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Hormones
Insect Biochemistry/Hormones P Y Scaraffia and R L Miesfeld, University of Arizona, Tucson, AZ, USA ã 2013 Elsevier Inc. All rights reserved.
Glossary Gonotrophic cycle Reproductive process of ovarian development and egg laying in blood-feeding insects.
Overview of Insects Insects are the most abundant multicellular organisms on planet Earth and are thought to account for >70% of all species. So far, over 1 million insect species have been identified, with estimates putting the total number of insect species worldwide at 6–10 million. Insects are invertebrates that represent a class of arthropods characterized by three pairs of jointed legs; a three-part body consisting of a head, thorax, and abdomen; compound eyes; a chitin exoskeleton; and two antennae. Holometabolous insects hatch from eggs and progress through three distinct life stages. Newly hatched eggs produce larvae that go through metamorphosis, which is separated by molts. Since the chitin exoskeleton cannot accommodate the growing insect, molting provides a way for insects to shed their exoskeleton multiple times until they reach the pupal stage in which they complete metamorphosis in a protected shell called a pupal case. In the final developmental stage, the adult insect emerges from the pupal case, and within a few hours or days, females and males mate to produce fertilized eggs. In some other insects (hemimetabolous), the adult form from the hatching can be reached gradually with successive molts during a process called incomplete metamorphosis (cockroaches and grasshoppers). In this type of development, the immature stages (nymphs, or when aquatic, naiads) are like the adults except in size, flight, and sexual maturity. Endocrine regulation in insects relies on a large number of peptide and nonpeptide hormones, several of which have been studied in great detail. Three of the best-characterized insect hormones are ecdysone, juvenile hormone (JH), and prothoracicotropic hormone (PTTH), all of which play important roles in insect metamorphosis and reproduction. Ecdysone, and most likely JH, bind to receptor proteins that function as transcription factors, which directly or indirectly regulate the expression of specific target genes. In contrast, PTTH binds to a membranebound receptor tyrosine kinase in the prothoracic gland of immature insects and initiates an intracellular phosphorylation cascade that activates ecdysone synthesis. Ecdysone synthesis in adult insects occurs primarily in the ovaries in response to the brain neuropeptide ovarian ecdysteroidogenic hormone.
Insect Biochemistry Insect Metabolomics Insects provide an ideal model system to investigate metabolic regulation in multicellular organisms because they can often
590
Hemolymph The circulatory fluid of insects, similar to blood or lymphatic fluid in other organisms. Holometabolous insects Insects that undergo complete metamorphosis from eggs to larvae to pupae to adults.
be easily manipulated for biochemical studies, and for some of the most important insect species, whole genome sequence information is readily available, which greatly facilitates molecular genetic approaches. Metabolomics is the systematic study of small molecules that are utilized or generated by metabolic processes in living cells. The most common type of metabolomic study involves collecting biochemical samples from cell or tissue cultures, or whole organisms, under various conditions that can be quantitatively and qualitatively analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. Based on bioinformatic analysis of genome sequences, it is often possible to predict what metabolic pathways in insects are responsible for changes in metabolite profiles as a consequence of environmental signals or genomic variations in metabolic enzymes. One example of how insect biochemistry can be investigated using a metabolomics approach is the mass spectrometrybased study of the rosy (ry) mutation in Drosophila melanogaster. The ry mutation maps to the xanthine oxidase gene that encodes an enzyme required to convert xanthine, a product of purine degradation, to uric acid. In D. melanogaster, loss of xanthine oxidase causes a reddish brown eye color due to a deficiency in red pigment, which is how this mutation was first identified. In humans, xanthine oxidase deficiency leads to a condition called xanthinuria that is characterized by xanthine accumulation in the urinary tract and kidney. Using wholebody extracts of D. melanogaster wild-type and ry mutant flies, mass spectrometric analysis revealed significant increases in xanthine and hypoxanthine, as well as higher levels of guanine metabolites and biopterins. Surprisingly, the ry mutant defect in xanthine oxidase was associated with a 15-fold increase in glycerophosphocholine levels, presumably due to osmotic stress caused by the inability to excrete uric acid. Another example of how metabolomics have been used to investigate biochemical processes in insects, was a recent study in which larvae of the Antarctic midge Belgica antarctica were exposed to high heat, freezing temperatures, or desiccation, and the levels of 75 metabolites were analyzed by mass spectrometry. It was found that the steady-state level of a variety of sugars, amino acids, and citric acid cycle intermediates were differentially affected by these extreme environmental conditions. Specifically, levels of alanine, aspartate, mannitol, and urea were elevated after 6 h of freezing conditions (–10 ºC), whereas glycine and serine levels were decreased. Similarly, it was found that levels of a-ketoglutarate and putrescine were elevated after 1 h at 30 ºC, but glycerol, serine, and glucose
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
levels were decreased after a 1 h heat shock. Since serine levels decreased under both conditions of extreme cold and heat, as well as when the larvae were subjected to desiccating conditions, it was proposed that decreased serine levels may be a marker of environmental stress in this insect species.
