH signaling pathways, respectively. These findings provide new insights into the potential function of ALKBH4 in insects.