GENE-39664; No. of pages: 8; 4C: Gene xxx (2014) xxx–xxx

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Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium nipponense Shubo Jin a,b, Sufei Jiang b, Yiwei Xiong b, Hui Qiao b, Shengming Sun b, Wenyi Zhang b, Fajun Li b, Yongsheng Gong b, Hongtuo Fu a,b,⁎ a

Wuxi Fishery College, Nanjing Agricultural University, Wuxi 214081, People's Republic of China Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, People's Republic of China

b

a r t i c l e

i n f o

Article history: Received 25 December 2013 Received in revised form 26 April 2014 Accepted 4 May 2014 Available online xxxx Keywords: Macrobrachium nipponense Slow-tonic S2 tropomyosin and Slow tropomyosin isoform Sex-determination Expression pattern Crustacean

a b s t r a c t This paper reports that Slow-tonic S2 tropomyosin (Sst) and Slow tropomyosin isoform (Sti) was highly expressed in androgenic gland transcriptome of Macrobrachium nipponense, which may play crucial roles in sexual differentiation to maleness. In this study, two Sst and Sti gene homologues designated as Mnsst and Mnsti were cloned and characterized from a freshwater prawn M. nipponense. The full-length cDNA of Mnsst and Mnsti consists of 997 bp and 1926 bp, respectively, with an ORF of 852 bp encoding 284 amino acids, and the similarity in ORF reached to 95.82%. The deduced amino acid sequences of Mnsst and Mnsti shared the highest identity with Slow-tonic S2 tropomyosin and Slow tropomyosin isoform of Homarus americanus. Real-time quantitative RT-PCR showed that the Mnsst and Mnsti genes were expressed in different tissues with the highest level of expression in the androgenic gland, implying that these two genes may be related to sex-determination in M. nipponense. Real-time quantitative RT-PCR revealed that in addition, Mnsst and Mnsti were speculated to be related with embryonic organogenesis of M. nipponense, especially for the formation of complete mouthpart and digestive organ and stimulating larval changes of morphology and initiate metamorphosis, the results of present study implied that the two genes may play complex and important roles in sex differentiation of M. nipponense. Thus, we isolated two candidate genes that may advance the studies of sex-determination mechanism in M. nipponense and even the whole crustacean species, as well as promoting the all-male population culture of M. nipponense. © 2014 Published by Elsevier B.V.

1. Introduction The oriental river prawn, Macrobrachium nipponense (Crustacea; Decapoda; Palaemonidae), is an important commercial prawn species that is widely distributed in freshwater and low-salinity estuarine regions of China and other Asian countries (Cai and Shokita, 2006; De Grave and Ghane, 2006; Feng et al., 2008; Ma et al., 2011; Mirabdullaev and Niyazov, 2005; Salman et al., 2006; Yu and Miyake, 1972). It is considered as an important fishery resource in China with an aquaculture production of 205,010 tons annually (Bureau of

Abbreviations: M. nipponense, Macrobrachium nipponense; Sst, Slow-tonic S2 tropomyosin; Sti, Slow tropomyosin isoform; T, testis; O, ovary; B, brain; AG, androgenic gland; H, heart; I, intestine; M, muscle; L, liver; ES, eyestalk; VD, vasa deferentia; CS, cleavage stage; BS, blastula stage; GS, gastrul stage; NS, nauplius stage; PS, protozoea stage; ZS, zoea stage; L1, the first day larva after hatching; L5, the fifth day larva after hatching; L10, the tenth day larva after hatching; L14, the fourteenth day larva after hatching. ⁎ Corresponding author at: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, People's Republic of China. E-mail address: [email protected] (H. Fu).

Fishery, Ministry of Agriculture, P.R.C., 2009). Facing stiff market competition, Macrobrachium producers require improvements in river prawn production techniques and river prawn genetic traits to obtain more profit. As with many other Macrobrachium species, the male counterparts grow faster and gain more weight at harvesting time than female river prawns. Thus, culture of all-male populations would be beneficial for economic purposes. Therefore, the long-term goals of the M. nipponense aquaculture industry are to make genetic selections and attain a better understanding of sex-differentiation mechanism in this species. The sex-determination and differentiation mechanism of M. nipponense were still unclear. Fortunately, many sex-determination homologous genes, shared high similarity with sex-determination genes in other species, were carried out from testis cDNA libraries and androgenic gland transcriptome (Jin et al., 2013; Qiao et al., 2012), and sexlethal, transformer-2 and extra sex comb may be involved in sexdifferentiation and external sexual forms in M. nipponense based on the quantitative real-time PCR analysis (Zhang et al., 2013a,b,c). The androgenic gland is found in most crustacean species. It produces hormones that play crucial roles in sexual differentiation to maleness, including the development of the testes and male sexual

http://dx.doi.org/10.1016/j.gene.2014.05.014 0378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

