Juvenile Hormone Esterase Gene Research Articles

Technique to deliver dsRNA in Bemisia tabaci nymphs and its validation through silencing of juvenile hormone esterase gene

AbstractRNA interference (RNAi) is a technique employed to investigate the functions of various genes in whitefly and other insects. The effectiveness of the RNAi technique depends upon the delivery of dsRNA into the insects and it has been achieved through microinjection, oral feeding, egg soaking, transfection, topical application, spraying with nanoparticles, leaf‐mediated dsRNA feeding or plant‐mediated/ transgenic plants. In Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), most of the studies on RNAi were conducted on the adult through either injection or oral feeding. There is a need to develop a system to deliver dsRNA in whitefly nymphs because the first instar nymphs after hatching find a suitable place on the leaf and feed on the same place till adult emergence. So, we evaluated nine different techniques using different sponges, tubes, and, Petri plates for feeding nymphs. The detached leaf survived for maximum number of days (8.25 days) with minimum nymphal mortality (12.01%) in the Whitefly Nymphal Feeding System‐1 (WNFS‐1) treatment; therefore, WNFS‐1 was selected for the delivery of dsRNA to B. tabaci nymphs. In WNFS‐1, blue food dye was used to confirm that nymphs were being fed, and the results were evidenced by blue‐coloured bodies. The juvenile hormone esterase (jhe) gene was silenced in order to successfully deliver dsRNA to nymphs using WNFS‐1 and resulting in much higher nymphal mortality (68.36%). Therefore, WNFS‐1 is a reliable method of delivering dsRNA for the purpose of silencing the genes in the nymphal stages of whiteflies. The WNFS system may also be used extensively to evaluate any chemistry (insecticides, hormones, biopesticides, etc.) in nymphs of whitefly.

Novel miR-108 and miR-234 target juvenile hormone esterase to regulate the response of Plutella xylostella to Cry1Ac protoxin

Insect hormones, such as juvenile hormone (JH), precisely regulate insect life-history traits. The regulation of JH is tightly associated with the tolerance or resistance to Bacillus thuringiensis (Bt). JH esterase (JHE) is a primary JH-specific metabolic enzyme which plays a key role in regulating JH titer. Here, we characterized a JHE gene from Plutella xylostella (PxJHE), and found it was differentially expressed in the Bt Cry1Ac resistant and susceptible strains. Suppression of PxJHE expression with RNAi increased the tolerance of P. xylostella to Cry1Ac protoxin. To investigate the regulatory mechanism of PxJHE, two target site prediction algorithms were applied to predict the putative miRNAs targeting PxJHE, and the resulting putative miRNAs were subsequently verified for their function targeting PxJHE using luciferase reporter assay and RNA immunoprecipitation. MiR-108 or miR-234 agomir delivery dramatically reduced PxJHE expression in vivo, whilst only miR-108 overexpression consequently increased the tolerance of P. xylostella larvae to Cry1Ac protoxin. By contrast, reduction of miR-108 or miR-234 dramatically increased PxJHE expression, accompanied by the decreased tolerance to Cry1Ac protoxin. Furthermore, injection of miR-108 or miR-234 led to developmental defects in P. xylostella, whilst injection of antagomir did not cause any obvious abnormal phenotypes. Our results indicated that miR-108 or miR-234 can be applied as potential molecular targets to combat P. xylostella and perhaps other lepidopteran pests, providing novel insights into miRNA-based integrated pest management.

Transcriptome Analysis and Knockdown of the Juvenile Hormone Esterase Gene Reveal Abnormal Feeding Behavior in the Sugarcane Giant Borer.

The sugarcane giant borer (SGB), Telchin licus licus, is a pest that has strong economic relevance for sugarcane producers. Due to the endophytic behavior of the larva, current methods of management are inefficient. A promising biotechnological management option has been proposed based on RNA interference (RNAi), a process that uses molecules of double-stranded RNA (dsRNA) to specifically knock down essential genes and reduce insect survival. The selection of suitable target genes is often supported by omic sciences. Studies have shown that genes related to feeding adaptation processes are good candidates to be targeted by RNAi for pest management. Among those genes, esterases are highlighted because of their impact on insect development. In this study, the objective was to evaluate the transcriptome responses of the SGB’s gut in order to provide curated data of genes that could be used for pest management by RNAi in future studies. Further, we validated the function of an esterase-coding gene and its potential as a target for RNAi-based control. We sequenced the gut transcriptome of SGB larvae by Illumina HiSeq and evaluated its gene expression profiles in response to different diets (sugarcane stalk and artificial diet). We obtained differentially expressed genes (DEGs) involved in detoxification, digestion, and transport, which suggest a generalist mechanism of adaptation in SGB larvae. Among the DEGs, was identified and characterized a candidate juvenile hormone esterase gene (Tljhe). We knocked down the Tljhe gene by oral delivery of dsRNA molecules and evaluated gene expression in the gut. The survival and nutritional parameters of the larvae were measured along the developmental cycle of treated insects. We found that the gene Tljhe acts as a regulator of feeding behavior. The knockdown of Tljhe triggered a forced starvation state in late larval instars that significantly reduced the fitness of the larvae. However, the mechanism of action of this gene remains unclear, and the correlation between the expression of Tljhe and the levels of juvenile hormone (JH) metabolites in the hemolymph of the SGB must be assessed in future research.

