Our understanding of JH biosynthesis has significantly changed in the last years. In this review I would like to discuss the following topics: (1) the progresses in understanding the JH biosynthesis pathway. Access to genome sequences has facilitated the identification of all the genes encoding biosynthetic enzymes and the completion of comprehensive transcriptional studies, as well as the expression and characterization of recombinant enzymes. Now the existence of different flux directionalites, feed-back loops and pathway branching points in the JH biosynthesis pathways can be explored; (2) the new concepts in the modulation of JH synthesis by allatoregulators. The list of putative JH modulators is increasing. I will discuss their possible role during the different physiological states of the CA; (3) the new theoretical and physiological frameworks for JH synthesis analysis. I will discuss the bases of the flux model for JH biosynthesis. JH plays multiple roles in the control of ovary development in female mosquitoes; therefore, the CA presents different physiological states, where JH synthesis is altered by gating the flux at distinctive points in the pathway; (4) in the final section I will identify new challenges and future directions on JH synthesis research.
Juvenile hormone (JH) regulates development and reproductive maturation in insects [
Our understanding of JH biosynthesis has significantly changed over the past few years. Access to genome sequences has facilitated the identification of all the genes encoding JH biosynthetic enzymes [
This review will emphasize the work that has been done on the biosynthesis of JH III in the mosquito
JHs are lipophilic molecules commonly produced and released into the hemolymph by the CA, generally a pair of endocrine glands connected to the brain [
The JHs are involved in reproduction, caste determination, behavior, stress response, diapause, and several polyphenisms [
To form active transcription factors, functionally specialized bHLH-PAS proteins, such as Met, pair with a partner of their family. JH-dependent interaction between Met and its partner Taiman/SRC requires the hormone to be bound to a specific ligand-binding site. Met binds JH and its mimics with high affinity through a well-conserved hydrophobic pocket within its PAS-B domain [
In mosquitoes, JH acts via Met to regulate posteclosion development of the fat body and plays a dual role. Thousands of genes are active when the JH titer is low and then are suppressed by the rising JH; other genes appear specifically when the JH titer is high [
JH is synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life [
JH biosynthesis pathway. The biosynthesis of JH III involves 13 enzymatic reactions that can be conventionally divided into early (MVAP) and late (JH-branch) steps. Metabolites are shown in bold and enzymes in italic. Chemical structures are in [
The early steps follow the MVAP to form farnesyl pyrophosphate (FPP) [
FPP synthases have been identified from several insects and are typically active as homodimers [
The enzymes of the MVAP are well conserved in eukaryotes; in insects all the MVAP enzymes seem to be encoded by single-copy genes, and identification of predicted amino acid sequences was possible based on sequence homology [
In the late steps of JH synthesis, conversion of FPP to farnesol (FOL) is catalyzed in
The order of the last two biosynthetic steps, methyl esterification and epoxidation, catalyzed by a JH acid methyltransferase (JHAMT) and an epoxidase (EPOX), differs between insect species [
The late steps of JH biosynthesis were generally considered to be JH-specific [
On the contrary, the last two enzymes of the pathway (JHAMT and EPOX) are encoded by single genes in most insect species and are expressed predominantly in the CA [
The development of simple methods for detailed analysis of enzymatic activities derived from insect CA is critical. Fluorescence approaches are simplifying the study of the ability of CA extracts and recombinant enzymes to metabolize MVAP and JH-branch intermediates
Regulatory signals control the CA at least at three different levels [
The correct allocation of nutrients between competing needs such as reproduction, growth, maturation, or flight is a vital component of an insect’s life-history strategy [
Three sources of nutrients provide energy and building blocks for the three distinct phases of ovarian development in
JH biosynthesis rates and ovarian development in female mosquitoes. Top panel: representative images of the progression of ovary development from emergence to 24 h after blood feeding. The inset in 96 h shows the lipid content of follicles from females fed 3% sugar (top) and 20% sugar (bottom). Colors for the panels match the colors for the nutrition-dependent physiological states of the CA shown in the panel below. Bottom panel: JH biosynthesis by CA dissected from pupa, sugar-fed, and blood-fed adult females. Hours represent times before (pupa) and after adult emergence (sugar-fed), or after blood feeding (BF).