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S. Jin et al. / Gene xxx (2014) xxx–xxx

characteristics (Sagi et al., 1990). In Macrobrachium rosenbergii, males underwent sex reversal to females after their androgenic gland was ablated from the males. The all-male population was generated when the “reversal females” were mating with normal male M. rosenbergii (Sagi and Cohen, 1990; Sagi et al., 1986). Considering the fact that ablation or implantation of the androgenic gland at certain stages of development can result in sex reversal to male or female (Malecha et al., 1992; Manor et al., 2004; Nagamine et al., 1980; Sagi et al., 1990), the expression pattern of androgenic gland hormone genes in crustaceans has received much attention in recent years (Ma et al., 2013; Mareddy et al., 2011; Sook et al., 2011; Tomer and Sagi, 2012; Tomer et al., 2009; Tomer et al., 2011). Two genes which were reported to be significantly expressed in the androgenic gland were the Slow-tonic S2 tropomyosin (Sst) and Slow tropomyosin isoform (Sti). There were no expression of these genes in vasa deferentia, ovary, and testis (Jin et al., 2013). Slow-tonic S2 tropomyosin and Slow tropomyosin isoform belongs to tropomyosin family. Tropomyosins control the function of actin filaments in both muscle and non-muscle cells. They are often divided into muscle and non-muscle tropomyosin isoforms. In the muscle sarcomere, muscle tropomyosin isoforms regulate interactions between actin and myosin, playing a pivotal role in the regulation of muscle contractions. Non-muscle tropomyosin isoforms are found in all cells. They are important in the control and regulation of cell cytoskeleton and other key cellular functions (Gunning et al., 2005). In crustaceans, the Slow-tonic S2 tropomyosin and Slow tropomyosin isoform cDNA sequence was only deposited in Homarus americanus in GenBank so far. In the current study, our objective was to isolate, characterize, and express these three genes in M. nipponense with the aim of developing new genetic tools for breeding an all-male population. The fulllength cDNA sequences of these three genes from M. nipponense were cloned, and their structural characteristics were analyzed. A phylogenetic analysis was carried out in order to establish their orthology. Furthermore, the expression level of these genes in different adult tissues and developmental stages in embryo and larvae of M. nipponense was determined by real-time PCR. These results are the foundations for further research of the functions of Slow-tonic S2 tropomyosin and Slow tropomyosin isoform. 2. Methods and materials 2.1. Prawns and tissue preparation A total of 30 healthy adult male M. nipponense with wet weight of 3.78–5.26 g were obtained from Tai lake in Wuxi, China (120° 13′ 44″ E, 31° 28′ 22″ N). These specimens were transferred to a 500 L tank in lab condition and maintained in aerated freshwater at room temperature (26 °C) for 72 h prior to tissue collection. The androgenic glands were extracted under an Olympus SZX16 microscope. The ovaries, testes, muscles, hearts, brains, livers, androgenic glands, eyestalks, vasa deferentia and intestines were collected from mature prawns. The samples were treated with phosphate buffer saline (PBS), and immediately frozen in liquid nitrogen until used for RNA extraction, in order to prevent total RNA degradation. 2.2. Rapid amplification of cDNA ends (RACE) Total RNA was extracted from androgenic glands using RNAiso Plus Reagent (TaKaRa), following the protocol of the manufacturer. The isolated RNA was treated with RNase-free DNase I (Sangon, Shanghai, China), in order to eliminate possible genomic DNA contamination. The concentration of the total RNA sample was measured by BioPhotometer (Eppendorf), and 1% agarose gel was used to measure the RNA quality. The RACE technique was utilized to clone the full-length cDNA sequence of a gene, based on the known middle fragment. The first strand 3′ cDNA and 5′ cDNA synthesis for gene cloning was performed using a M-MLV reverse transcriptase by the 3′-full RACE core