RNA interference mediated knockdown of juvenile hormone esterase gene in Bemisia tabaci (Gennadius): Effects on adults and their progeny

Abstract Juvenile hormone is responsible for regulating metamorphosis and reproduction in insects. Analysis of key elements of juvenile hormone regulation would enhance the understanding of this complex mechanism. Juvenile hormone esterase plays an important role in maintaining juvenile hormone titres in insects. In this study, effects of knockdown of juvenile hormone esterase gene (jhe) in Bemisia tabaci were studied using RNA interference (RNAi) technique. dsRNA corresponding to two conserved regions of jhe gene, substrate binding pocket site (jhe1), catalytic triad site (jhe2), green fluorescent protein gene (gfp) as control were synthesized. dsRNAs incorporated in artificial diet (20% sucrose solution) @ 2.5, 1.0, 0.5 and 0.1 μg/μl were fed to adult whiteflies for 48 h, followed by shifting whiteflies to live plants for next generation biology study. Based on qRT-PCR analyses, reduced jhe gene expression was observed in adult whiteflies after dsRNA feeding @ 2.5 and 1.0 μg/μl. jhe gene knockdown affects the survival and reproduction of whiteflies adversely in a dose-dependent manner. Moreover, oral feeding of dsRNA to adult whiteflies @ 2.5 and 1.0 μg/μl showed adverse effects on next generation of whitefly viz., lower egg hatchability and shortened egg incubation period. Minimum number of viable eggs (1.04 and 1.80 eggs/female) were observed when whiteflies were fed with highest concentration of dsjhe1 and dsjhe2 as compared to control (16.58 eggs/female). These data suggest that jhe gene acts as a major biological player in whitefly and its progeny and further indicate to be potential target for managing whitefly population.

Neofunctionalization of \u201cJuvenile Hormone Esterase Duplication\u201d in Drosophila as an odorant-degrading enzyme towards food odorants

Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called “juvenile hormone esterase duplication (JHEdup)” has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids.

Abnormal development in larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) resulting from baculovirus-mediated overexpression of a JHE-related gene (SnJHER)

The Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae) has a unique and recently multiplied juvenile hormone esterase gene family (SnJHER) with particular transcriptional profiles and functional characteristics. Unlike conventional juvenile hormone esterase genes (JHEs), the SnJHER gene family seems to have been recently evolved from a common ancestral JHER gene. SnJHERs seem to be regulated by both ecdysone agonists and xenobiotics, while their real role in development remains to be exploited. In this study we transiently expressed the major SnJHER isoform in Bm5 and Hi5 cell lines. The JHER-expressing cell lines showed increased toxicity when treated with the juvenile hormone analog methoprene. Moreover baculovirus-mediated transient gene transduction of the SnJHER gene in larvae of S. nonagrioides resulted in moulting abnormalities. These were more marked after the additional application of the juvenile hormone analog methoprene. Our results indicate a potential mechanism by which SnJHER interferes with normal JHE.

Functional characterization of a juvenile hormone esterase related gene in the moth Sesamia nonagrioides through RNA interference.

Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer’s larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.

