Elsevier

Gene

Volume 562, Issue 1, 10 May 2015, Pages 22-31
Gene

Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation

https://doi.org/10.1016/j.gene.2014.12.008Get rights and content

Highlights

  • Mn-Vg was cloned and characterized.

  • The expression in both male and female tissues was detected by real-time PCR.

  • The expression patterns in different embryo and ovary stages were investigated.

  • Eyestalk ablation was carried out to illustrate the hormonal regulation of Vg.

  • RNAi was employed to demonstrate the delay role of Vg in ovary cycle of M. nipponense.

Abstract

Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis.

Introduction

The oriental river prawn Macrobrachium nipponense (Crustacea; Decapoda; Palaemonidae) is an important commercial prawn species that is widely distributed in freshwater areas of China and other Asian countries. The total fishing production reaches 230,248 t per year in China (Bureau of Fishery, 2011), with an annual production value of more than 100 million RMB. As the scale of production expanded, “sexual precocity” began to appear. This term refers to early male and female gonad development, which leads to excessive propagation and overpopulation; in the M. nipponense population this occurs especially in autumn. Precocity results in the coexistence of multiple generations, intensive breeding density, and lack of oxygen, which can lead to short life span and low market value of the product. Thus, this phenomenon is restricting the sustainable development of M. nipponense. Understanding the reproductive process and the mechanisms that regulate ovarian maturation in M. nipponense is crucial to improving production of this commercially important species.

Vitellogenin (Vg), which is the precursor of vitellin (Vn), is synthesized by female shrimp during gonad maturation. In the mature female prawn, gonad maturity depends on the rapid synthesis and accumulation of Vg in the oocytes during the breeding season (Wilder et al., 2010). In many oviparous vertebrate and invertebrate animals, Vn provides the substrate and energy for embryonic and ovarian development (e.g., carbohydrates, amino acids, lipids, vitamins, phosphorus, sulfur, and trace elements) (Matozzo et al., 2008). The complete cDNA sequence encoding Vg has been cloned for many species of decapod crustacean. In addition, the molecular characteristics and the regulatory mechanism of Vg have been widely studied (Tsutsui et al., 2000, Yang et al., 2000, Raviv et al., 2006, Okumura et al., 2007, Jia et al., 2013). However, molecular studies of M. nipponense Vg (Mn-Vg) are needed to better understand the mechanisms involved in the reproductive process of this species.

In crustaceans, the site and the process of Vg synthesis are still controversial. Mainly have two kinds: extra-ovarian sources, namely by the organ beyond ovary synthesis precursor, Vg was considered to be taken into the developing oocytes from the hemolymph by the vitellogenin receptor (VgR) via receptor-mediated endocytosis. The mechanisms for endocytotic internalization of Vg have been well studied in certain oviparous vertebrates (Schneider, 1992) and insects (Sappington and Raikhel, 1998), but such studies in crustaceans are limited. Vg synthesis also may be endogenous (i.e., auto-synthesis), whereby the oocyte itself produces Vg with participation from relevant organelles. For example, the ovary was found to be the site of Vg synthesis in Penaeus semisulcatus (Browdy et al., 1990) and Callinectes sapidus (Lee and Watson, 1995). However, the Vg gene was uniquely expressed in the hepatopancreas of Macrobrachium rosenbergii (Yang et al., 2000) and Pandalus hypsinotus (Tsutsui et al., 2004). Further studies indicated that both the ovary and hepatopancreas were the Vg synthesis sites in Marsupenaeus japonicus (Okumura et al., 2007), Fenneropenaeus merguiensis (Phiriyangkul et al., 2007), Litopenaeus vannamei (Raviv et al., 2006), Penaeus japonicus (Tsutsui et al., 2000), Metapenaeus ensis and Penaeus monodon (Tiu et al., 2006a, Tiu et al., 2006b).