set ver. 2.0 kit and the 5′-full RACE kit (Takara Bio Inc., Japan), respectively. Reaction conditions were recommended by the manufacturer. The synthesized cDNAs were kept at −20 °C, used for the 3′/5′-RACE PCR with 3′ gene-specific primer (3GSP1, 3GSP2) or 5′ GSP (5GSP1, 5GSP2) (Table 1). The 3′ GSP and 5′ GSP were designed on the basis of EST sequence of Slow-tonic S2 tropomyosin and Slow tropomyosin isoform from the M. nipponense androgenic gland transcriptome. The PCR products were purified using gel extraction kit (Sangon, Shanghai, China), following the manufacturer's instruction. Amplified cDNA fragments were transferred into the pMD18-T vector (Takara Bio Inc., Japan). Recombinant bacteria were identified by blue/white screening and confirmed by PCR. Nucleotide sequences of the cloned cDNAs were determined using an automated DNA sequencer (ABI Biosystem, USA). The nucleotide sequence similarities were examined by BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/). 2.3. Nucleotide sequence and bioinformatics analyses The 5′ and 3′ sequences from RACEs were assembled with the partial cDNA sequence corresponding to each fragmental sequence by DNAMAN 5.0. Sequences were analyzed based on the nucleotide and protein databases using the BLASTX and BLASTN search program (http://www.ncbi. nlm.nih.gov/BLAST/) of GenBank. The protein prediction was performed using the ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein function sites were analyzed by PredictProtein software (http:// www.Predictprotein.org/). Multiple sequence alignment was created using the ClustalW1.81. And the phylogenetic trees based on the amino acid sequences were performed by the neighbor-joining method using Molecular Evolutionary Genetics Analysis, MEGA4.0. All primers used in this experiment were designed using the primer designing tool (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/). 2.4. Embryo, larvae and various tissue expression by real-time quantitative PCR The relative mRNA expression of these three genes at different stages from embryo to larva, and various adult tissues was measured by real-time quantitative PCR (RT-qPCR). Total RNA was isolated from different stages of embryo and larvae, as well as various tissues of

Table 1 Universal and specific primers used in this study. Primer name

Nucleotide sequence (5′ → 3′)

Purpose

Sst-3GSP1 Sst-3GSP2 Sst-5GSP1 Sst-5GSP2 Sti-3GSP1 Sti-3GSP2

GGCTGAGAAGACTGAGGAGGAG AATACCAAGCTTGAGGAGAAAG GAGCGGTTCTCGAGTACCTTG CCTCTCGAGATCTTCCTCAAGC GCTGATCTTGAAAGAGCTGAGG TTGAGGTGTCTGAAGAGAAGG

Sti-5GSP1 Sti-5GSP2

GAGCGGTTCTCGAGTACCTTG CCTCTCGAGATCTTCCTCAAGC

3′ RACE OUT 3′ RACE IN

TACCGTCGTTCCACTAGTGATTT

FWD first primer for 3′ RACE FWD second primer for 3′ RACE RVS first primer for 5′ RACE RVS second primer for 5′ RACE FWD first primer for 3′ RACE FWD second primer for 3′ RACE RVS first primer for 5′ RACE RVS second primer for 5′ RACE RVS first primer for 3′ RACE

CGCGGATCCTCCACTAGTGATTTCA CTATAGG CATGGCTACATGCTGACAGCCTA

RVS second primer for 3′ RACE FWD first primer for 5′ RACE

Sst-RTF Sst-RTR Sti-RTF Sti-RTR β-ActinF

CGCGGATCCACAGCCTACTGATGA TCAGTCGATG GCAGAGAGATCTGTGCAGAAGC GAACCCTGACAGTTCAGAGAAC GCAGAGAGATCTGTGCAGAAGC GTAGCCAGACAGTTCGCTGAAT TATGCACTTCCTCATGCCATC

β-ActinR

AGGAGGCGGCAGTGGTCAT

FWD second primer for 5′ RACE FWD primer for Sst expression RVS primer for Sst expression FWD primer for Sti expression RVS primer for Sti expression FWD primer for β-actin expression RVS primer for β-actin expression

5′ RACE OUT 5′ RACE IN

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

S. Jin et al. / Gene xxx (2014) xxx–xxx

adult prawns, using RNAiso Plus Reagent (TaKaRa) and following the manufacturer's instructions. Experiments were performed in triplicates and at least three shrimps were analyzed for each type of tissue. Approximately 1 μg of total RNA from each tissue was used for firststrand cDNA synthesis using iscript™ cDNA Synthesis Kit Perfect Real Time (BIO-RAD). The cDNA of RT-qPCR was kept at −20 °C. The SYBR Green RT-qPCR assay was carried out in the Bio-Rad iCycler iQ5 RealTime PCR System. Amplification of β-actin as an internal reference was also carried out in the same sample (the primer's sequences are shown in Table 1). DEPC-water for the replacement of template was used as negative control. All samples were run in triploid (each duplicate for Slow-tonic S2 tropomyosin, Slow tropomyosin isoform and β-actin gene). The relative copy number of each gene was calculated according to the 2− ΔΔCT comparative CT method (Livak and Schmittgen, 2001). The RT-qPCR primers of each gene were designed based on the open reading frame (Figs. 1 and 2).

