Archives of Insect Biochemistry and Physiology 64:175–185 (2007) © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20168 Published online in Wiley InterScience (www.interscience.wiley.com) cDNA Characterization and Expression Analysis of Two Arylphorin-Like Hexameric Protein Genes From the Diamondback Moth, Plutella xylostella (L.) Muhammad Ashfaq, Shoji Sonoda,* and Hisaaki Tsumuki We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investi- gated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin- like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the devel- opmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome. Arch. Insect Biochem. Physiol. 64:175–185, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: arylphorin; cDNA; gene expression; JH-analog; Plutella xylostella; Lepidoptera Research Institute for Bioresources, Okayama University, Okayama, Japan Contract grant sponsor: Grant-in-Aid for Encouragement of Young Scientists from the Japan Society for the Promotion of Science (JSPS); Contract grant number: 16780036; Contract grant sponsor: Ohara Foundation. *Correspondence to: Shoji Sonoda, Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan. E-mail: sonodas@rib.okayama-u.ac.jp Received 25 July 2006; Accepted 10 November 2006 INTRODUCTION Hexameric storage proteins (SPs) are prime components of hemolymph in the immature life stages of insects. SPs are produced in the fat body, released in hemolymph during larval development, and subsequently sequestered in fat body during the prepupal period where they serve as a source of amino acids for later life stages (Tojo et al., 1981; Telfer and Kunkel, 1991). The proteins generally consist of six identical or similar subunits of ap- proximately 80 kDa (Markl and Decker, 1992; Ryan et al., 1985). Insect SPs are categorized into two main groups: arylphorin-like, with combined phe- nylalanine/tyrosine content more than 15%, and methionine-rich, with a methionine content more than 3.5% (Haunerland, 1996). Some SPs, how- ever, are neither arylphorin-like nor methionine- rich in nature (Jones et al., 1990). Arylphorins are not sex specific as they are present in males and females; however, their synthesis may fluctuate dur- ing larval development (Webb and Riddiford, 1988). A single gene encoding arylphorin-like pro- tein has been characterized from some lepidopter- ans (Willott et al., 1989; Fujii et al., 1989; Zheng et al., 2000). In general, both arylphorin and methionine-rich proteins are considered sources of nitrogen and amino acids for post-prepupal stages, as, for ex- ample, production of oocytes and yolk proteins (Pan and Telfer, 2001; Telfer and Pan, 2003). Arylphorins are crucial as well in the synthesis of other somatic tissues, like adult integument (Pan and Telfer, 1996) and thus may be functionally di-