Molecular cloning, characterization, and temporal expression of the clock genes period and timeless in the oriental river prawn Macrobrachium nipponense during female reproductive development

Abstract

The circadian clock is crucial for sustaining rhythmic biochemical, physiological, and behavioral processes in living creatures. In this study, we isolated and characterized two circadian clock genes in Macrobrachium nipponense, period (Mnper) and timeless (Mntim). The complete Mnper cDNA measures 4283 bp in length with an open reading frame encoding 1292 amino acids, including functional domains such as PER-ARNT-SIM (PAS), cytoplasmic localization domain (CLD), TIM interaction site (TIS), and nuclear localization signal (NLS). The deduced Mntim protein comprises1540 amino acids with functional domains such as PER interaction site (PIS), NLS, and CLD. Tissue distribution analyses showed that the two genes were highly expressed in the eyestalk and brain in both males and females, as well as being expressed in the ovary. The expression profiles of Mnper and Mntim were determined in the eyestalk, brain, and ovary under simulated breeding season and non-breeding season conditions. The expression profiles of both Mnper and Mntim appeared to be unaffected in the eyestalk. However, the expression of both genes exhibited significant seasonal variations in the brain, and thus we assumed the brain to be their functional location. The expression profiles under different simulated seasons and the variations during different ovarian stages indicate that both genes might be involved with female reproduction. Especially the mRNA levels in the brain varied greatly during these stages indicating that the clock function in the brain is closely related to ovarian development and female reproduction. And the reproductive roles of clock genes need to be elucidated.

Introduction

In various organisms, physiological and behavioral events are regulated by circadian rhythms controlled by endogenous clocks (Ikeno et al., 2011, Danbara et al., 2010, Chahad-Ehlers et al., 2012). Circadian rhythms are remarkably well conserved in insects and mammals, where they are maintained by autoregulatory feedback loops involving a set of so-called clock genes. In recent years, many clock genes have been cloned and characterized in mammals and insects, such as clock (clk), period (per), timeless (tim), cycle (cyc), cryptochrome (cry), vrille (vri), and Bmal1 (Abruzzi et al., 2011, Outa et al., 2013, Pozo et al., 2012, Crosthwaite and Heintzen, 2010, Gu et al., 2014). Expression analyses have shown that these clock genes are rhythmically expressed in the classical pacemaker structures (the hypothalamic suprachiasmatic nucleus in mammals, and the retina and pineal gland in non-mammalian vertebrates), but also in various peripheral tissues, including the liver, muscle, kidney, and heart (Martín-Robles et al., 2011, Mazzoccoli et al., 2012, Akhtar et al., 2002, Durgan et al., 2005, Yoo et al., 2004). The circadian rhythms of various biological phenomena have been described in several crustacean species, but few investigations have considered the regulation of neuronal elements identified in the endogenous circadian clocks (Strauss and Dircksen, 2010).

Reproductive physiology is influenced profoundly by circadian rhythms (Boden et al., 2013, Casper and Gladanac, 2014, Amaral et al., 2014, Shimizu et al., 2012). Clock gene expression has been observed in tissues in the hypothalamic–pituitary–gonadal axis. In mammals, the circadian clock has effects on the serum concentrations of many reproductive hormones (Sellix and Menaker, 2008, Nakamura et al., 2010, Ratajczak et al., 2012). In invertebrates, clock gene expression has been observed in the ovaries of the silkworm (Sakamoto and Shimizu, 1994), prawn (Yang et al., 2006), desert locust (Tobback et al., 2011), and fruit fly (Beaver et al., 2003, Liu and Zhao, 2014). However, circadian rhythms and/or the functional consequences of clock gene expression have only been described in the fruit fly. Mutant flies lacking functional period or timeless gene expression produce fewer mature oocytes and progeny after mating with a wild-type male (Hardin, 2005, Beaver et al., 2003). More evidence is required regarding the importance of the circadian clock in crustacean ovaries.

The oriental river prawn Macrobrachium nipponense is an important commercial species in China with an annual production value of nearly 200 million RMB (Bureau of Fishery et al., 2014). M. nipponense exhibits circadian changes during the ovary maturation cycle. The photoperiod and temperature play critical roles in controlling the entry of the ovaries into a period of rapid development with a short maturation cycle during the breeding season. However, the molecular mechanisms that connect external factors and gonad development are still unknown. In our previous study (Qiao et al., 2015), we found two homologous clock genes (period and timeless) based on gene expression profile analyses of the testis and ovary. In the present study, we isolated and characterized the period and timeless genes from M. nipponense, and investigated the tissue-specific distributions of these two genes in male and female individuals. We examined the effects of the photoperiod and temperature on female reproduction and clock genes by simulating the breeding and non-breeding seasons (light: dark (LD) 14:10 at 25 °C and LD 10:14 at 15 °C, respectively). The expression patterns of the two genes were investigated during different ovary stages to determine the regulatory roles of clock genes in ovary development. The results of this study should help to understand the relationships between the Mnper and Mntim genes and female reproduction in crustaceans.