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region (UTR) of Mnsst contains 50 bp and 1021 bp, respectively. Both of the two clones included poly (A) tail. The nucleotide and deduced amino acid sequences of the full-length cDNAs are shown in Figs. 1 and 2, respectively. The cDNA sequences of Mnsst and Mnsti have been submitted to GenBank with accession nos. KF910722 and KF910723, respectively. 3.2. Bioinformatics analysis PredictProtein software from Colombia University was used for structure and protein functional site analysis. It showed that the secondary structure of both Mnsst and Mnsti contained a total of 98.94% helix and 1.06% loop because of the high similarity of amino acid. Additionally, the analysis showed that Mnsst and Mnsti contained a N-glycosylation site, protein kinase C phosphorylation site, casein kinase II phosphorylation site, leucine zipper pattern and tropomyosin signature. Mnsti protein has more casein kinase II phosphorylation site than Mnsst protein.

2.5. Statistical analysis 3.3. Similarity comparison and phylogenetic analysis Quantitative data were expressed as mean ± SD. Statistical differences were estimated by one-way ANOVA followed by LSD and Duncan's multiple range test. All statistics were measured using SPSS Statistics 13.0. A probability level of 0.05 was used to indicate significance (P b 0.05). 3. Results 3.1. Sequence analysis of Mnsst and Mnsti The full length of Mnsst and Mnsti cDNA sequence was 977 bp and 1926 bp, respectively, and both of these genes included a 852 bp open reading frame (ORF) encoding a 284 amino acid protein. Mnsst displayed an estimated molecular mass of 32.698 kDa and a theoretical pI of 4.40. The 5′ and 3′ untranslated region (UTR) of Mnsst contains 50 bp and 72 bp, respectively. Mnsti displays an estimated molecular mass of 32.713 kDa and a theoretical pI of 4.40. The 5′ and 3′ untranslated

The comparison of Mnsst and Mnsti amino acid sequences with the sequences of previously reported different types of tropomyosin showed that Mnsst and Mnsti protein shared the highest identity to Slow-tonic S2 tropomyosin (AAS98885.1) and Slow tropomyosin isoform (O44119.1) of H. americanus. Because of the extraordinary similarity between Mnsst and Mnsti, both of their proteins share high resemblance to Erimacrus isenbeckii (BAF47269.1), Paralithodes camtschaticus (BAF47265.1), Eriocheir sinensis (ABO71783.1), Oratosquilla oratoria (BAF95206.1) and M. rosenbergii (ADC55380.1). The amino acid sequence of Mnsst and Mnsti showed more than 90% similarity with above species. Neighbor-joining method was used to construct a condensed phylogenetic tree using the complete different types of tropomyosin proteins deposited in NCBI by MEGA 5.0, in order to study the relationship between Mnsst and Mnsti with other well-defined arthropod tropomyosin. The NJ tree placed the sequences for Mnsst and Mnsti in a branching

Fig. 1. Nucleotide and deduced amino acid sequence of Mnsst. The nucleotide sequence is displayed in the 5′–3′ directions and numbered at the left. The deduced amino acid sequence is shown in a single capital letter amino acid code. 3′ UTR and 5′ UTR are shown with lowercase letters. Codons are numbered at the left with the methionine (ATG) initiation codon, an asterisk denotes the termination codon (TGA). RACE and real-time qPCR primers are marked with arrows.

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

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S. Jin et al. / Gene xxx (2014) xxx–xxx

Fig. 2. Nucleotide and deduced amino acid sequence of Mnsti. The nucleotide sequence is displayed in the 5′–3′ directions and numbered at the left. The deduced amino acid sequence is shown in a single capital letter amino acid code. 3′ UTR and 5′ UTR are shown with lowercase letters. Codons are numbered at the left with the methionine (ATG) initiation codon, an asterisk denotes the termination codon (TGA). RACE and real-time qPCR primers are marked with arrows.

order that reflected the phylogenetic relationship of the respective species. According to the NJ tree, Mnsst and Mnsti have the closest relationship with the Slow type of tropomyosin in known species, while the relationship with other type of tropomyosin in the other species was separated (Figs. 3, 4).

