An immune-induced Reeler protein is involved in the Bombyx mori melanization cascade

Abstract

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.

Introduction

Insects rely on an innate immunity response to defend themselves against microbial infection. The melanization cascade is an important component of the insect immune defense system, and it is involved in numerous defense responses, including nodulation, phagocytosis and melanotic encapsulation (Cerenius and Soderhall, 2004, Kanost et al., 2004, Mavrouli et al., 2005, Wilson et al., 1999).

Nodule formation is the earliest and predominant cellular defense response to bacterial infections in insects (Bedick et al., 2000). Nodulation is thought to begin with the formation of hemocyte aggregates and to end with melanization, forming darkened nodules. The nodulation process contributes to clearance of the bacteria from the circulating hemolymph (Miller and Stanley, 2000). Noduler is a novel immune-induced protein that was purified from bacteria-challenged hemolymph in wild silkworm, Antheraea mylitta, and it was first reported to mediate the nodulation response (Gandhe et al., 2007). Several genes that share sequence similarity with A. mylitta noduler have been identified from immunized larval fat bodies of the fall webworm, Hyphantria cunea (Shin et al., 1998), and from Manduca sexta (Zhu et al., 2003), Samia cynthia ricini (Bao et al., 2003), Lonomia obliqua (Veiga et al., 2005) and Bombyx mori (GenBank accession no. DQ898221). The deduced amino acid sequences of these proteins include a characteristic reeler domain (Zhu et al., 2003). The reeler domain was initially identified in the mouse reelin protein (D’Arcangelo et al., 1995), a secreted glycoprotein that plays a pivotal role in the development of the central nervous system in mammals (Quattrocchi et al., 2002). The reeler domain-containing genes identified in lepidopteran insects may have immune-related functions, as shown in studies reporting that bacterial challenge induced their expression in S. c. ricini, A. mylitta, H. cunea and M. sexta larvae (Bao et al., 2003, Gandhe et al., 2007, Shin et al., 1998, Zhu et al., 2003). However, the detailed functions of these reeler domain-containing molecules in the insect defense system remain largely unknown. The silkworm, B. mori, is a major model for insect biochemical research and the first lepidopteran insect for which draft genome sequences became available (Xia et al., 2004). However, the functions of the B. mori reeler proteins have not yet been investigated. Two reeler domain-containing genes are predicted in the silkworm genome (http://silkworm.genomics.org.cn/). One contains the complete open reading frame (ORF) sequence (BGIBMGA014360-TA), and the other lacks some coding sequence at the 3′ terminus of the ORF (BGIBMGA005981-TA). It is interesting to know if both of the reeler genes were all expressed in B. mori and if they had the expression differences in response to bacterial challenge.

Gandhe et al. (2007) reported that Noduler in A. mylitta functions in nodule formation in the cellular immune response to bacterial infection. Further study is needed to understand how this reeler domain-containing protein participates in the nodulation response. Melanization is of central importance in the nodulation and capsulation responses in some lepidopteran and dipteran insects, such as Pseudoplusia and Drosophila, and results in melanin formation (Lavine and Strand, 2002, Lavine and Strand, 2003, Sideri et al., 2008). Prophenoloxidase (proPO) activation is an important step in the insect melanization cascade; proPO is activated by microbial infection and leads to the proteolytic conversion of proPO into phenoloxidase (PO) in the hemolymph (Ji et al., 2004). PO catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine (dopa) and the oxidation of dopa to dopaquinone (Ashida and Brey, 1998, Kan et al., 2008), which allows for wound healing and the melanization of foreign microorganisms that are trapped in hemocyte nodules (Kanost et al., 2004). Dopamine present in hemolymph is also a significant substrate for PO. PO-based oxidation of dopamine produces dopaminequinone and consequently the cross-linking and melanization of proteins (Sideri et al., 2008). A relationship between Reeler protein and the proPO activation system has not yet been reported in insects; therefore, a functional analysis of Reeler is of considerable interest. In this report, we investigated the possible relationship between reeler gene expression and PO activity. Our findings reveal that the Reeler protein is a necessary component of the insect melanization cascade.