Potential role of Methoprene-tolerant (Met) in methyl farnesoate-mediated vitellogenesis in the Chinese mitten crab (Eriocheir sinensis)

https://doi.org/10.1016/j.cbpb.2020.110524Get rights and content

Highlights

  • A novel Met gene was isolated and characterized from Eriocheir sinensis.

  • Effect of MF on EsMet and EsVg expression in vitro and vivo were analyzed.

  • A positive correlation of EsMet and EsVg expression levels with hemolymph MF titer.

  • EsMet possibly act as a MF receptor in MF-mediated vitellogenesis.

Abstract

Methoprene-tolerant (Met) belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of nuclear transcriptional regulators and is a leading candidate receptor for juvenile hormone (JH III) in insects. Methyl farnesoate (MF) is a de-epoxide form of JH III that regulates many developmental processes in crustaceans, including reproduction, molting, and morphogenesis, much like JH III in insects. In this study, the full-length cDNA for Met was cloned from the Chinese mitten crab (Eriocheir sinensis) (EsMet). The amino acid sequence of EsMet contains three conserved domains (bHLH, PAS-A, and PASsingle bondB) characteristic of the bHLH-PAS family, having six conserved amino acid residues specifically responsible for JH or MF binding. Tissue distribution analysis revealed that EsMet mRNA is highly expressed in the hepatopancreas. In addition, EsMet and EsVg expression in the hepatopancreas were found to be significantly increased in early endogenous vitellogenic oocytes (stage II) during ovarian development, and the hemolymph MF titer was significantly increased in late exogenous vitellogenic oocytes (stage III), indicating that EsMet is involved in vitellogenesis regulation. In vitro, MF addition markedly upregulated EsMet and EsVg expression in hepatopancreatic tissue, but only EsVg was induced in ovarian tissue. In vivo, EsMet and EsVg expression in the hepatopancreas were both significantly and synchronously increased after MF injection, but not in the ovaries. In addition, EsMet and EsVg expression were upregulated in the hepatopancreas after eyestalk ablation, while only EsVg expression was induced in the ovaries. Thus, our results indicate that Met may act as a receptor for MF in MF-mediated vitellogenesis in crustaceans.

Introduction

The Chinese mitten crab (Eriocheir sinensis) is of significant economic importance in the Chinese freshwater aquaculture industry (Wang et al. 2018). In recent years, precocious puberty has become recognized as a serious problem in crab aquaculture in that lead to animals with low growth rates, poor survival, short life spans, and thus low commercial value (Li et al. 2011a). Accordingly, increasing research attention is being dedicated to the endocrine factors regulating gonadal development in crabs (Li et al. 2011b; Wang et al. 2016).

For most decapods, female gonad maturation is characterized by maturation of the ovary, with gradual growth resulting from the uptake of the yolk protein precursor, vitellogenin (Vg) (Tiu et al. 2009). In crustaceans, Vg is mainly synthesized in the hepatopancreas and ovaries under the control of various endocrine factors (Li et al. 2006; Subramoniam, 2000, Subramoniam, 2010). One such endocrine factor is methyl farnesoate (MF), a sesquiterpenoid hormone that is secreted from crustacean mandibular organs (MOs) (Purna and Nagaraju 2007). Briefly, MF is a de-epoxidated form of insect juvenile hormone III (JH III) and, like JH III, it regulates numerous developmental processes (Miyakawa et al. 2014), including reproduction (Qu et al. 2018; Reddy et al. 2004; Volkova and Zadereev 2012), molting (Gismondi and Joaquim-Justo 2019), and morphogenesis (Kamimura 2004; Laufer et al. 2005; Purna and Nagaraju 2007).

Abundant evidence indicates that MF can stimulate ovarian maturation and vitellogenesis in crustaceans, much like JH III does in insects. For instance, MF addition stimulates ovarian maturation in Litopenaeus vannamei (Alnawafleh et al. 2014), Oziotelphusa senex senex (Girish et al. 2018; Reddy et al. 2004), and Procambarus clarkia (Laufer et al. 1998). Furthermore, the mean oocyte diameter in the prawn Macrobrachium malcolmsonii is significantly increased after MF addition (Nagaraju et al. 2003). MF treatment also stimulates vitellogenin synthesis in Cherax quadricarinatus (Medesani et al. 2012), Oziotelphusa senex senex (Girish et al. 2018), and Portunus trituberculatus (Liu et al., 2016a, Liu et al., 2016b). In addition, eyestalk ablation treatment (ESA) increases the hemolymph concentration of MF in Homarus americanus (Tsukimura and Borst 1992) and Litopenaeus vannamei (Alnawafleh et al. 2014), while bilateral ablation has been demonstrated to induce vitellogenesis in Sicyonia ingentis (Paran et al. 2010). However, although the role of MF in crustacean gonad development is clearly important, the molecular mechanism of its signaling action is much less studies than that of JH in insects.

Methoprene-tolerant (Met) is an intracellular receptor for JH III in insects, and the role of Met in JH-mediated insect ovarian development is well established (Liu et al., 2016a, Liu et al., 2016b; Villalobos-Sambucaro et al. 2015; Zhu et al. 2010). Met belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of transcriptional regulators and has three independent typical conserved domains (Jindra et al. 2013; Li et al. 2011a). When JH III binds to the C-terminal PAS domain (PASsingle bondB) of Met, Met forms a heterodimer with another bHLH-PAS protein, steroid receptor coactivator (SRC). This dimer then enters the nucleus with the help of the molecular chaperone Heat shock protein 83 (Hsp83) and then activates downstream genes such as Kruppel homolog 1 (Kr-h1) (Charles et al. 2011; He et al. 2014; Kayukawa et al. 2012; Li et al. 2011a; Zhang et al. 2011; Li et al. 2014). In the nucleus, the heterodimer interacts with the DNA sequence of the promoter region of Kr-h1, affecting growth and development in insects. A previous study on the microcrustacea Daphnia demonstrated that its Met responds to MF in the same way as insect Met responds to JH III (Miyakawa et al. 2013). Furthermore, in the Japanese blue crab (Portunus trituberculatus), Met transcriptionally regulates Vg synthesis and potentially serves as an MF receptor, and amino acid sequencing has indicated that Met in crustaceans belongs to the bHLH-PAS family (Liu et al., 2016a, Liu et al., 2016b; Miyakawa et al. 2013). Thus, these findings indicate that JH signaling mechanisms may be conserved between insects and crustaceans. However, further studies are needed.

