Construction Of CDNA Library From Daphnia Carinata Pathenogenetic And Studies On Chemonsensory Gene

Daphnia are arthropod zooplankton,which are ubiquitous in fresh water. They play an important role in the aquatic ecosystem,as they are the primary food source of fish and invertebrate predators,and have long been used as a signal organism that can indicate toxicity in the environment(Watanabe,T.,et al. 2005).They have two different reproductive modes,parthenogenesis and sexual reproduction.The switch between these two modes is determined by environmental factors such as community density,temperature,food availability and salinity(Kleiven,L.,et al.1992).Therefore,Daphnia are suitable organisms for studying the mechanism of reproductive mode switching.Presently,little is know regarding the molecular details of Daphnia reproductive biology,and such data is key to understanding their unique reproductive program.Detailed understanding of this subject will have an important economical and practical significance in the large-scale aquaculture of fish,shrimp,and crabs.Moreover, an understanding of Daphnia reproduction could yield important insight into the reproduction and development of animals and humans alike.EST analysis,which surveys sequences contained in cDNA libraries,is a powerful approach for discovering new genes and analyzing gene expression profiles in tissues or cells(Tassanakajon,K.,et al.2006).It has already been successfully used in development and reproduction biology studies,especially for identifying sex-biased genes.For example,female-specific genes of Culex pipiens pallens(Chen,ZH.,et al.2007),sex-related genes in giant tiger shrimp (Preechaphol,L.,et al.2007),and reproductive switching genes from Rhopalosiphum padi were all studied using EST-based strategies(Tagu,P.L.,et al.2004).The first Daphnia cDNA library was created from Daphnia magna, and the expression profiles were analyzed using ESTs(Tagu,P.L.,et al.2004). The cDNA clones and EST sequence information were useful in subsequent functional analysis of Daphnia biology,as well as investigation of the links between ecology and genomics(Shaw,C,et al.2007).Based on information from ESTs,important sex determination dsx/mab-3 domain(DM domain) genes have already been cloned from Daphnia magna(Sagawa,Y.,et al.2005;Kato, K.,et al.2008).In order to study sex-biased genes in Daphnia pulex,a cDNA library from Daphnia pulex was also constructed(Eads,C,et al.2007).Despite these analyses,genes that are related to the switch between the two reproductive modes are still uncharacterized,and information regarding Daphnia genetics in the National Center for Biotechnology Information(NCBI) public database is limited.In addition,comparison between genes expressed across Daphnia species could be helpful in identifying the most important players in the Daphnia reproductive cycle.Furthermore,our present findings provide fundamental and significant information for future studies of Cladocerans reproductive biology.Specifically,these data provide novel molecular and cellular information on the life cycle of Cladocerans.Our results and conclusions were listed as following:1.Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea Daphnia carinataThe water flea Daphnia carinata(D.carinata) reproduces both sexually and parthenogenetically,yet little is known about the genes involved in these processes.To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation,we have generate.d and characterized an expressed sequence tag(EST) dataset from D.carinata.A set of 1495 clusters was generated from sequencing 3072 randomly chosen clones from a parthenogenetic,juvenile water flea cDNA library.The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities,including metabolism, translation,development and reproduction,as well as genes involved in sensing environmental factors.We speculate that genes involved in development and reproduction,along with genes that allow the organism to sense changes in the environment,play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation.Additionally, 86%of the D.Carinata unique sequences could be stringently mapped to the D. pulex genome,of which 125 mapped to intergenic and intronic regions on the current assembly.Our results provide practical insight into crustacean reproductive biology,in addition to establishing a new animal model for reproductive and developmental biology.2.Cloning of Chemonsensory from Daphnia carinata and analysis of the differences of expression when it is involved in the response to chemical and physical stimulations and the effects to the reproduction and development.In this research,after inducing by different environmental factors,we detected and analysed many biochemical and physiological data to discuss the effects of the environmental factors on the reproduction conversion and the role of Chemonsensory in this process.The results of the temperature experiment showed that high temperature accelerates reproduction;low temperature restrains both growth and reproduction.The results of the toxicological experiment of Cd²⁺ showed thatD.carinata lower Cd²⁺ concentrations accelerates growth of D.carinata;wherea higher Cd²⁺concentrations restrains it.In addition,Cd²⁺ can reduce the reproductive capability apparently.These results are consistent with the results of the expression of Chemonsensories which came from daphnias under the same culture condition.All the environmental factors except high temperature can induce high expressions of Chemonsensories.That indicates in unsuitable conditions,Chemonsensories play an important role.It is reported that CSPs belong to a conserved protein family and can sense chemicals and changes in the environment,they may play a crucial role in the Daphnia life cycle.With all of this data in mind,we speculate that CSPs are sensitive and responsive to environmental factors,and could trigger a series of signal transduction events and metabolic changes.So CSPs may play important roles in regulating the transformation of reproduction.