3.4. Tissue distribution of Mnsst and Mnsti mRNA Tissue distribution analysis might reflect physiological function of a protein. The expression levels of Mnsst and Mnsti were significantly expressed in androgenic gland, which are dramatically higher than the expression in other tissues based on RT-qPCR analysis. Mnsst has the lowest expression level in ovary such that there was 8286.50 fold greater expression of Mnsst in the androgenic gland than in the ovary. The gene expression level in the testis and vasa deferentia was 62.45 and 619.60 fold greater than that in the ovary, respectively (Fig. 5). According to the statistical analysis, the expression of Mnsst in androgenic gland was significantly different from those of other tissues, whereas there is no expression difference between the tissues except the androgenic gland. Mnsti had the lowest expression level in the testis. The androgenic gland had a 463.95 fold greater expression of Mnsti than that of the testis. The expression level of Mnsti in the ovary and vasa deferentia had a 2.07 and 5.19 fold greater expression than that in the testis, respectively (Fig. 5). According to the statistical analysis, the expressions of Mnsti in androgenic gland and vasa deferentia were significantly different from

those of other tissues, whereas there is no expression difference between the tissues except the androgenic gland and vasa deferentia. 3.5. Expression analysis of Mnsst and Mnsti mRNA during the embryo and larvae The temporal expressions of Mnsst and Mnsti during embryonic and larval development were analyzed by RT-qPCR. Mnsst and Mnsti have the same expression pattern during the developmental stage. During embryonic development, both of Mnsst and Mnsti had the lowest expression level at cleavage stage (CS). The expression level gradually increased to nauplius stage (NS) and then abruptly increased and peaked at zoea stage (ZS). During larval development L5, neither Mnsst nor Mnsti was expressed. However, expression of these two genes increased dramatically during L10 (233.01 folds and 293.31 folds, respectively). Finally, both of these two genes decreased during L14 (Fig. 6). According to the statistical analysis, the expression of Mnsst in ZS was significantly different from those of other developmental stages, whereas the expressions of Mnsti in ZS and L10 were significantly different from those of other developmental stages. 4. Discussion Three candidate novel genes were detected from androgenic gland of M. nipponense, including Slow-tonic S2 tropomyosin and Slow tropomyosin isoform. In the present study, we have cloned and identified these three genes which were named Mnsst and Mnsti in M. nipponense.

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

S. Jin et al. / Gene xxx (2014) xxx–xxx 61

62

5

Penaeus monodon Fenneropenaeus merguiensis Sinonovacula constricta

86 96 42

Tyrophagus putrescentiae

Metapenaeus ensis Macrobrachium rosenbergii

22 91

Crangon crangon Portunus pelagicus

68

Homarus americanus fast

54 54

Procambarus clarkii Oratosquilla oratoria 99

Squilla oratoria Melicertus latisulcatus Erimacrus isenbeckii

49

Eriocheir sinensis Macrobrachium nipponense slow

100

Homarus americanus slow

38 51

Paralithodes camtschaticus

2

Fig. 3. The phylogenetic tree of sst from different organisms based on amino acid sequence comparisons. Species names and types of sst are listed on the right of the tree. Their accession numbers in GenBank are as follows: Macrobrachium nipponense (in this study), Homarus americanus slow-tonic S2 tropomyosin (AAS98885.1), Erimacrus isenbeckii (BAF47269.1), Paralithodes camtschaticus (BAF47265.1), Penaeus monodon (ADM34184.1), Tyrophagus putrescentiae (ABQ96644.1), Eriocheir sinensis (ABO71783.1), Portunus pelagicus (AGE44125.1), Homarus americanus fast tropomyosin isoform (AAC48288.1), Squilla oratoria (ABQ57495.1), Melicertus latisulcatus (AGF86397.1), Procambarus clarkii (ACN87223.1), Fenneropenaeus merguiensis (ADC55381.4), Metapenaeus ensis (Q25456.1), Sinonovacula constricta (ABL14250.1), Oratosquilla oratoria (BAF95206.1), Macrobrachium rosenbergii (ADC55380.1) and Crangon crangon (ACR43473.1).