Blood Meal Metabolism in Mosquitoes Because of their role as biological vectors for many of the arthropod-borne infectious diseases in humans, mosquitoes have been extensively studied at the biochemical level. Mosquitoes are responsible for transmitting not only malarial parasites and a variety of arboviruses, but also nematode worms that cause lymphatic filariasis. It is estimated that 40% of the world’s population is at risk of being infected by malaria, with 300 million people being infected each year and over 1 million deaths. Malaria is primarily transmitted by the mosquito species Anopheles gambiae in Africa, Anopheles albimanus in South America, and by Anopheles stephensi in Asia. The other major mosquito-borne disease affecting humans is Dengue fever, which can develop into Dengue hemorrhagic fever and be fatal. Aedes aegypti mosquitoes transmit Dengue virus and are one of the most rapidly spreading human disease vectors in the world. It is estimated that up to 3 billion people in the Americas, Africa, and Asia are at risk of being infected by Dengue virus, and that of the nearly 50 million new Dengue infections each year, 20 000 deaths will occur from Dengue hemorrhagic fever. Newly emerged Ae. aegypti female mosquitoes feed on nectar for several days until they are able to take their first blood meal (males do not blood feed). The blood meal is required for Ae. aegypti egg development and results in the deposition of 100 fertilized eggs within 72 h of feeding. This process of blood digestion, oocyte maturation in the ovaries, and the laying of fertilized eggs is called the gonotrophic cycle. In the course of each gonotrophic cycle, blood meal metabolism requires efficient retrieval of nutrients, and rapid excretion of toxic products such as ammonia (defined here as NH3, NHþ 4, or combination of both), in order to produce viable eggs. This is an amazing accomplishment considering the mosquito’s size. A typical female mosquito weighs 2 mg and can consume a blood meal of 2ml in few minutes. Taking into account the mass of water ( 1.6mg), protein ( 400 mg), lipid ( 10 mg), and carbohydrate ( 1 mg) in this 2-ml blood meal, it can be
calculated that the female mosquito consumes her own body weight in a single feeding. This feat would be equivalent to a 125 lb woman drinking a 12 gallon smoothie that contains 25 lbs of hamburger meat, 0.5 lb of butter, and 2 tbls of sugar. As metabolically challenging as blood meal digestion must be, the female Ae. aegypti mosquito is highly capable and typically completes 4–7 gonotrophic cycles to produce up to 400 mosquito progeny over a 1-month period. The vast majority of nutrients, in a typical mosquito blood meal, are derived from blood meal proteins, of which three constitute 80% of the total protein. These are hemoglobin ( 330mg), serum albumin ( 50 mg), and Igg antibodies ( 15 mg). Biochemical and molecular genetic studies have shown that mosquito blood meal digestion is initiated by endoprotease and exopeptidase enzymes that are secreted into the lumen by midgut epithelial cells within 30 min of feeding. Although mosquitoes require 10 of the 20 naturally occurring amino acids in their diet (essential amino acids), metabolic labeling studies using 14C-protein revealed that only 14% of the ingested protein is recycled into newly synthesized maternal proteins or egg proteins (Figure 1). In contrast, 70% of the amino acids retrieved from the blood meal are deaminated to generate reduced carbon that is used for immediate energy needs or excreted as waste. Blood meal-derived amino acids are also used for lipogenesis to supply stored energy for the female mosquito (10%), and converted to egg lipids that are used to complete oocyte maturation ( 6%).
