RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say)

https://doi.org/10.1016/j.jinsphys.2013.08.002Get rights and content

Highlights

  • A putative LdSAHase gene was cloned from L. decemlineata.

  • dsLdSAHase ingestion caused negative effects on larval performance.

  • Pyriproxyfen application partially rescued the negative effects.

  • This suggests that LdSAHase plays a critical role in JH biosynthesis.

Abstract

In Leptinotarsa decemlineata, juvenile hormones (JHs) play primary roles in the regulation of metamorphosis, reproduction and diapause. In JH biosynthetic pathway in insect corpora allata, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a potent feedback inhibitor S-adenosyl-L-homocysteine (AdoHcy). Rapid removal of AdoHcy is hypothesized to be essential for JH synthesis. AdoHcy hydrolase (SAHase) is the only eukaryotic enzyme catalyzing the removal. In the present paper, we firstly cloned a putative LdSAHase gene from L. decemlineata. The cDNA consists of 1806 bp and encodes a 525 amino acid protein. LdSAHase was expressed in all developmental stages. The gene had the highest and the lowest level of transcription respectively in the 3rd- and 4th-instars’ heads that contain corpora allata, which was positively correlated with JH titer in the haemolymph and the mRNA level of a JH early-inducible gene, the Krüppel homolog 1 gene (Kr-h1). Secondly, dietary ingestion of bacterially-expressed LdSAHase-dsRNA significantly decreased LdSAHase and LdKr-h1 mRNA levels, reduced JH titer, and caused the death of the larvae, and the failure of pupation and adult emergence. After continuous exposure for 12 days, 42% of the larvae died, 65% of the prepupae failed to pupate and 100% of the pupae failed to emerge. Moreover, RNAi-mediated LdSAHase knockdown also reduced larval developing time, and decreased larval weight. Lastly, application of JH analogue pyriproxyfen to LdSAHase-dsRNA-exposed larvae did not greatly increase LdSAHase expression level and JH content, but up-regulated LdKr-h1 mRNA level. Expectedly, pyriproxyfen application could partially rescue the negative effects on the survival and the development. Thus, our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Introduction

Juvenile hormones (JHs) are a family of sesquiterpenoids, and regulate metamorphosis and reproduction in insects (Daimon et al., 2012, Noriega et al., 2006, Riddiford et al., 2013). JHs are produced and secreted from the corpora allata (CA), a pair of small endocrine organs just posterior to the brain (Riddiford, 2012, Riddiford et al., 2013). The biosynthetic pathway of JHs is conventionally divided into two main components, the early steps and the late steps (Belles et al., 2005). The early steps of JH biosynthesis follow the mevalonate pathway from acetyl-CoA to farnesyl pyrophosphate (FPP). During the late steps, FPP is first hydrolyzed to farnesol (Cao et al., 2009) and then oxidized successively to farnesal and farnesoic acid (FA) (Belles et al., 2005). FA is finally converted to the active JH by means of an epoxidation (C10, 11) and a methyl transfer (Marchal et al., 2010, Mayoral et al., 2009). The last two steps diverge depending on the insect order. In Lepidoptera, epoxidase may have higher affinity for FA, so FA is first epoxidized to JH acid, and then methylated to JH. In contrast, in insect species in Orthoptera, Diptera, Coleoptera and Dictyoptera, FA is first esterified to form methyl farnesoate (MF), and then MF was epoxidized to JH (Belles et al., 2005, Defelipe et al., 2011, Mayoral et al., 2009).