Slow-tonic S2 tropomyosin and Slow tropomyosin isoform belongs to tropomyosin family, which controls the function of actin filaments in both muscle and non-muscle cells. Mammals possess more than 40 different tropomyosin isoforms (Gunning et al., 2005). Many other types of tropomyosin had been identified from crustacean (Heeley et al., 1995; Donald et al., 1998; Karl and Dirk, 1990; Liang et al., 2008; Motoyama et al., 2007), whereas Slow-tonic S2 tropomyosin and Slow tropomyosin isoform was only researched in H. americanus in NCBI (Medler et al., 2004). The full length of Mnsst and Mnsti cDNA sequence was 977 bp and 1926 bp, respectively, and both of these genes included an 852 bp open reading frame (ORF) encoding a 284 amino acid protein. According to the nucleotide sequence analysis, Mnsst and Mnsti shared 95.82% of similarity in open reading frame. The main differences in open reading frame between Mnsst and Mnsti were from 824 bp to 905 bp, and Mnsst lacks 949 bp at the 3′-termination. Both of Mnsst and Mnsti contained 284 deduced amino acid sequences and the difference between the amino acid sequences was mainly at C-termination from 269 bp to 284 bp, which is the same as that in H. americanus. Moreover, the protein functional site analysis revealed that Mnsst and Mnsti proteins had some differences in casein kinase II phosphorylation site, which might have contributed to the differences in signal transduction and regulation of activities between Mnsst and Mnsti.

BLASTP similarity comparisons showed that the Mnsst and Mnsti protein shared the highest identity to Slow-tonic S2 tropomyosin and Slow tropomyosin isoform of H. americanus, respectively, and the identity reaches to 98%. The identities to other known species were also higher than 90%, implying that tropomyosin family in different species was very conserved. The prominent similarities between the Mnsst and Mnsti sequence with Slow-tonic S2 tropomyosin and Slow tropomyosin isoform in H. americanus suggest that the sequences obtained in this study were the Slow-tonic S2 tropomyosin and Slow tropomyosin isoform in M. nipponense. The NJ tree showed that Mnsst and Mnsti had the closest relationship with Slow-tonic isoform tropomyosin, including H. americanus, Erimacrus isenbeckii, Eriocheir sinensis and Paralithodes camtschaticus, while the relationship with other types of tropomyosin in other species was separated, which was consistent with the BLASTP. The exact orthologous relation for Mnsst and Mnsti needs further study when more slow types of tropomyosin are cloned from crustacean. According to the previous studies, Slow-tonic S2 tropomyosin was only detected in single fibers from the superficial flexor and extensor muscles combined with S1 and S2 myosin heavy chain (MHC) (Donald et al., 1998). The expression pattern of Slow tropomyosin isoform was only detected in developing human quadriceps muscle.

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

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S. Jin et al. / Gene xxx (2014) xxx–xxx

61 Penaeus monodon 62

Fenneropenaeus merguiensis Sinonovacula constricta

87 96

Tyrophagus putrescentiae

56

Metapenaeus ensis 38

Macrobrachium rosenbergii Portunus pelagicus

70

Homarus americanus fast

57 52

Procambarus clarkii

100

Oratosquilla oratoria 99

Squilla oratoria Melicertus latisulcatus

Macrobrachium nipponense Homarus americanus slow 66 74

Eriocheir sinensis Portunus sanguinolentus

93

Paralithodes camtschaticus 46

Erimacrus isenbeckii

2 Fig. 4. The phylogenetic tree of sti from different organisms based on amino acid sequence comparisons. Species names and types of sti are listed on the right of the tree. Their accession numbers in GenBank are as follows: Macrobrachium nipponense (in this study), Homarus americanus Slow tropomyosin isoform (O44119.1), Homarus americanus fast tropomyosin isoform (AAC48288.1), Penaeus monodon (ADM34184.1), Erimacrus isenbeckii (BAF47269.1), Fenneropenaeus merguiensis (ADC55381.4), Paralithodes camtschaticus (BAF47265.1), Squilla oratoria (ABQ57495.1), Melicertus latisulcatus (AGF86397.1), Procambarus clarkii (ACN87223.1), Portunus pelagicus (AGE44125.1), Eriocheir sinensis (ABO71783.1), Oratosquilla oratoria (BAF95206.1), Metapenaeus ensis (Q25456.1), Sinonovacula constricta (ABL14250.1), Tyrophagus putrescentiae (ABQ96644.1), Macrobrachium rosenbergii (ADC55380.1) and Portunus sanguinolentus (ABL89183.1).