Urea Synthesis in Ae. aegypti Excess nitrogen cannot be stored in cells, and therefore, nitrogen balance must be highly regulated to prevent ammonia toxicity, while at the same time, providing sufficient nitrogen for the biosynthesis of amino acids, nucleotide bases, and coenzymes. Aquatic organisms excrete most of their excess nitrogen directly into water as ammonia, whereas animals that spend all or part of their life cycle on land excrete nitrogen as uric acid or urea. Mammals synthesize urea via a metabolic pathway called the urea cycle that derives the two nitrogen atoms in urea from carbamoyl phosphate and aspartate. Certain amphibians and some fish do not express urea cycle enzymes; however, the urea they produce is the result of uric acid degradation by the uricolytic pathway (Figure 2). The
Blood meal
CO2 Energy conversion NH3 Reduced carbon
Protein + Lipid + Carbohydrates
Amino acids Protein synthesis
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Lipid synthesis
Oxidative metabolism and waste (~70%)
Maternal lipids (~10%) Egg lipids (~6%)
Egg proteins (~4%) Maternal proteins (~10%)
Figure 1 Blood meal metabolism in female Aedes aegypti mosquitoes. The major nutrient in the mosquito blood meal is protein, which is converted to amino acids that are either deaminated and used as reduced carbon for energy conversion and lipid synthesis, or used directly for the synthesis maternal and egg proteins.
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Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
O H N
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2 H2O + O2
CO2 + H2 O2 NH2
O
H N
N H
N H
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O Urate oxidase
N N H H Uric acid
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O
Allantoin H2O
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COOH + CH O
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NH2 + O H2 N
Urea
O Urea
Allantoicase O 2 H2O
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COOH NH2
N N H H Allantoic acid
O
Figure 2 The uricolytic pathway converts uric acid into urea. Feeding experiments in Aedes aegypti mosquitoes with 15NH4Cl have been used to quantitate uricolytic pathway intermediates by mass spectrometry (bolded N atoms were 15N-labeled in these experiments). RNAi-mediated knockdown of urate oxidase expression in blood-fed mosquitoes confirmed that the uricolytic pathway is functional under normal physiological conditions.
uricolytic pathway converts uric acid to urea and glyoxylic acid through a three-step process requiring the enzymes urate oxidase, allantoinase, and allantoicase. Early metabolic studies of nitrogen metabolism in blood-fed Ae. aegypti mosquitoes showed that excess nitrogen produced by blood meal digestion is excreted as ammonia, amino acids, uric acid, and urea. Based on studies in vertebrates, it was assumed that the urea present in mosquito feces was produced by converting excess dietary arginine into urea via the arginase reaction. However, by using a combination of 15 N-isotope labeling experiments, bioinformatic analysis of the Ae. aegypti genome sequence, and RNAi-mediated knockdown of urate oxidase expression, it was recently shown that a portion of this excreted urea is in fact derived from the uricolytic pathway. As illustrated in Figure 2, mass spectrometric analysis of uric acid containing 15N-labeled in two positions revealed that uric acid is metabolized to 2 mol of 15N-labeled urea, thereby providing biochemical evidence that the uricolytic pathway is functional in Ae. aegypti mosquitoes. In addition, RNAimediated knockdown of urate oxidase expression in 15NH4Clfed mosquitoes was found to significantly decrease the amount of 15N-urea in the feces. Similarly, knocking down urate oxidase expression in blood-fed mosquitoes resulted in a buildup of uric acid in mosquito tissues. Given the unusually high-protein content of the mosquito blood meal, it is likely that the uricolytic pathway provides a mechanism for the adult female mosquito to maximize nutrient uptake while minimizing the risk of ammonia toxicity.
Insect Hormones Endocrine regulation in insects is complex with many different types of hormones involved, including steroids (ecdysone), lipid compounds (JH), multiple peptide hormones (PTTH; ovarian ecdysteroidogenic hormone, OEH; adipokinetic hormone, AKH), and biogenic amines (serotonin, DOPA). The discussion here focuses on the two major gonadotropins, ecdysone and JH, and the peptide hormone PTTH, which
regulates ecdysteroidogenesis in the larval and pupal prothoracic gland. Insect endocrinology has been extensively studied in D. melanogaster, Manduca sexta, and Ae. aegypti, primarily with regard to the structure and function of 20-hydroxyecdysone (20E), the biologically active form of ecdysone, JH-III, the most abundant JH found in insects, and a variety of peptide hormones, of which PTTH is one of the best characterized at the biochemical level.