In decapods, vitellogenesis is hormonally regulated, and it can be inhibited by the occurrence of hormones in the neurosecretory cells of the X-organ/sinus gland. For example, gonad-inhibiting hormone (GIH), which is synthesized in the X-organ/sinus complex, is thought to play an inhibitory role for the initiation of vitellogenesis in the ovary (Kleijn et al., 1994, De Kleijn et al., 1998, Gu et al., 2002). Adiyodi and Adiyodi (1970) reported that eyestalk ablation removed the inhibition of neuropeptides and accelerated the accumulation of Vg. In addition, Jayasankar et al. (2002) found that eyestalk ablation increased Vg synthesis in the hepatopancreas of the giant freshwater prawn M. rosenbergii. In P. japonicus, Vg mRNA transcripts were measured both in the hepatopancreas and the ovary in normal and eyestalk-ablated adult shrimp. An obvious increment of mRNA levels was revealed in the ovary, whereas mRNA levels were negligible in the hepatopancreas (Tsutsui et al., 2005). Overall, existing data indicate that the mechanisms for hormonal regulation of Vg synthesis vary among crustaceans. Thus, molecular characterization and functional studies of Vg are critical to understand the reproductive mechanisms in M. nipponense. Such information can be used to improve aquaculture production in practice.

In this study, we cloned the cDNA encoding the Vg gene from M. nipponense (Mn-Vg) and conducted structural and phylogenetic analyses. The expression profiles of different tissues and development stages (embryo and larvae) were determined using quantitative real-time PCR (qPCR). qPCR was also used to evaluate the effects of eyestalk ablation to gain a better understanding of the hormonal regulation mechanism involved in Vg synthesis. RNAi technology was firstly applied to investigate the expression pattern of Vg in ovary cycles. The results of this study should be helpful for developing methods to cope with the problem of sexual precocity in the aquaculture setting.

Section snippets

Experiment animal

Adult healthy M. nipponense were obtained from Tai lake in Wuxi, China (120°13′44″E, 31°28′22″N). The body weight of the female/male prawns ranged from 1.26 to 4.25 g. Individuals, feed with paludina twice per day, were acclimatized in a recirculating water aquarium system filled with aerated freshwater (25–28 °C) before tissues and embryos were collected. A variety of tissues including: ovary, heart, hepatopancreas, muscle, hemocytes, gill, eyestalk, gut and brain were dissected out from mature

Molecular cloning and structural analysis of the Mn-Vg gene

The full-length Mn-Vg gene is 7804 base pairs (bps) long and includes a 5′-terminal untranslated region (UTR) of 34 bp, a 7611 bp open reading frame (ORF) encoding 2536 amino acid (aa) residues, and a 159 bp 3′-terminal UTR (excluding the poly(A) + tail). The Mn-Vg cDNA sequence was submitted to GenBank under the accession number KJ768657. Fig. 1 shows the sketch map of the deduced amino acid sequences, and the specific sequence information for Mn-Vg is provided in Fig. 1s. Mn-Vg has an estimated

Discussion

In this study, we identified the complete Mn-Vg transcript sequence, which is approximately 8 kb in size with 2536 aas encoded by its ORF. The deduced amino acid sequences revealed the common characteristic sequence of insect Vg (Chen et al., 1997, Sappington and Raikhel, 1998, Sappington et al., 2002), with considerable conservation, particularly in the N-terminus. The vitellogenin N-terminal and VYWD domains are widely found in insects and vertebrates (Baker, 1988). The GLLG motif within the

Acknowledgments

The project was supported by the Freshwater Fisheries Research Center, China Central Governmental Research Institutional Basic Special Research Project from the Public Welfare Fund (2013JBFM15), the National Natural Science Foundation of China (Grant No. 31272654), the National Science & Technology Supporting Program of the 12th Five-year Plan of China (Grant No. 2012BAD26B04), Jiangsu Provincial Natural Science Foundation for Young Scholars of China (Grant No. BK2012091), the Science &

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