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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 . [ABSTRACT FROM AUTHOR]

In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata , designated DapcaDsx1 , was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Daphnia (water fleas) are small crustaceans that undergo an unusual switch from asexual to sexual reproduction that is dependent on environmental conditions. In this study, a senescence-associated protein (SAP) from the common freshwater species Daphnia pulex was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). Real-time PCR was employed to quantify the expression of D. pulex SAP (DpSAP) in individual organisms. The role of DpSAP in the reproductive transformation was further investigated in both parthenogenetic and sexual females by using digoxin-labeled SAP RNA probes and RNA whole-mount in situ hybridization. DpSAP was more highly expressed in sexual females, indicating a role in growth and reproduction. Cellular localization studies using RNA whole-mount in situ hybridization showed specific expression in the second tentacle joints. These expression patterns suggest an important role for DpSAP in the reproductive transformation of D. pulex. [Copyright &y& Elsevier]