Juvenile hormone synthesis: "esterify then epoxidize" or "epoxidize then esterify"? Insights from the structural characterization of juvenile hormone acid methyltransferase.

Juvenile hormones (JHs) play key roles in regulating metamorphosis and reproduction in insects. The last two steps of JH synthesis diverge depending on the insect order. In Lepidoptera, epoxidation by a P450 monooxygenase precedes esterification by a juvenile hormone acid methyltransferase (JHAMT). In Orthoptera, Dictyoptera, Coleoptera and Diptera epoxidation follows methylation. The aim of our study was to gain insight into the structural basis of JHAMT's substrate recognition as a means to understand the divergence of these pathways. Homology modeling was used to build the structure of Aedes aegypti JHAMT. The substrate binding site was identified, as well as the residues that interact with the methyl donor (S-adenosylmethionine) and the carboxylic acid of the substrate methyl acceptors, farnesoic acid (FA) and juvenile hormone acid (JHA). To gain further insight we generated the structures of Anopheles gambiae, Bombyx mori, Drosophila melanogaster and Tribolium castaneum JHAMTs. The modeling results were compared with previous experimental studies using recombinant proteins, whole insects, corpora allata or tissue extracts. The computational study helps explain the selectivity toward the (10R)-JHA isomer and the reduced activity for palmitic and lauric acids. The analysis of our results supports the hypothesis that all insect JHAMTs are able to recognize both FA and JHA as substrates. Therefore, the order of the methylation/epoxidation reactions may be primarily imposed by the epoxidase's substrate specificity. In Lepidoptera, epoxidase might have higher affinity than JHAMT for FA, so epoxidation precedes methylation, while in most other insects there is no epoxidation of FA, but esterification of FA to form MF, followed by epoxidation to JH III.

Comparative genomics of insect juvenile hormone biosynthesis.

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).

Midgut exopeptidase activities in Aedes aegypti are induced by blood feeding.

Midgut extracts from Aedes aegypti females exhibited hydrolytic activities against synthetic substrates for carboxypeptidase A, carboxyopeptidase B and leucine-aminopeptidase. The three activities showed a broad pH optimum, with maximum activities at pH between 6.5 and 8.5. Enzymatic activities were further characterized by testing the effects of a variety of protease inhibitors. Captopril and 1-10-phenantroline inhibited the activities of carboxypeptidases A and B, while leuhistin, amastatin and bestatin inhibited aminopeptidase activity. Exopeptidase activities were induced by a blood meal and the highest activities were found during the peak of trypsin activity, about 20-24h after feeding. An amino acid meal failed to induce significant increases in any of the three exopeptidase activities. The amounts of exopeptidase activities induced were proportional to the protein concentration of the meal. The addition of soy-trypsin inhibitor to the protein meal blocked the post-feeding induction of exopeptidases. The features of the induction of synthesis of the three exopeptidase activities resembled the induction of synthesis of late trypsin during the second phase of digestion.

Neuroendocrine factors affecting the steady-state levels of early trypsin mRNA in Aedes aegypti.

Transcription of the early trypsin gene occurs in the midgut after adult emergence under control of juvenile hormone (JH). We tested the hypothesis that factors that affect the steady-state levels of early trypsin mRNA do so by influencing the levels of JH. We investigated the effect of ingesting different meals on early trypsin mRNA levels as well as on JH levels. We also studied how early trypsin mRNA levels changed when the midgut was isolated from different components of the neuroendocrine system by abdominal ligation and decapitation. Early trypsin transcripts levels are high in unfed females; feeding different meals had three distinct effects on the changes of steady-state levels of early trypsin mRNA: (1) blood and protein meals caused the level to decrease drastically and remained low for at least 24 h; (2) amino acid meals caused a transient decrease in the mRNA level, but it returned to high levels after 12-18 h; and (3) sugar, latex and saline meals had no effect on the early trypsin mRNA steady-state levels. The changes in JH levels after ingesting blood and amino acid meals show profiles resembling the changes in early trypsin mRNA levels for the corresponding meal. Decapitation at 1, 2 and 3 days after emergence does not affect the steady-state levels of early trypsin in unfed females. In contrast, 24 h after feeding, transcript levels were significantly higher in decapitated females when compared with non-decapitated fed females. We propose that the changes in the steady-state levels of early trypsin mRNA observed after the ingestion of different meals, ligations and decapitations are generated by changes in the levels of juvenile hormone.