Comprehensive studies of transcripts, enzyme activities, and metabolites delimited four distinct nutrition-dependent CA physiological conditions that we named as follows: inactive, active, modulated, and suppressed CA (Figure
Recent detailed studies in sugar-fed females revealed a previously undetected peak of maximum JH synthesis 12 h after adult emergence (Figure
If mosquitoes are nutritionally stressed, by 48–72 h JH synthesis is significantly reduced. This period represents the beginning of the ORS and female mosquitoes often ingest sugar meals to supplement their partially depleted preimaginal reserves. During the ORS, if nutrients are scarce, the brain directs the CA to “adjust” to the new adult nutritional condition; in mosquitoes fed a restricted diet such as 3% sugar, JH synthesis decreases to a low 12 fmol/h, triggering the resorption of ovarian follicles [
A coordinated expression of most JH biosynthetic enzymes has been previously described in mosquitoes and silkworms [
There are factors that can stimulate (allatotropins) or inhibit (allatostatins) CA activity [
Three families of allatostatins have been identified in insects: cockroach allatostatins (YXFGL-amide or type-A), cricket allatostatins (W2W9 or type-B), and
The insulin/TOR signaling network is evolutionarily conserved in most eukaryotes and plays a central role in the transduction of nutritional signals that regulate cell growth and metabolism [
The
Application of bovine insulin on the mosquito CA-CC incubated
Starvation effects on insulin signaling components and JH synthesis in the CA of mosquitoes. This scheme summarizes starvation-related changes of insulin/TOR pathway components and JH synthesis. Molecules in red color are downregulated (↓), while those in green are upregulated (↑). Phosphoinositide 3-kinase (PI3K) and TOR are involved in the transduction of insulin signaling in the CA [
Several additional factors have been proposed to be involved in the regulation of JH biosynthesis by the CA, including biogenic amines, 20-hydroxyecdysone (20E), ecdysis triggering hormone (ETH), and short neuropeptide F (sNPF) [
ETH is a small C-terminally amidated peptide, known as a major regulator of ecdysis in insects [
The short neuropeptide F (sNPF), among other functions, modulates feeding, metabolism, reproduction, and stress responses in insects [
In mosquitoes, the role of each of these endocrine regulators might be limited to particular periods of CA activity. Developmental modulators such as ETH might play important roles during pupal maturation of the CA; insulin and/or allatotropin may well be the brain activators acting on the CA of the newly emerged female, while allatostatin-C and insulin could play a role in the nutritional modulation of JH synthesis during the “state of arrest,” as well as during the suppression of JH synthesis after a blood meal. In the CC-CA of mosquitoes, the expression of the following receptors has been detected: ETH A and B, ecdysone A and B, insulin, ultraspiracle A and B, allatotropin, AST-C A and B, and the short neuropeptide F. It is possible that signals from all these modulators are integrated in the CA, which suggests that the regulation of JH synthesis is extremely complex (Figure
Effect of modulators on JH biosynthesis in female mosquitoes. Schematic representation of some of the tissues and molecules involved in JH biosynthesis regulation in mosquitoes. PG: prothoracic gland. OV: ovaries. CC:
JH synthesis is controlled by the rate of flux of isoprenoids, which is the outcome of a complex interplay of changes in precursor pools, enzyme levels, and external modulators such as nutrients and allatoregulatory factors [
A schematic representation of a model for the control of the flux of precursors in the JH biosynthetic pathway. Precursor pools (S2, S3, etc.) are represented by circles and connected by arrows (MVA: mevalonic acid, 5P-MVA: mevalonate 5-phosphate). E: enzymes are followed by a number that refers to the position in the pathway (E3 = MK: mevalonate kinase). Regulatory factors might be affecting both precursor pool sizes and enzymatic activities (e.g., AST-C: allatostatin-C). JH: juvenile hormone, from [
The JH synthetic pathway involves 13 discrete enzymatic steps organized in an obligatory sequence. Each product represents the substrate for the next “downstream” enzyme. Enzymes are connected by metabolite pools that are common to them; for example, FOL is the product of the FPPase activity and the substrate for farnesol dehydrogenase. The pools are in fact the
Branch point regulation is an important mechanism controlling carbon flow in the MVAP; the FPP produced by the MVAP can be shunted to many metabolic branches for the synthesis of critical molecules such as ubiquinone, dolichol, or prenylated proteins [
Compartmentalization of the enzymatic steps might add an additional level of complexity. Studies in plants have emphasized the importance of compartmentalization in the control of terpene biosynthesis [