In insects, methylation of FA or JH acid using S-adenosyl-L-methionine (AdoMet) releases a byproduct S-adenosyl-L-homocysteine (AdoHcy) (Feyereisen and Farnsworth, 1987, Noriega et al., 2006, Tehlivets et al., 2013). AdoHcy inhibits a number of AdoMet-dependent methyltransferases in vitro, such as mammalian DNA (cytosine-5-)-methyltransferase, mRNA cap (guanine-N7-)-methyltransferase, tRNA (uracil-5-)-methyltransferase, protein isoprenylcysteine carboxylmethyltransferase and phospholipid methyltransferases (Bacolla et al., 1999, Baron and Casey, 2004, Hausmann et al., 2005, Mao et al., 1995), and in vivo, such as DNA (cytosine-5-)-methyltransferase and phospholipid methyltransferases (Castro et al., 2005, Malanovic et al., 2008). Rapid conversion of AdoHcy to adenosine and homocysteine prevents the feedback inhibition. AdoHcy hydrolase (SAHase, EC 3.3.1.1) is the only eukaryotic enzyme catabolizing AdoHcy (De La Haba and Cantoni, 1959, Luka et al., 2009). In the cockroach Diploptera punctata CAs, SAHase activity is high enough to rapidly hydrolyze AdoHcy and thus removes the inhibitory effect of AdoHcy on the methylation of FA (Feyereisen and Farnsworth, 1987). Accordingly, it can be hypothesized that SAHase is essential for JH biosynthesis in other insect species.

Leptinotarsa decemlineata (Say) is a notorious defoliator of potato, and often causes extremely large potato yield loss (Jiang et al., 2012). The beetle has a complicated and diverse life history (Alyokhin, 2009, Alyokhin et al., 2008). JH plays primary roles in the regulation of several most important events in the life cycle in L. decemlineata, including metamorphosis (Vermunt et al., 1999), reproduction (De Loof and De Wilde, 1970, Dortland, 1979, Lefevere and De Wilde, 1984) and diapause (De Loof and De Wilde, 1970, De Wilde and De Boer, 1969, Kramer, 1978, Lefevere and De Wilde, 1984, Schooneveld et al., 1977). Moreover, dietary ingestion of bacterially-expressed dsRNA can effectively knock down target genes in L. decemlineata (Zhu et al., 2011). Thus, L. decemlineata provides an excellent coleopteran model for studying the endocrine control of life history.

In the present paper, we first cloned a putative SAHase gene in L. decemlineata. We then studied the influence of bacterially-expressed LdSAHase dsRNA on the performance of the larvae. Since JH activity of pyriproxyfen has been proven in L. decemlineata (De Kort et al., 1997, Koopmanschap et al., 1989, Yi and Adams, 2000), we also tested the combined effect of pyriproxyfen and LdSAHase-dsRNA on the larvae. Our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Section snippets

Insect culture

Post-diapause L. decemlineata adults were collected from potato field in spring at Urumqi city (43.82N, 87.61E), Xinjiang Uygur autonomous region in China. Insects were routinely reared in an insectary according to a previously described method (Shi et al., 2013).

Molecular cloning

Total RNA was extracted from the 3rd-instar larvae using TRIzol reagent (Invitrogen) according to the manufacturer′s instructions, and was treated for 30 min at 37 °C with RNase free DNase I (Ambion, Austin, TX) to eliminate traces of

Identification of a putative LdSAHase

A cDNA of a putative SAHase containing the complete coding sequence was isolated from L. decemlineata. The cDNA consists of 1806 bp containing a 1575 bp protein-coding region. Its predicted protein has 525 amino acid residues, with a calculated molecular weight of 58.18 kDa and an isoelectric point of 7.46. The predicted protein is designated LdSAHase (Fig. S1). SAHase is an exceptionally well-conserved enzyme (Stepkowski et al., 2005). Of the SAHase-like proteins, LdSAHase shares great identities

Discussion

In JH biosynthesis pathway, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a byproduct AdoHcy (Feyereisen and Farnsworth, 1987, Noriega et al., 2006, Tehlivets et al., 2013). It is hypothesized that rapid removal of AdoHcy is essential for JH biosynthesis in insect CA (Feyereisen and Farnsworth, 1987). SAHase is the only eukaryotic enzyme catabolizing AdoHcy (De La Haba and Cantoni, 1959, Luka et al., 2009). In the present paper, therefore, we cloned a putative

Acknowledgments

This research was supported by the National Natural Sciences Foundation of China (31272047) and a nationally special fund of China for agri-scientific research in the public interest (201103026). We are very grateful to Mr. Jiang He, Mrs. Wei-Hua Jiang, Mr. Zhi-Tian Wang, Mrs. Man-Hui Xiong, Mr. Wei-Ping Lu, and Mrs. Ping Liu for help in insect rearing and sample collection. We would like to thank other field entomologists and technicians at Urumqi city in Xinjiang Uygur autonomous region in

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