Cloning, expression and localization of DacaCSP2 and DacaCSP3 during different reproductive stages in Daphnia carinata

• We cloned full-length cDNA of two chemosensory genes (CSP2/3) from Daphnia carinata.
• CSP2/3 protein levels varied between males, and sexual or parthenogenetic females.
• CSP2/3 are expressed in the ovaries, summer eggs, thoracic limbs, rectum and second antennae.
• CSP2/3 may be the trigger for females to transform into males.
• CSP2/3 mRNA expression levels increase in response to temperature changes.

Daphnia carinata are unique freshwater crustaceans that undergo both sexual and asexual reproduction, depending on environmental factors. While the molecular mechanism behind the reproductive transformation has been unknown, chemosensory proteins (CSPs) may be involved. We have cloned the cDNA sequences of two CSP genes from D. carinata using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). The full-length cDNA of DacaCSP2 (GenBank accession no: KM624608) was 632 bp, with an ORF (open reading frame) of 330 bp encoding a 12.02 kDa protein; and the full-length cDNA of DacaCSP3 (GenBank accession no: KM624609) was 935 bp, with an ORF of 342 bp encoding a 12.78 kDa protein. Both CSPs encoded an N-terminal signal peptide, four conserved cysteines, an OS-D superfamily domain, a 2Fe–2S ferredoxin domain, an anaphylatoxin domain and an EGF-like domain. DaCaCSP2 and DaCaCSP3 proteins were most closely related to CSPs from Daphnia pulex and were more distantly related to CSPs from other insects. Using quantitative PCR, we found expression levels of DaCaCSP2 and DaCaCSP3 mRNA were highest in sexual females, followed by parthenogenetic females, and lowest in males. The expression levels of DaCaCSP2 and DaCaCSP3 mRNA also increased at lower temperatures, which suggested they could respond to environmental cues. Whole mount in situ hybridization (ISH) showed that DaCaCSP2 and DaCaCSP3 were expressed mainly in the ovaries, summer eggs, thoracic limbs, rectum and second antennae in sexual females; while they were expressed mainly in the ovaries, thoracic limbs, rectum and second antennae in parthenogenetic females. Together, these results suggest that DacaCSP2 and DacaCSP3 may respond to environmental cues and control the reproductive switch from sexual to asexual reproduction in D. carinata.