Recombinant juvenile hormone esterase, an effective tool for modifying juvenile hormone-dependent expression of the early trypsin gene in mosquitoes.

The study of the changes in the steady-state levels of the early trypsin (ET) messenger RNA (mRNA) was used as a sensitive assay for measuring the effects of recombinant juvenile hormone esterase (rJHE) on juvenile hormone (JH)-dependent gene expression in mosquitoes. ET is a female-specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first few hours after ingestion of a blood meal. Transcription of the early trypsin gene is part of the normal postemergence maturation of the midgut in the adult female, and it is regulated by JH. JHE was cloned from Heliothis virescens and expressed in a baculovirus vector. Injection of rJHE into mosquitoes resulted in an increase of JHE activity in the haemolymph. Injection of rJHE into newly emerged adult females delayed the normal increase in steady-state levels of ET mRNA observed in controls. Topically applied methoprene (a JH analogue) reversed the effect of rJHE. Injection of increasing concentrations of rJHE into 3-day-old unfed females resulted in a dose-dependent decrease in the steady-state levels of ET mRNA after 24 h. The effect of rJHE was transient, once the enzyme was cleared (72 h after injection), the steady-state levels of ET mRNA were restored. The injection of rJHE is an effective tool for modifying JH-dependent expression of the early trypsin gene in mosquitoes.

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti.

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.

Increase in the size of the amino acid pool is sufficient to activate translation of early trypsin mRNA in Aedes aegypti midgut.

Early trypsin is a female-specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first 4-6 h after ingestion of a blood meal. Transcription of the early trypsin gene occurs after adult emergence under control of juvenile hormone, but the transcript remains untranslated before feeding. Early trypsin was in vitro translated using mRNA extracted from midguts of unfed and fed females, indicating that there are not structural features in the early trypsin mRNA that impede translation in vitro. Eight single protein meals exhibiting different molecular weights and amino acid composition, as well as ingestion of several amino acid mixtures of different complexity, had the ability to prompt early trypsin translation. In contrast, ingestion of saline, latex or midgut-filling sugars were unable to induce early trypsin mRNA translation. In addition intra-thoracic injection of an amino acid solution induced early trypsin translation, while injection of saline or albumin failed. In summary an increase in the size of the midgut amino acid pool by feeding or injection of an amino acid solution was sufficient to induce translation of early trypsin mRNA; 35S-labeled amino acids, fed with a protein meal, were incorporated into newly synthesized early trypsin; the first phase of trypsin synthesis is likely induced by an initial rapid increase in the concentration of amino acids in the midgut cells after ingestion of a blood meal.

A molecular view of trypsin synthesis in the midgut of Aedes aegypti.

Ingestion of a blood meal induces two phases of trypsin synthesis in the midgut of Aedes aegypti females. The first phase, which encompasses the first 4-6 hours following a blood meal, is characterized by the presence of small amounts of early trypsin. The second phase, which occurs between 8 and 36 hours after blood feeding, is characterized by the presence of large amounts of late trypsin. A specific form of regulation of trypsin synthesis characterizes each phase: early trypsin synthesis is regulated at the translational level, while late trypsin synthesis is regulated at the transcriptional level.The enzymatic activity of early trypsin plays a unique and critical role in the regulation of late trypsin synthesis. Early trypsin acts like a "sensor". It carries out limited proteolysis of the ingested proteins and, somehow, the products of this limited proteolysis induce synthesis of late trypsin, which is the protease responsible for the majority of the endoproteolytic cleavage of the meal proteins.Transcription of the early trypsin gene starts a few hours after adult emergence and is under control of juvenile hormone. However, the early trypsin mRNA is stored in the midgut epithelium and remains untranslated until a blood meal is taken. The exact mechanism responsible for initiating translation is presently unknown, but an increase in the size of the amino acid pool in the midgut is sufficient to activate translation of early trypsin mRNA.The transcription of the late trypsin gene is regulated by uncharacterized proteolysis products generated by the action of early trypsin on the blood meal proteins. Once transcription has been activated, the rate of transcription of the late trypsin gene is proportional to the amount of protein present in the meal. In addition, the amount of late trypsin protein translation is controlled by the amount of amino acid released during digestion. Regulation at both transcriptional and translational levels allows the midgut to adjust the amount of late trypsin with remarkable flexibility in response to a particular meal.

cDNA cloning and pattern of expression of an adult, female-specific chymotrypsin from Aedes aegypti midgut.