Molecular cloning and characterization of a juvenile hormone esterase gene from brown planthopper, Nilaparvata lugens

Juvenile hormone (JH) plays key roles in the regulation of growth, development, diapause and reproduction in insects, and juvenile hormone esterase (JHE) plays an important role in regulating JH titers. We obtained a full-length cDNA encoding JHE in Nilaparvata lugens (NlJHE), the first JHE gene cloned from the hemipteran insects. The deduced protein sequence of Nljhe contains the five conserved motifs identified in JHEs of other insect species, including a consensus GQSAG motif that is required for the enzymatic activity of JHE proteins. Nljhe showed high amino acid similarities with Athalia rosae JHE (40%) and Apis mellifera JHE (39%). Recombinant NlJHE protein expressed in the baculovirus expression system hydrolyzed [ 3H] JH III at high activity and yielded the specificity constants ( k cat/ K M = 4.28 × 10 6 M −1 s −1) close to those of the validated JHEs from other insect species, indicating that Nljhe cDNA encodes a functional JH esterase. The Nljhe transcript was expressed mainly in the fat body and the expression level reached a peak at 48 h after ecdysis of the 5th instar nymphs. In the 5th instar, macropterous insects showed significantly higher Nljhe mRNA levels and JHE activities, but much lower JH III levels, than those detected in the brachypterous insects soon after ecdysis and at 48 h after ecdysis. These data suggest that NlJHE might play important roles in regulation of JH levels and wing form differentiation.

Tissue‐specific regulation of juvenile hormone esterase gene expression by 20‐hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.

Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti

Juvenile hormone esterase (JHE) plays an important role in regulating juvenile hormone titers. Recent sequencing and annotation of the Aedes aegypti genome identified ten putative jhe gene sequences. Analysis of these ten putative jhe gene sequences showed that only three of them, EAT43357, EAT43353 and EAT43354 contained GQSAG motif and showed high sequence similarity with the sequences of jhe genes identified from other insect species. To determine which putative jhe gene(s) code for functional JHE, the mRNA profiles of EAT43357, EAT43353 and EAT43354 were measured during the final instar larval and pupal stages by using quantitative real-time reverse transcriptase polymerase chain reaction (PCR). The mRNA for EAT43357 was detected during the late final instar larval stage. In contrast, EAT43354 mRNA was detected only during the pupal stage and EAT43353 mRNA was detected only during the larval stage. The mRNA of EAT43357 was detected in both fat body and midgut tissues. JHE enzyme levels gradually increased during the final instar larval stage reaching a peak at 42 h after ecdysis into the final instar larval stage. The mRNA expression profiles of EAT43357 correlate with the developmental expression profiles of JHE enzyme activity suggesting that this gene may encode for a functional JHE. The EAT43357 and EAT43354 cDNA were expressed in a baculovirus system. Proteins isolated from Sf9 cells infected with recombinant baculovirus expressing EAT43357 but not EAT43354 gene exhibited JHE activity confirming that EAT43357 gene codes for a functional JHE enzyme.

Interactions of ultraspiracle with ecdysone receptor in the transduction of ecdysone‐ and juvenile hormone‐signaling

Analyses of integration of two-hormone signaling through the vertebrate nuclear hormone receptors, for which the retinoid X receptor is one partner, have generated a number of mechanistic models, including those described as 'subordination' models wherein ligand-activation of one partner is subordinate to the liganded state of the other partner. However, mechanisms by which two-hormone signaling is integrated through invertebrate nuclear hormone-binding receptors has not been heretofore experimentally elucidated. This report investigates the integration of signaling of invertebrate juvenile hormone (JH) and 20-OH ecdysone (20OHE) at the level of identified nuclear receptors (ultraspiracle and ecdysone receptor), which transcriptionally activate a defined model core promoter (JH esterase gene), through specified hormone response elements (DR1 and IR1). Application of JH III, or 20OHE, to cultured Sf9 cells transfected with a DR1JHECoreLuciferase (or IR1JHECoreLuciferase) reporter promoter each induced expression of the reporter. Cotreatment of transfected cells with both hormones yielded a greater than additive effect on transcription, for especially the IR1JHECoreLuciferase reporter. Overexpression in Sf9 cells of recombinant Drosophila melanogaster ultraspiracle (dUSP) fostered formation of dUSP oligomer (potentially homodimer), as measured by coimmunoprecipitation assay and electrophoretic mobility assay (EMSA) on a DR1 probe, and also increased the level of transcription in response to JH III, but did not increase the transcriptional response to either 20OHE treatment alone or to the two hormones together. Inapposite, overexpression of recombinant D. melanogaster ecdysone receptor (dEcR) in the transfected cells generated dUSP/dEcR heterodimer [as measured by EMSA (supershift) on a DR1 probe] and increased the transcriptional response to 20OHE-alone treatment, but did not increase the transcriptional response to the JH III-alone treatment. Our studies provide evidence that in this model system, JH III-activation of the reporter promoter is through USP oligomer (homodimer) that does not contain EcR, while the 20OHE-activation is through the USP/EcR heterodimer. These results also show that the integration of JH III and 20OHE signaling is through the USP/EcR heterodimer, but that when the EcR partner is unliganded, the USP partner in this system is unable to transduce the JH III-activation.

