The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis.

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT-PCR using degenerate primers based on the N-terminal amino acid sequence of purified hJHBP and a conserved region near the C-terminus of other lepidopteran hJHBPs. 5'- and 3'-ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP-mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS-PAGE with a Mr of 32 kDa and showed a Kd of 4.5 x 10-7 M with JH III, both similar to those of native hJHBP.

Dissecting insect development: baculovirus-mediated gene silencing in insects.

A novel concept applying baculovirus-mediated gene silencing to study insect gene function and regulation is described in this paper. A recombinant baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), was constructed with the juvenile hormone esterase (JHE) gene from the tobacco budworm Heliothis virescens in the antisense orientation, driven by the viral p10 promoter. Infection with this recombinant greatly reduced the haemolymph JHE level and resulted in aberrant morphogenesis of final-instar H. virescens larvae. The body organization remained larval, although the cuticle became hard and brown, similar to pupal cuticle. These results demonstrated that baculovirus-mediated gene silencing can be accomplished and utilized to dissect insect development and to design a new class of baculovirus insecticides.

Expression of the juvenile hormone esterase gene in the Colorado potato beetle, Leptinotarsa decemlineata: photoperiodic and juvenile hormone analog response.

Metamorphosis and reproduction in insects are controlled by juvenile hormone (JH). One of the factors, which regulate the JH titer in the hemolymph, is the activity of juvenile hormone esterase (JHE). JHE from the Colorado potato beetle, Leptinotarsa decemlineata, consists of two 57kDa subunits. In this study, the JHE-cDNA was used as a probe to examine where and when the gene is transcribed as well as how gene expression responds to photoperiodic treatment and to topical application with a JH analog, pyriproxyfen. JHE transcripts were almost exclusively found in RNA extracts from fat body tissue in both larvae and adults. JHE-mRNA levels in the fat body correlated positively with levels of JHE activity in the hemolymph. In the last larval instar, high levels of JHE-mRNA were found in the feeding stage. In adults, reared under short-day conditions, JHE-mRNA levels were high between day 2 and day 9, which correlated with high JHE activity in the hemolymph. During these conditions, the JH titer decreases in preparation for pupation and diapause, respectively. The JHE-mRNA levels and JHE activity in the hemolymph were higher in short-day than in reproductive long-day adults. If the JH analog pyriproxyfen was applied to animals of the last larval instar on day 0 or day 3, JHE gene expression was enhanced. In contrast, if pyriproxyfen was applied to short-day adults on day 1 or day 4, the mRNA levels and the JHE activity in the hemolymph were suppressed to levels similar to those found in long-day adults. Thus, transcription of JHE is dependent on developmental stage, tissue, photoperiod and the level of its substrate JH.

Evidence for two juvenile hormone esterase-related genes in the Colorado potato beetle.

Juvenile hormone esterase (JHE) activity in the haemolymph of the Colorado potato beetle is necessary to initiate pupation in larvae as well as diapause in adults. The enzyme appears in the haemolymph as a dimer consisting of two 57 kDa subunits. The sequence of an encoding cDNA, JHE.A, is distinct from lepidopteran JHEs. In this study, RT-PCR using primers designed on the basis of the 5'- and 3'-ends of the coding region revealed the existence of a related gene, JHE.B. The presence of two JHE-related genes was also shown by PCR amplification on genomic DNA from different individual beetles followed by restriction enzyme analysis. Both forms, probably paralogues, were transcribed since they could be amplified on messenger RNA from fat bodies. The size of the PCR products generated with mRNA and genomic DNA were both 1.6 kb, suggesting the absence of introns in the genomic JHE coding sequence. The sequence of a genomic clone, which encoded JHE.B, was 77% identical and 82% similar in amino acids compared to JHE.A. No introns were found in the coding sequence of these coleopteran JHE-related genes, in contrast to lepidopteran JHE genes. Southern blot analysis of digested genomic DNA confirmed the presence of two JHE-related genes.

Influence of Pyriproxyfen on the Expression of Haemolymph Protein Genes in the Colorado Potato Beetle, Leptinotarsa decemlineata.

Pyriproxyfen, a potent juvenile hormone analogue for the Colorado potato beetle, Leptinotarsa decemlineata, was applied topically to last-instar larvae and short-day adults at different times after moulting. The effect of the hormone analogue on concentration and composition of protein in the haemolymph was studied at different intervals after pyriproxyfen application. The hormone analogue had little effect on total protein concentration of the haemolymph, but affected protein composition. Diapause protein 1 was prevented from being synthesized if pyriproxyfen was applied before the gene was activated and disappeared from the haemolymph if applied after the gene had been expressed. It therefore inactivated the gene for diapause protein in both larvae and adults. Pyriproxyfen also induced appearance of vitellogenin at both stages, indicating induction of expression of the vitellogenin gene. It also affected the stability of mRNA for diapause protein. The analogue caused mRNA for diapause protein 1 to disappear untimely compared to controls in last-instar larvae and short-day adults. The response of adults to the JHA was much more pronounced than that of larvae, although the analogue had a strong biological effect on last-instar larvae because it prevented metamorphosis at low doses. Copyright 1997 Elsevier Science Ltd. All rights reserved

Purification and characterization of juvenile hormone esterase from hemolymph of the Colorado potato beetle.

In the Colorado potato beetle (Leptinotarsa decemlineata), low juvenile hormone (JH) titers are necessary to initiate metamorphosis and diapause. Low JH titers coincide with high activities of JH esterase, which occur mainly in the hemolymph. The specific activity of JH esterase appeared to be highest in the last larval instar, at day 3 after the molt, and reached a value of 13.5 nmol/min/mg. JH esterase was purified from hemolymph collected at this stage be a sequence of separation systems, including preparative nondenaturing PAGE, isoelectric focusing, and SDS-PAGE. The enzyme had a molecular weight of 120,000 and was composed of two subunits with molecular weights of 57,000, which were not linked by disulphide bridges. Isoelectric focusing revealed two forms of the enzyme with isoelectric points of 5.5 and 5.6. The Km and kcat of the purified enzyme were determined. The major form with pl 5.6 had a Km of 1.4 x 10(-6) M and a kcat of 0.9 s-1 and the minor form with pl 5.5 had a Km of 2.2 x 10(-6) M and a kcat of 1.9 s-1. The quaternary structure of L. decemlineata JH esterases, as differs from JH esterases in other species, which are monomers.

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle.

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.