A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the mosquito Aedes aegypti. The 938 base sequence codes for a 268 amino acid protein, which contains an 18-residue signal peptide and a seven-residue activation peptide. The deduced amino acid sequence contains several features typical of chymotrypsin proteases, including the catalytic triad of serine proteases and the residues that determine the chymotrypsin substrate specificity pocket. The chymotrypsin mRNA, absent in larvae, pupae, males and newly emerged females, reaches detectable levels within 24 h post-emergence and attains a maximum level 3-7 days after emergence. Translation of the chymotrypsin mRNA is induced by feeding a protein meal, and there is a dramatic increase in midgut chymotrypsin enzymatic activity after feeding. Chymotrypsin activity remained high during protein digestion, but chymotrypsin protein levels and enzymatic activity were almost undetectable once digestion was completed, 48 h after feeding.

Juvenile hormone controls early trypsin gene transcription in the midgut of Aedes aegypti.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first few hours after ingestion of a blood meal. The enzymatic activity of early trypsin plays an essential role in the transcriptional activation of the late trypsin gene, which encodes the major midgut endoprotease involved in blood meal protein digestion. Transcription of the early trypsin gene is part of the normal post-emergence maturation of the midgut in the adult female. Abdominal ligation within 1 h of emergence completely prevented the transcription of the early trypsin gene. Topically applied JH III or methoprene induced transcription of the early trypsin gene in ligated abdomens to levels similar to those observed in non-ligated females. The induction of early trypsin transcription by JH is dose-dependent and 'head-independent', suggesting that factors coming from the neuro-secretory axis are not required.

Identification of three allatostatins and their cDNA from the mosquito Aedes aegypti.

Three allatostatins have been isolated from the mosquito Aedes aegypti. These peptides have the following structures: Ser-Pro-Lys-Tyr-Asn-Phc-Gly-Leu-amide, Leu-Pro-His-Tyr-Asn-Phe-Gly-Leu-amide, and Arg-Val-Tyr-Asp-Phe-Gly-Leu-amide. A cDNA encoding these peptides was isolated from an abdominal ganglia cDNA library and sequenced. It was found to encode two additional allatostatins: Ala-Ser-Ala-Tyr-Arg-Tyr-His-Phe-Gly-Leu-amide and Leu-Pro-Asn-Arg-Tyr-Asn-Phe-Gly-Leu-amide. Northern analysis of whole mosquito mRNA revealed a single prepro-allatostatin message of around 3,000 bases. Identification of a partial prepro-allatostatin cDNA from a midgut cDNA library shows that the same gene is also expressed in the mosquito midgut.

Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, aminoterminal sequence determination, and cloning and sequencing of the gene.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. It plays an essential role in the transcriptional activation of the late trypsin form, the major midgut endoprotease involved in the blood meal digestion. Early trypsin is the most abundant midgut polypeptide isolated by benzamidine-sepharose affinity chromatography 3 h after feeding. The amino-terminal sequence of the early trypsin protein matches that of the 3a1 cDNA for a putative trypsinogen described by Kalhok et al. (Insect. Molec. Biol., 2, 71-79, 1993). The early trypsin cDNA was over expressed in Escherichia coli. Polyclonal antibodies generated against this recombinant protein were used to show that the enzyme was present in the midgut during the first 4 h after feeding. A 2.5 kb genomic clone of the early trypsin was isolated, mapped and subcloned. A 1.56 kb subclone, corresponding to 1303 bp of the upstream regulatory region and 265 bp of the coding region, was sequenced. The gene contains a 64 nucleotide intron which interrupts the codon for Val at position 18 of the protein. This Val is located toward the end of the putative signal sequence of the protein.

Aedes aegypti midgut early trypsin is post-transcriptionally regulated by blood feeding.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal.

Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti.

Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility.

Dietary control of late trypsin gene transcription in Aedes aegypti.