Developmental and hormonal regulation of juvenile hormone esterase gene in Drosophila melanogaster

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to study developmental expression and hormonal regulation of the juvenile hormone esterase gene ( DmJhe) in the fruit fly, Drosophila melanogaster. The levels of DmJhe mRNA were low during the embryonic stage. A peak of Dmjhe mRNA was detected in the first, second and third instar larvae. The Dmjhe mRNA levels also increased soon after pupal ecdysis. The Dmjhe mRNA was detected in both male and female adult flies. The peaks of Dmjhe mRNA observed in the larvae coincided with the peaks of juvenile hormone (JH). In contrast, the mRNA for ecdysone-induced transcription factor , Drosophila hormone receptor 3 (DHR3) showed peaks of expression that coincided with the ecdysteroid peaks in embryo, larva and pupa. JH III induced Dmjhe mRNA but not DHR3 mRNA in explanted tissues cultured in Grace's medium. 20-hydroxyecdysone induced DHR3 mRNA and suppressed JH III induction of DmJhe mRNA. These studies show that the expression of jhe in D. melanogaster is regulated by both JH and 20E.

Identification and Characterization of a Juvenile Hormone (JH) Response Region in the JH Esterase Gene from the Spruce Budworm, Choristoneura fumiferana

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.

Identification of a juvenile hormone esterase gene by matching its peptide mass fingerprint with a sequence from the Drosophila genome project

Juvenile hormone esterase (JHE, EC 3.1.1.1) from whole Drosophila melanogaster prepupae has previously been purified by selective precipitations, isoelectric focussing and two column chromatography steps. JHE bands from dried silver-stained SDS–PAGE gels of that material were digested with trypsin. The masses of the tryptic digest peptides were determined by MALDI–TOF mass spectrometry. Only one predicted gene product (CG8425) from the D. melanogaster genome matches the JHE tryptic fingerprint with high confidence. This predicted JHE sequence includes features that are conserved among all active members of the serine carboxylesterase multigene family as well as features peculiar to JHEs from other species. Also we show that this JHE can be purified by an alternative method using anion exchange chromotography followed by trifluoromethylketone affinity chromatography. A cDNA encoding this JHE was isolated using 3′ and 5′ RACE. This sequence is in agreement with the Drosophila genome project's prediction except that the sixth predicted intron is not removed; instead there is a stop codon followed by a polyadenylation signal and a polyA tail.

Regulation of the juvenile hormone esterase gene by a composite core promoter

Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5' region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5'-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the 'downstream promoter element' described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.

Regulation of the juvenile hormone esterase gene by a composite core promoter

Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5ʹ region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5ʹ-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the ‘downstream promoter element’ described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.

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 57 kDa 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.

Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif.

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the 'downstream element') that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.

Molecular Population Genetics of Sodium Channel and Juvenile Hormone Esterase Markers in Relation to Pyrethroid Resistance in Heliothis virescens (Lepidoptera: Noctuidae)

Pyrethroid insecticide resistance in strains of tobacco budworm, Heliothis virescens (F.), has been linked to a locus hscp , which encodes the target of action of pyrethroids, a voltage-gated sodium channel (α subunit). Using denaturing gradient gel electrophoresis, we examined allelic variation for the DNA marker locus Hpy within this gene, and for an additional marker locus Hejs within a juvenile hormone esterase gene, for population samples from the southern United States in 1990 and 1995. FST, the estimate of genetic variance among populations for Hpy was 0.041 (SE = 0.005), significantly greater than the FST of 0.001 (SE = 0.003) for Hejs , and other estimates reported for allozyme loci in a 1989 collection over a similar geographic range. The greater variance for Hpy suggests that selection had been acting at or near the hscp locus. There was a significant excess of Hpyl, 3 , and 5 homozygotes in the 1990 collection, relative to Hardy–Weinberg expectation. However, there was also excess homozygosity for Hejs alleles, for reasons unknown. Hpy3 , the 3rd most common allele of Hpy , significantly increased after pyrethroid selection on Texas and Louisiana samples of 1990, and on progeny of a Louisiana sample of 1995. This indicates positive linkage disequilibrium between Hpy3 and presumed resistance-conferring allele(s) at another locus. Disequilibrium has persisted from 1990 to 1995. We conclude that one or more mutations elsewhere in the hscp gene confer resistance to pyrethroids in populations of H. virescens .