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.

A Peptide from the Male Accessory Gland in Leptinotarsa decemlineata: Purification, Characterization and Molecular Cloning.

Our interest in the male accessory glands (MAGs) of Leptinotarsa decemlineata was raised recently by our finding that certain cells produce a secretory substance that is recognized by one of our monoclonal antibodies (MAC-18), developed for the immunohistochemical demonstration of peptidergic neurons in the brain. We undertook to isolate this substance, presumably a peptide, to find out more about its role in the post-mating physiology of the recipient of this peptide, the mated female. This paper describes the purification and chemical characterization of the immunoreactive peptide from 100 pairs of male accessory glands. The peptide was purified by two subsequent reversed-phase-HPLC runs, and fractions were analyzed on Western blots that were immunostained by MAC-18. This indicated the presence of an 8 kDa peptide in the MAG. Partial analysis of the N-terminal amino acids by automated Edman degradation revealed a sequence of 40 amino acid residues. To obtain the full amino acid sequence of this peptide, the technique of reverse transcriptase PCR (3'RACE) was used. A PCR product of 350 bp was obtained, which encoded the 3'-end of the mRNA. After cloning and sequencing, this product contained most of the genetic information of the MAG peptide. The PCR product was also used as a probe for screening a cDNA library constructed from mRNA extracted from MAGs. The nucleotide sequence coding for the signal peptide was elucidated by 5'RACE. The cDNA and 5'RACE clones were analyzed and sequenced. The sequence of the cDNA clone contained an insert of 411 bp, which agreed well with the mRNA size measured by Northern blotting. Translation of the DNA sequences confirmed the data from partial amino acid sequence analyses and also predicted the remainder of the amino acid sequence. The entire peptide, designated Led-MAGP, consists of 74 residues; its mass was calculated and confirmed by mass spectrometry at 7971 Da. The peptide contains seven imperfect hexa-repeats, and this hexa-repeat sequence shows remarkable similarity to the hexa-repeat section of the chicken prion protein. The physiological function of the peptide has yet to be determined, but the hexa-repeat motif has recently been identified as the signal that induces internalization of the prion protein by coated-pit mediated endocytosis. Possible implications for the control of reproductive activities in L. decemlineata are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved

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

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.

The relative importance of nervous and humoral pathways for control of corpus allatum activity in the adult Colorado potato beetle, Leptinotarsa decemlineata (Say).

To elucidate corpus allatum (CA) regulatory mechanisms under the influence of photoperiod and starvation in Leptinotarsa decemlineata, gland activities were measured in vitro by the short-term radiochemical assay. This data was substantiated with juvenile hormone (JH) titer determinations by using a physicochemical method or a radioimmunoassay. Under short-day conditions both neural and humoral factors may be involved in CA inhibition. This was indicated by the temporary activation of short-day glands after denervation in early prediapause and the gradual inhibition of active CA from long-day females implanted into short-day hosts. Studies with farnesenic acid as a precursor indicated that the last two steps in the biosynthetic pathway of JH are also diminished during humoral inhibition of the CA. Transfer of short-day beetles to long-day photoperiod completely activated the CA and this process was independent of CA innervation. Starvation leads to neural inhibition of intact glands but to possible stimulation of the denervated CA, since implanted glands in starved hosts were fully activated irrespective of the photoperiod.