Elsevier

Gene

Volume 582, Issue 1, 10 May 2016, Pages 59-68
Gene

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

https://doi.org/10.1016/j.gene.2016.01.048Get rights and content

Highlights

  • 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.

Abstract

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.

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.

References (55)

  • H.L. Qiao et al.

    Expression analysis and binding experiments of chemosensory proteins indicate multiple roles in Bombyx mori

    J. Insect Physiol.

    (2013)
  • A. Tuccini et al.

    Putative odorantbinding proteins in antennae and legs of Carausius morosus (Insecta, Phasmatodea)

    Insect Biochem. Mol.

    (1996)
  • X. Yi et al.

    Identification of a novel interacting partner of the chemosensory protein 1 from Plutella xylostella L.

    Int. J. Biol. Macromol.

    (2014)
  • M.Q. Zhang et al.

    Cloning, expression and cellular localization of the doublesex gene in the water flea, Daphnia carinata, during different developmental stages

    Gene

    (2014)
  • J.Y. Zhu et al.

    Identification and expression profiling of six chemosensory protein genes in the beet armyworm, Spodoptera exigua

    J. Asia Pac. Entomol.

    (2015)
  • V. Alekseev et al.

    Maternal control of resting egg production in Daphnia

    Nature

    (2001)
  • M.N. Andersson et al.

    Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae)

    BMC Genomics

    (2013)
  • M.N. Andersson et al.

    Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae)

    BMC Genomics

    (2014)
  • S. Angeli et al.

    Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria

    Eur. J. Biochem.

    (1999)
  • M. Averof et al.

    Hox genes and the diversification of insect and crustacean body plans

    Nature

    (1995)
  • K.N. Baer et al.

    Evaluation of selected endocrine disrupting compounds on sex determination in Daphnia magna using reduced photoperiod and different feeding rates

    Bull. Environ. Contam. Toxicol.

    (1999)
  • L.P. Ban et al.

    Chemosensory proteins of Locusta migratoria

    Insect Mol. Biol.

    (2003)
  • W.Q. Cao et al.

    Effect studies of some environmental conditions on reproductive conversion of Moina mongolica, a salt water cladocera

    J. Oceanogr. Taiwan Strait

    (2001)
  • G.R. Carvalho et al.

    The effect of food availability, female culture-density and photoperiod on ephippia production in Daphnia magna Straus (Crustacea: Cladocera)

    Freshw. Biol.

    (1983)
  • E. Danty et al.

    Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein

    J. Neurosci.

    (1999)
  • L. Gong et al.

    Cloning and characterization of three chemosensory proteins from Spodoptera exigua and effects of gene silencing on female survival and reproduction

    Bull. Entomol. Res.

    (2012)
  • W. Guo et al.

    CSP and takeout genes modulate the switch between attraction and repulsion during behavioral phase change in the migratory locust

    PLoS Genet.

    (2011)
  • Cited by (6)

    1

    These authors contributed equally to this work.

    View full text