cDNA cloning and characterization of Bombyx mori juvenile hormone esterase: an inducible gene by the imidazole insect growth regulator KK-42

Abstract

The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed ${}^3\text{H}$-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.

Introduction

The post-embryonic development of insects is regulated by two hormones: ecdysteroids, mainly 20-hydroxyecdysone (20E), and sesquiterpenoids, juvenile hormones (JHs). JH modulates 20E action, preventing metamorphosis at the larval stage and stimulating reproductive maturation at the adult stage (Riddiford, 1996). At times critical to metamorphosis, JH is believed to disappear from hemolymph, allowing the insect to enter metamorphosis. JH titer in hemolymph must thus be strictly controlled. The JH titer is controlled by at least 2 steps — biosynthesis and degradation. Two JH metabolizing enzymes, JH esterase (JHE) and JH epoxide hydrolase, have been identified. One of them, JHE (EC 3.1.1.1), a member of the carboxylesterase family, hydrolyzes the highly stable α/β unsaturated methylester of JH into biologically inactive JH acid (Hammock, 1985).

JHEs from many orders of insects have been extensively studied because of their importance in insect development and potential use as an insecticide. JHE proteins have been characterized and purified from Heliothis virescens (Hanzlik et al., 1989), Leptinotarsa decemlineata (Vermunt et al., 1997a), Manduca sexta (Venkatesh et al., 1990), Trichoplusia ni (Hanzlik and Hammock, 1987), Tenebrio moliter (Thomas et al., 2000) and Bombyx mori (Shiotsuki et al., 2000). JHE cDNA clones have been isolated from H. virescens (Hanzlik et al., 1989), L. decemlineata (Vermunt et al., 1997b) and Choristoneura fumiferana (Feng et al., 1999). In T. ni, a cDNA encoding JHE-related protein (JHER) has been isolated (Jones et al., 1994). Among these, JHE cDNAs from H. virescens (Hammock et al., 1990, Ward et al., 1992, Bonning et al., 1995) and C. fumiferana (Feng et al., 1999) were expressed using baculovirus and the JHE activity of recombinant proteins has been confirmed. Recently, Hajos et al. (1999) reported that infection of a recombinant baculovirus harboring JHE cDNA of H. virescens in the antisense orientation with H. virescens larvae greatly reduced hemolymph JHE titer and resulted in aberrant morphogenesis at the final instar. This indicates how important JHE is for insect development.

In addition to insect hormones and their metabolizing enzymes, several classes of insect growth regulators (IGRs) are reported to affect insect development. Among them, imidazole IGR KK-42 possesses many types of biological activity in several orders of insects. One of the unique activities is an induction of precocious metamorphosis in B. mori (Kuwano et al., 1985). In this insect, JHE activity in the hemolymph is quite low until the penultimate (4th) instar and increases at the final instar, thereby decreasing JH titer and inducing metamorphosis. When 4th instar larvae were topically applied with KK-42, JHE activity in hemolymph was induced, as seen in the final instar (Shiotsuki et al., 1999).

As an initial step to understand molecular action of KK-42 on JHE, we cloned JHE cDNA from B. mori (BmJHE), produced recombinant BmJHE by using baculovirus and confirmed its function as JHE. Tissue distribution and developmental expression of BmJHE mRNA and JHE protein in precocious, normal metamorphosing and extra-molted larvae were investigated by Northern and Western blotting, respectively.