Research paperCloning, expression and localization of DacaCSP2 and DacaCSP3 during different reproductive stages in Daphnia carinata
Introduction
Daphnia carinata are small species of freshwater crustaceans belonging to the Cladocera order, commonly known as water fleas. Due to their small size, fast breeding, ease of cultivation and environmental sensitivity, this species is often used for water monitoring and as a source of protein for the fish breeding industry (Yang, 1994, Qi et al., 1991, Rao et al., 2002). D. carinata are unique in that they can undergo both sexual and asexual reproduction, depending on environmental factors. When conditions are favorable, they reproduce parthenogenetically to enable rapid population growth; but as conditions deteriorate, sexual reproduction is initiated and both males and sexual females are produced (Kleiven et al., 1992, Baer and Owens, 1999, Cao et al., 2001). Eggs fertilized by males develop into resting eggs that lie dormant until conditions improve, thus ensuring the continued survival of the population (Carvalho and Hughes, 1983). Reproductive mode research has primarily focused on the effects of environmental factors (Alekseev and Lampert, 2001, Cao et al., 2001, Innes, 1997, Khmeleva et al., 1995, Lu and Qian, 1992, Lu et al., 1999, Martinez-Jeronimo et al., 1994, Pedrozo and Bohrer, 2003, Shi and Shi, 1996, Wang et al., 2000, Yang, 2004). However, to date, the molecular mechanism of the reproductive transformation from asexual to sexual reproduction remains largely unknown.
Chemosensory proteins (CSPs) may be important for controlling this reproductive switch. The CSP family of proteins was well conserved across evolution. CSPs were first discovered and named as olfactory specific protein D (OS-D) in Drosophila melanogaster (McKenna et a1., 1994), and soon after, many members of the same family were isolated and cloned in several insect species of different orders, such as Lepidoptera (Maleszka and Stange, 1997, Picimbon et al., 2000), Hymenoptera (Ishida et al., 2002), Orthoptera (Angeli et al., 1999, Picimbon et al., 2000, Ban et al., 2003, Zhou et al., 2013) and Phasmids (Tuccini et al., 1996, Mameli et al., 1996, Marchese et al., 2000). CSPs were not only found in external sensory organs but in external and internal non-sensory tissues (Danty et al., 1999; Maleszka et al., 2007, Guo et al., 2011; Andersson et al., 2013, Andersson et al., 2014), including reproductive organs (e.g., in Locusta migratoria) (Zhou et al., 2013). They were multifunctional proteins that were involved in both insect chemoreception (Angeli et al., 1999) and in insect embryo development (e.g., in the honeybee) (Foret et al., 2007, Maleszka et al., 2007). We hypothesize that CSPs could have multiple chemosensing and reproductive functions, they may be responsible for sensing environmental factors and inducing the reproductive switch in D. carinata.
In this study, we cloned the full-length cDNA of two CSP genes (DacaCSP2 and DacaCSP3) from D. carinata. We investigated the relative expression levels of DacaCSP2 and DacaCSP3 mRNA during different reproductive phases, and at different temperatures, using quantitative polymerase chain reaction (qPCR). We also determined the cellular localization of these RNAs using whole mount in situ hybridization (ISH). This study provides basic information for elucidating the molecular mechanisms of the reproductive switch in D. carinata.
Section snippets
Sample preparation
D. carinata were obtained from the channel at the Minhang Campus, East China Normal University. Healthy parthenogenetic organisms were identified and cultivated by feeding with cladoceran nutrient solution (1.5 g cow dung, 2 g vegetables, 20 g fertile soil in 1 l water, boiled and filtered). D. carinata were incubated at 25 ± 1 °C under a 14 h light/10 h dark photoperiod. For 2–3 weeks, sexual and parthenogenetic adults were collected, frozen in liquid nitrogen and stored at − 80 °C for RNA extraction.
Results and discussion
CSPs were first discovered in the Artemia franciscana of crustaceans. However, as most researches have been conducted in insects, we still have known little about the function of CSPs in crustaceans. Here, we successfully have cloned and characterized the full-length cDNA of two CSP genes from the crustacean D. carinata by RT-PCR and RACE (both 5′ and 3′).
Conclusions
In this study, we successfully cloned and characterized the full-length cDNA of two CSP genes from D. carinata, the expression were investigated by qPCR, and cellular localization were studied using RNA whole-mount in situ hybridization. The results showed that DacaCSP2 and DacaCSP3 mRNA were expressed distinctively and specifically in asexual and sexual reproduction stages, suggesting that DacaCSP2 and DacaCSP3 could sense environmental changes and control the reproductive switch in D. carinata
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study was financially supported by grants from the National Natural Science Foundation of China (No. 31172043 and 31572221), the Natural Science Foundation of Zhejiang, China (No. LY12C19003), Shanghai Science Board Basic Foundation 332 (No. 13DJ1400100), the National Water Pollution and Control Foundation of China 333 (2012ZX07403002) and the Post Doctor Program of Yangpu, Shanghai. Parts of the study were conducted in the Instruments Sharing Platform of School of Life Sciences, ECNU.
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These authors contributed equally to this work.