In Aedes aegypti the levels of midgut trypsin activity after feeding are directly proportional to the protein concentration in the meal. The mechanisms of this up-regulatory event were investigated by analyzing the expression of the late trypsin gene under different dietary conditions. Transcription of the gene was dependent on both the quality and quantity of protein in the meal. As measured by Northern blot analysis, the levels of late trypsin gene expression increased up to 100-fold 24 h after feeding on gamma-globulin, hemoglobin or albumin (100 mg/ml). In contrast, gelatin, histone, amino acids, saline or agarose were very poor inducers of transcription. The rates of late trypsin transcription induced during the first 24 h were directly proportional to the concentration of protein in the meal. These data further support the suggestion that the primary mechanism that regulates the synthesis of trypsin in the mosquito midgut is transcriptional regulation of the gene. This regulatory mechanism enables the midgut to maintain the appropriate balance between protease synthesis and the protein content of the meal.

A comparison of three methods for isolating RNA from mosquitoes.

The two most common protocols for RNA isolation, Chirgwin et al., 1979, and Chomczynski & Sacchi, 1987, were compared with a protocol using glass powder. The quality of the recovered RNA was tested using agarose gels and Northern hybridization. With the glass powder protocol, we consistently obtained intact RNA, whereas with the other protocols we often obtained degraded RNA. Using specific radioactive probes, we measured ribosomal RNA and trypsin mRNA recovered from fed adult Aedes aegypti females. Our protocol gave a 40- and a 3-fold increase in recovery when compared with the Chirgwin and Chomczynski protocol, respectively. The method using glass powder is simple, fast, reproducible and gives a high yield of intact RNA.

Autogeny in three species of Triatominae: Rhodnius prolixus, Triatoma rubrovaria, and Triatoma infestans (Hemiptera: Reduviidae).

Autogeny, the capacity of a female to lay eggs without having ingested any blood meal in the adult stage, was studied in three species of Triatominae: Triatoma infestans, Triatoma rubrovaria, and Rhodnius prolixus. When nymphs of T. rubrovaria and R. prolixus were fed chicken blood, autogeny occurred frequently, even with only one meal. In T. infestans autogeny was frequent as well, but demanded at least two nymphal blood meals. Total number of autogenic eggs was positively correlated with the body weight of the adult female. We conclude, therefore, that autogeny is common in Triatominae.

Ecdysteroids: biochemical mechanism of 20-hydroxyecdysone inactivation in Triatoma infestans (Hemiptera, Reduviidae).

When 3H-20-hydroxyecdysone was injected into Triatoma infestans during the fifth nymphal instar, it was converted to very polar metabolites (peak 1). The polar metabolites were glucuronoconjugates of 20-hydroxyecdysone. Acetylation of the conjugates followed by hydrolysis with glucuronidase gave a major labelled product: 20-hydroxyecdysone and minor peaks corresponding to a mixture of 20-hydroxyecdysone acetates. These results indicated that the 2,3,22,25-glucuronoconjugates of 20-hydroxyecdysone were the principal products in the inactivation process.

Ecdysteroids: biochemical mechanism of 20-hydroxyecdysone inactivation in Triatoma infestans (Hemiptera, Reduviidae).

Al inyectar 20-OH-ecdisona-3H en el 5o.estadio ninfal de T. infestans, se observo la transformacion de la misma a metabolitos muy polares (pico I). La mayor parte de los constituyentes del pico I evidenciaron ser glucurono-conjugados al ser analizados por hidrolisis enzimaticas. La acetilacion de los conjugados seguida por hidrolisis enzimatica permitio identificar las posiciones ocupadas por el acido glucuronico al inactivar la 20-OH-ecdisona.Al inyectar los conjutados radiactivos aislados (pico I) en ninfas del 5o. estadio, se observo que no se revierte el proceso de inactivacion

Ecdysteroids: biochemical mechanism of 20-hydroxyecdysone inactivation in Triatoma infestans (Hemiptera, Reduviidae).

Al inyectar 20-OH-ecdisona-3H en el 5o.estadio ninfal de T. infestans, se observo la transformacion de la misma a metabolitos muy polares (pico I). La mayor parte de los constituyentes del pico I evidenciaron ser glucurono-conjugados al ser analizados por hidrolisis enzimaticas. La acetilacion de los conjugados seguida por hidrolisis enzimatica permitio identificar las posiciones ocupadas por el acido glucuronico al inactivar la 20-OH-ecdisona.Al inyectar los conjutados radiactivos aislados (pico I) en ninfas del 5o. estadio, se observo que no se revierte el proceso de inactivacion