Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase

Abstract

Insect growth and development are intricately regulated by the titers of juvenile hormones (JHs) and ecdysteroids (and͞or their metabolites) in the insect hemolymph. Hydrolysis of the methyl ester of JH by a JH-specific esterase (JHE) is a key pathway for the degradation of JH. Here, we generate transgenic silkworm strains that overexpress JHE by using the binary GAL4͞UAS system. Overexpression of JHE from the embryonic stage resulted in larval-pupal metamorphosis after the third stadium, two stadia earlier than that observed in wild-type insects. This precocious metamorphosis suggests that JHs are not critical for normal development of embryo or larva before the second molt in Lepidoptera (moths and butterflies). Our transgenic approach allowed us to dissect the function of key physiological events that occur from embryogenesis. Until now, these types of studies were possible only in later larval stadia by using physical techniques such as allatectomy or the application of JH analogues. We believe that our system will allow further pioneering studies in insect physiology.Bombyx mori ͉ juvenile hormones ͉ transgenesis T wo major groups of nonpeptidic hormones, juvenile hormones (JHs) and ecdysteroids, play critical roles in the regulation of insect growth and development (1). The presence of JH during exposure to the ecdysteroids ensures a molt to a like stage, whereas the absence of JH during an ecdysteroid pulse allows metamorphosis. Concentrations of both hormones are regulated precisely by biosynthesis and degradation. Two definitive primary pathways for JH metabolism, hydrolysis of the methyl ester of JH by soluble esterases and hydration of the epoxide by microsomal epoxide hydrolases, have been described so far (2-5). In several insect orders, the key route of JH metabolism is by means of the hydrolysis of the methyl ester, and JH esterase (JHE) is considered the principal enzyme responsible for this function (2). Because of their importance in insect development, JHEs have been isolated and characterized from many insect orders such as Coleoptera (6), Diptera (7), and Lepidoptera (8-10), including the silkworm, Bombyx mori (11).As the primary degradative enzyme of JH, JHE has been used as an attractive tool for the study of JH action. Inhibition of JHE activity by a variety of 3-substituted 1,1,1-trifluoropropanone sulfides reduces the rate of JH degradation, thus delaying metamorphosis and resulting in giant larvae of Manduca sexta (12). By contrast, recombinant baculoviruses expressing JHE can reduce JH levels in Lepidoptera (13). However, difficulties in establishing stable and prompt in vivo expression of JHE in Lepidoptera have halted further studies.The mechanism of JH action in insects still remains a mystery despite a great deal of knowledge about its biological actions (5). Using Drosophila as a model for the study of JH action meets with difficulties because, in contrast to most insects, including those in Lepidoptera, Drosophila displays few morphogenetic actions that can be easily bioassayed ...

Ras1CA overexpression in the posterior silk gland improves silk yield

“…Based on this method, a binary GAL4/UAS system for targeted gene expression was developed [11] and was further applied to analyze gene function of juvenile hormone esterase [12]. A major advantage of the GAL4/UAS system is its ability to target gene expression in a tissue-or stage-specific manner [15].…”

“…Because the posterior silk gland grows dramatically during larval life and its protein synthesis capacity is extremely high at the last instar stage [2,3], we hypothesized that Ras activity might be involved in regulating fibroin production and silk yield. Here, we report that, using the binary GAL4/UAS transgenic system [11,12], overexpression of the Ras1 CA oncogene specifically in the Bombyx posterior silk gland increases cell size and protein synthesis in the posterior silk gland and leads to silk yield improvement. Combining properties of both hybrid and molecular breeding techniques in Bombyx may produce a new breakthrough in sericulture.…”

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

“…Recent genetic studies also have shown that deprivation of JHs cannot induce precocious metamorphosis during the early larval instars. For example, overexpression of a JH-degrading enzyme JH esterase (JHE) in transgenic Bombyx resulted in precocious metamorphosis after three instars (8). In addition, the dimolting (mod) mutant larvae of Bombyx with a null mutation in a JH biosynthetic enzyme, JH epoxidase CYP15C1, metamorphosed into miniature pupae after the third or fourth instar (7).…”

“…It is widely considered that this "status quo" action of JH is required to maintain the larval status throughout the larval stage. Interestingly, however, several attempts to deplete JHs using classic and modern techniques have failed to induce precocious metamorphosis during the very early larval instars (6)(7)(8)(9)(10)(11)(12).…”

When inordinate tissue growth is beneficial: Improving silk production by increasing silk gland size

“…In the case of B. mori, which had been reluctant to genetic transformation for long time, an equivalent system for germline transgenesis using a piggyBack transposon-derived vector was developed in 2000 [8]. It was successfully used, for example, by Takahiro Shiotsuki and associates to study the regulatory role of juvenile hormone (JH) esterase on the degradation of JH in B. mori [9]. The results were spectacular: overexpression of JH esterase from the embryo stage resulted in steady degradation of JH and, as a consequence, in larval-pupal metamorphosis after the third larval instar, two instars earlier than in wildtype silkworms [9].…”