Accordingly, in the present study, we cloned the full-length cDNA of Met from the Chinese mitten crab (Eriocheir sinensis) (EsMet). qPCR was used to determine gene expression, and hemolymph MF titer values were used to explore the correlation, if any, between Met and Vg expression. To explore the effect of MF on Met expression, the relative expression levels of Met and Vg genes in vitro, in vivo, and in eyestalk ablation experiments were determined. Thus, our study provides a foundation for further exploration of the roles of Met in the regulation of E. sinensis development and valuable information for the development of techniques to control precocious puberty in aquaculture.

Section snippets

Experimental animals

Wild female adult Chinese mitten crabs (body weight: 80–100 g) were purchased from aquaculture farms in Xuancheng, Anhui in November 2018 for tissue distribution analyses. Tissues including hepatopancreas, ovary, muscle, gill, stomach, intestines, and heart were sampled, quickly frozen in liquid nitrogen, and stored at −80 °C until total RNA extraction.

For expression analysis, female adult crabs were obtained monthly from the farms, and their ovarian developmental stage was determined based on

Molecular characterization of EsMet

Full-length cDNA encoding Met was cloned from the Chinese mitten crab (E. sinensis) (Fig. 1). The GenBank accession number is MN829820. The cDNA of EsMet comprises 3383 bp including a 5′-untranslated region (UTR) of 202 bp, a 3′-UTR of 190 bp containing polyA, and an ORF of 2991 bp encoding 996 amino acids. The deduced amino acids were predicted to encode a protein with a molecular weight of 110.36 kDa and a theoretical isoelectric point of 5.94. There are no signal peptide cleavage sites

Discussion

In this study, cDNA encoding the Met protein was cloned from the Chinese mitten crab (E. sinensis). Similarly to Met in insects, the deduced protein was determined to belong to the family of bHLH-PAS nuclear transcriptional regulators, containing three independent typical conserved domains (bHLH, PAS-A, and PASsingle bondB) a hallmark of the bHLH-PAS protein family (Jindra et al. 2013; Li et al. 2011a). Previous studies have shown that the bHLH domain is very important in certain developmental pathways,

Declaration of Competing Interest

The authors declare that they have no conflicts of interests.

Acknowledgments

This work was supported by National Natural Science Foundation of Anhui Province [1808085MC61], Anhui Higher Institutions Natural Science Foundation [KJ2018A0137], Innovation Fund for the Postgraduates of Anhui Agricultural University [No. 2020ysj-28], and Anhui Agricultural University College Students Innovation and Entrepreneurship Training Program Innovative Training Project Foundation [201810364022].

References (49)

  • H. Miyakawa et al.

    Comparison of JH signaling in insects and crustaceans

    Curr. Opin. Insect Sci.

    (2014)
  • G.P.C. Nagaraju

    Is methyl farnesoate a crustacean hormone?

    Aquaculture.

    (2007)
  • Z. Qu et al.

    Juvenile hormone and sesquiterpenoids in arthropods: biosynthesis, signaling, and role of MicroRNA

    J. Steroid Biochem. Mol. Biol.

    (2018)
  • S.L. Tamone et al.

    Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro

    Gen. Comp. Endocrinol.

    (1993)
  • T. Tao et al.

    Cloning of two carboxylesterase cDNAs from the swimming crab Portunus trituberculatus: molecular evidences for their putative roles in methyl farnesotae degradation

    Comp. Biochem. Physiol. B Biochem. Mol. Biol.

    (2017)
  • S.H. Tiu et al.

    Cloning and expression study of the lobster (Homarus americanus) vitellogenin: conservation in gene structure among decapods

    Gen. Comp. Endocrinol.

    (2009)
  • B. Tsukimura et al.

    Regulation of methyl farnesoate in the hemolymph and mandibular organ of the lobster Homarus americanus

    Gen. Comp. Endocrinol.

    (1992)
  • M.J. Villalobos-Sambucaro et al.

    Genomic and functional characterization of a methoprene-tolerant gene in the kissing-bug Rhodnius prolixus

    Gen. Comp. Endocrinol.

    (2015)
  • Z. Zhang et al.

    Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant

    J. Biol. Chem.

    (2011)
  • J. Zhu et al.

    Identification of juvenile hormone target genes in the adult female mosquitoes

    Insect Biochem. Mol. Biol.

    (2010)
  • A.M. Abuhagr et al.

    Molt regulation in green and red color morphs of the crab Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

    J. Exp. Biol.

    (2014)
  • T. Alnawafleh et al.

    Stimulation of molting and ovarian maturation by methyl farnesoate in the Pacific white shrimp Litopenaeus vannamei (Boone, 1931)

    Fish. Aquat.Sci.

    (2014)
  • M. Ashok et al.

    Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • J.P. Charles et al.

    Ligand-binding properties of a juvenile hormone receptor, methoprene-tolerant

    Proc Natl Acad Sci U S A.

    (2011)
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    * These authors contributed equally to this paper.

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