Fig. 5. Expression characterization of Mnsst and Mnsti in the various adult tissues was revealed by real-time quantitative PCR. The amount of Mnsst mRNA was normalized to the β-actin transcript level. Data are shown as means ± SD (standard deviation) of three separate individuals in the tissues. Capital letters indicate expression difference of Mnsst in different adult tissues; lowercases indicate expression difference of Mnsti in different adult tissues. T—testis; O—ovary; B—brain; AG—androgenic gland; H—heart; I—intestine; M—muscle, L—liver, ES—eyestalk, VD—vasa deferentia.

Fig. 6. Expression characterization of Mnsst and Mnsti during the embryos and larvae before the metamorphosis was revealed by real-time PCR. The amount of Mnsst mRNAs was normalized to the β-actin transcript level. Data are shown as means ± SD of three replicates during the embryo and larvae. Capital letters indicate expression difference of Mnsst in different development stages; lowercases indicate expression difference of Mnsti in different development stages. CS—cleavage stage; BS—blastula stage; GS—gastrul stage; NS—nauplius stage; PS—protozoea stage; ZS—zoea stage. L1—the first day larva after hatching, and so on.

Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

S. Jin et al. / Gene xxx (2014) xxx–xxx

Slow tropomyosin isoform is not detected at 6 weeks of gestation. This gene then transiently appears between 12.5 weeks and 15 weeks of gestation and then disappears. Finally, slow tropomyosin isoform reexpresses itself at 36 weeks of gestation, and remains expressed into adulthood (Heeley et al., 1995). However, no previous reports were focused on the expression pattern of these genes in various tissues. In the present study, Mnsst and Mnsti genes were expressed in all detected tissues, indicating that these two genes might have wide biological functions in M. nipponense. However, they still showed certain distribution differences which may reflect different biological functions. According to the RT-qPCR analysis, the expression levels of Mnsst and Mnsti were significantly higher in androgenic glands than those in other tissues. The expression levels of Mnsst and Mnsti in sex glands, including the ovary, testis and vasa deferentia, were dramatically low. Thus, the RTqPCR analysis implies that these two genes may not be involved in the reproductive mechanism. Mnsst may be related to sex-determination mechanism or the formation of androgenic gland and maintain the normal function of androgenic gland. Although the expression level of Mnsti was dramatically higher in the muscle, there are no muscle cells in androgenic gland, implying that Mnsti may have similar role with Mnsst in M. nipponense. The actual functions of Mnsst and Mnsti need further study. Thus, Mnsst and Mnsti may dramatically influence in the breeding of all-male population of M. nipponense. Male M. rosenbergii undergoes sex reversal to “neofemales” through inhibition of the expression of a single insulin-like androgenic gland gene by RNAi technique. The “neofemales” can produce all-male progeny. The all-male population of M. rosenbergii was obtained when “neofemales” mated with male M. rosenbergii (Tomer et al., 2009). In this study, we provided another two candidate genes that may affect the formation of androgenic gland to form “neofemales”. This was done using RNAi technique to inhibit the expression of these two genes. To the best of our knowledge, no report was found to be focused on the research of expression level of Mnsst and Mnsti during the developmental stages in any species. We are the first to perform any research on this subject. Both of Mnsst and Mnsti are expressed at a low level during the early stages of embryonic development. Their expression level increased abruptly and reached the peak at zoea stage (ZS). Embryonic organogenesis of M. nipponense started from NS stage (Zhang et al., 2010). (ZS) has been confirmed to play an important role in the early stage of larval development, such as the complete body formation and many organ formation, especially for digestive organ (Chen et al., 2012). The expression of Mnsst and Mnsti during the embryonic development in this study suggests that Mnsst and Mnsti may play important roles in embryonic organogenesis of M. nipponense, especially for the formation of complete body and digestive organ. From hatching to metamorphosis for larvae of M. nipponense, it undergoes the molting process many times to accompany its morphological changes (Zhang et al., 2013a,b,c). Both of Mnsst and Mnsti were expressed at the late stage of larval development. During the larvae stage L5, Mnsst and Mnsti were not expressed. However, expression significantly increased and reached the peak at L10. Then it decreased slightly at metamorphic climax in M. nipponense. We speculated that the expression pattern of Mnsst and Mnsti during larval development may be necessary to stimulate larval changes of morphology and initiate metamorphosis. Insulin-like androgenic gland specific factor is believed to be similar to that of the isopod AG hormone, which was the first to be structurally elucidated and belongs to the insulin superfamily of proteins, considered as key regulator of male sex-determination (Sook et al., 2011). Mnsst and Mnsti have similar expression pattern with IAG in mature prawn tissues and embryonic development (Ma et al., 2013), implying that Mnsst and Mnsti may have similar function with IAG. This finding suggests that Mnsst and Mnsti are two novel genes in sex-determination mechanism, advancing the sexdetermination mechanism analysis in M. nipponense.