Metabolic enzymes that catalyze a series of successive reactions can form complexes on membranes or cytoskeletal structures [
What do integrated studies of CA transcripts, enzyme activities, and metabolites tell us about the coordination of MVAP and JH-branch activities? A comprehensive analysis of the JH biosynthetic pathway has been done in
Heat map representation of changes in JH biosynthetic enzyme mRNAs and activities in CA extracts. (a) Changes in mRNAs encoding JH biosynthetic enzymes. (b) Changes in activities of JH biosynthetic enzymes in CA extracts. Top: physiological stages are described as hours relative to adult emergence (0 h) or blood feeding (BF). Right side: enzyme names abbreviations: Acetoacetyl-CoA thiolase: thiolase; HMG-CoA synthase: HMGS; HMG-CoA reductase: HMGR; mevalonate kinase: MK; phosphomevalonate kinase: PMK; diphosphomevalonate decarboxylase: PPM-Dec; IPP isomerase: IPPI; FPP synthase: FPPS; farnesyl pyrophosphatase: FPPase; farnesol dehydrogenase: FOL-SDR; farnesal dehydrogenase: FALDH; juvenile hormone acid methyltransferase: JHAMT; and methyl farnesoate epoxidase: EPOX. Colors from white to red represent increases of transcript levels or enzymatic activities as percentages of the maximum value, from [
Schematic representation of the distinct four CA physiological conditions in reproductive female mosquitoes. The four CA phases and corresponding stages are as follows: inactive (early pupae), active (12–24 h sugar-fed females), modulated (48–96 h sugar-fed females), and suppressed (24 h blood-fed females). JH synthesis: the color and direction of the arrows reflect the following: low levels (arrows down and red), high levels (arrows up and black), or variable levels (arrow up and down). Changes in transcripts, activities, and metabolites are as follows: the direction of the arrows reflects the trend of the changes (increases: up and decreases: down); the size of the arrow reflects the magnitude of the changes, limiting factor: hypothetical critical factor limiting CA activity, from [
Further studies will be necessary to discover what enzymes restrict the flux into JH III at specific physiological conditions.
JH has long been the focus of intensive research intended to exploit its properties for the purpose of generating novel pest control products. Earlier research on JH biosynthesis was performed mainly on three insect models: cockroaches, locusts, and moths. These insects offered several advantages for JH synthesis studies, such as the size of the CA, the relatively high levels of JH synthesized, and the easiness of rearing them in the laboratory. Cockroaches, in particular
To answer the questions that remain unanswered, we need to identify some of the next challenges and future directions on JH synthesis research. Although the general features of JH biosynthesis seem to be conserved in most insects, there is clearly diversity in aspects such as the presence of particular JH homologues, the order of the final enzymatic steps, and the role of allatoregulators; therefore JH biosynthesis studies need to be extended beyond the classic model insects. The identification of all the genes encoding JH biosynthetic enzymes has allowed the completion of comprehensive transcriptional studies, as well as the expression and characterization of recombinant enzymes. New methods are currently facilitating the analysis of JH biosynthesis rates, enzymatic activities, and metabolite pool sizes in the CA. In the future, we should improve our understanding of the occurrence of different flux directionalities, feedback loops, and pathway branching points in the JH biosynthesis pathway. More research on compartmentalization of JH synthesis is necessary, as well as a better understanding of signaling pathways in the CA, including calcium signaling pathways and the interactions among the insulin and TOR pathways. The list of putative JH modulators continues to increase, and new concepts in allatoregulator-modulation of JH synthesis under different physiological frameworks are emerging. The utilization of new statistical approaches, theoretical models, and system biology approaches should continue to simplify the interpretation of JH synthesis rates changes.
In summary, integrative approaches using CA metabolomics, genomics, and proteomics are promising tactics to identify regulatory points in the flux of precursors in the JH synthesis pathway and unveil the molecular mysteries of a complex metabolic system such as the synthesis of juvenile hormone in the
The author declares that there is no conflict of interests regarding the publication of this paper.
The author thanks Dr. Mark Clifton, Dr. Martin Edwards, Dr. Crisalejandra Rivera Perez, and Mr. Pratik Nyati for critical reading of the paper. This work was supported by NIH Grant no. AI 45545 to Fernando G. Noriega.