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5. Conclusion In this study, we have cloned and characterized three novel genes from androgenic gland of M. nipponense, including Slow-tonic S2 tropomyosin and Slow tropomyosin isoform. Mnsst and Mnsti mRNAs were dramatically expressed in the androgenic gland, implying that Mnsst and Mnsti may be related to sex-determination mechanism or the formation of androgenic gland and maintenance of normal androgenic gland functions. Based on the RT-qPCR results of Mnsst and Mnsti in embryonic and larval development, we speculated that Mnsst and Mnsti may be related to embryonic organogenesis of M. nipponense, especially for the formation of complete body and digestive organ, and the stimulation of larval changes of morphology and initiate metamorphosis. Mnsst and Mnsti have similar expression pattern with IAG in different mature tissues and developmental stages, suggesting that Mnsst and Mnsti are two novel genes related to sex-determination mechanism. In this study, we confirmed two novel genes related to sex-determination in M. nipponense, advanced our understanding of the multiple biological functions of the Mnsst and Mnsti genes, and laid the foundation for further research on the regulation of sex determination pathway in M. nipponense. Further detailed investigations should be carried out to clarify the function of Mnsst and Mnsti in our future work. Conflict of interest There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the gene policies on sharing data and materials. Acknowledgments The project was supported by the National Natural Science Foundation of China (Grant No. 31272654), the Freshwater Fisheries Research Center, China Central Governmental Research Institutional Basic Special Research Project from the Public Welfare Fund (2013JBFT07), the Three Aquatic Projects of Jiangsu Province (D2013-6), the National Science & Technology Supporting Program of the 12th Five-year Plan of China (Grant No. 2012BAD26B04), the Jiangsu Provincial Natural Science Foundation for Young Scholars of China (Grant No. BK2012091) and the Science & Technology Supporting Program of Jiangsu Province (Grant No. BE2012334). References Bureau of Fishery, Ministry of Agriculture, P.R.C., 2009. Fisheries economic statistics. China Fishery Yearbook. Beijing China Agricultural Press, p. 236. Cai, Y., Shokita, S., 2006. Report on a collection of freshwater shrimps (Crustacea: Decapoda: Caridea) from the Philippines, with descriptions of four new species. Raffles Bulletin of Zoology 54, 245–270. Chen, Y., Zhu, Q., Chen, H., Zhu, X., Cui, Z., Qiu, G., 2012. The morphological and histological observation of embryonic development in the oriental river prawn Macrobrachium nipponense. Journal of Shanghai Ocean University 21 (1), 33–40. De Grave, S., Ghane, A., 2006. The establishment of the oriental river prawn, Macrobrachium nipponense (de Haan, 1849) in Anzali Lagoon, Iran. Aquatic Invasions 1, 204–208. Donald, L., Mykle, S., Juliel, S.C., Taniguchi, Hisaaki, Sano, Ken-Ichi, Maeda, Yuichiro, 1998. Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript. Journal of Muscle Research and Cell Motility 19, 105–115. Feng, J.B., Li, J.L., Cheng, X., 2008. Research progress on germplasm resource exploitation and protection of Macrobrachium nipponense. Journal of Shanghai Fisheries University 17, 371–376. Gunning, P.W., Schevzov, G., Kee, A.J., Hardeman, E.C., 2005. Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends in Cell Biology 15 (6), 333–341. Heeley, David H., Bieger, T., Waddleton, Deena M., Hong, C., Jackman, Donna M., et al., 1995. Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish. European Journal of Biochemistry 232, 226–234. Jin, S., Fu, H., Zhou, Q., Sun, S., Jiang, S., et al., 2013. Transcriptome analysis of androgenic gland for discovery of novel genes from the oriental river prawn, Macrobrachium nipponense, using Illumina Hiseq 2000. PLoS One 8 (10), e76840. http://dx.doi.org/ 10.1371/journal.pone.0076840.

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Please cite this article as: Jin, S., et al., Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.014

Molecular cloning of two tropomyosin family genes and expression analysis during development in oriental river prawn, Macrobrachium nipponense.

This paper reports that Slow-tonic S2 tropomyosin (Sst) and Slow tropomyosin isoform (Sti) was highly expressed in androgenic gland transcriptome of M...
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