20-Hydroxyecdysone The steroid hormone ecdysone is major regulator of molting, metamorphosis, and reproduction in insects. The site of ecdysone synthesis in insect nymphs or larvae is the prothoracic gland in response to PTTH signaling, whereas in the adult the majority of ecdysone is synthesized in the follicle cells of the ovaries. Ecdysone is secreted into the hemolymph and absorbed by fat body cells where it is converted to 20E by the enzyme ecdysone 20 monooxygenase, a P450 enzyme expressed in fat body tissue. The 20E product of this reaction is released into the hemolymph where it travels to target tissues by classic endocrine mechanisms. Physiological studies of 20E function in insects have shown that it controls the molting process during metamorphosis, as well as a number of other physiological processes including the wandering behavior of larvae, development of complex neuronal networks, and oocyte maturation in adult female insects. In adult female mosquitoes, 20E is required for completion of the gonotrophic cycle and production of viable eggs. Shortly after taking a blood meal, the neuropeptide OEH is released from the brain and is transported by the hemolymph to the ovaries where it stimulates the synthesis of ecdysone and 20E via a cyclic-AMP second-messenger signaling cascade. The biochemical response to 20E production is yolk protein synthesis in the fat body, and egg maturation in the ovaries, a process known as vitellogenesis (the various yolk proteins are named vitellogenins). The yolk proteins are secreted by fat body cells into the hemolymph where they are recovered by receptormediated endocytosis in the developing oocytes. Within 48 h
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
HO
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OH OH
H HO H
OH
HO H
(a)
CH3
H3C
CH3 O OCH3
H3C (c)
O
O
(b)
Figure 3 20-Hydroxyecdysone (20E) and juvenile hormone III (JH-III) control metamorphosis and reproduction in insects. (a) Chemical structure of 20E showing the four ring cholesterol core. (b) 20E binds to a hydrophobic pocked in the ligand-binding domain of the ecdysone receptor protein. Molecular structure based on PDB file 2R40. (c) Chemical structure of the terpenoid-derived hormone JH-III.
of blood feeding, 20E concentrations in the hemolymph decrease to previtellogenic levels, and JH titers rise sharply in preparation for the next blood meal and gonotrophic cycle. The chemical structure of 20E is shown in Figure 3(a) where it can be seen that it consists primarily of a central four-ring core derived from cholesterol, as is true of all steroidal hormones that have been characterized biochemically. Insects cannot synthesize cholesterol de novo, and therefore, ecdysone biosynthesis is dependent on dietary cholesterol uptake during the larval and adult stages. The mechanism of action of 20E is mediated through binding and activation of a heterodimeric transcription factor consisting of two DNAbinding nuclear receptor proteins called the ecdysone receptor (ECR) and ultraspiracle (USP). The ligand-binding domain of the ECR protein from Heliothis virescens (Tobacco budworm) contains numerous hydrophobic amino acids, such as phenylalanine, tyrosine, and tryptophan, that line the 20E-binding pocket (Figure 3(b)). Studies of the D. melanogaster ECR protein have shown that dimerization with USP occurs in the absence of 20E, but upon 20E binding, the ECR–USP complex is translocated to the nucleus where it interacts with specific DNA sequences that function as ecdysone response elements. The molecular structure of the DNA-binding domains of the D. melanogaster ECR and USP proteins bound to a pseudopalindromic ecdysone response element located upstream of the hsp27 gene promoter is shown in Figure 4. It can be seen that the USP-subunit binds within the major groove of DNA to the 50 half-site of the hsp27 response element and makes contacts through an a-helical region of the protein with nucleotides on both DNA strands. Similarly, the ECR subunit binds to the 30 halfsite sequence that also lies within the major groove of the DNA helix and makes several direct nucleotide contacts. The net result of this sequence-specific DNA-binding event is the recruitment of auxiliary transcription factors that bind to either adjacent DNA sequences, or through protein–protein interactions, to the ECR–USP complex. One of the most thoroughly characterized examples of 20E-mediated regulation of gene expression in insects is the induced expression of multiple vitellogenin genes in the fat body of blood-fed female Ae. aegypti mosquitoes. Using quantitative real-time polymerase chain reaction assays, it was shown that 20E signaling through
USP
ECR
5⬘-AGGGTTCAATGCACTTGT-3⬘ 3⬘-TCCCAAGTTACGTGAACA-5⬘ hsp27 Ecdysone response element Figure 4 20E signaling is mediated by a nuclear receptor heterodimer consisting of the ecdysone receptor (ECR) and the ultraspiracle (USP) proteins. The DNA-binding domains of ECR and USP make direct contacts to nucleotide bases contained in the two half-site sequences of the ecdysone response element in the Drosophila melanogaster hsp27 gene (bolded nucleotides). Molecular structure based on PDB file 2HAN.
the ECR–USP heterodimeric complex induces Ae. aegypti vitellogenin gene expression by as much as 10 000-fold within 24 h of blood feeding.
Juvenile Hormone (JH-III) Insects have a class of terpenoid compounds, collectively called JHs that regulate metamorphosis. The best-characterized and most abundant of these hormones is JH-III, the structure of which is shown in Figure 3(c). JHs were first discovered in the kissing bug Rhodnius prolixus in 1934 by Vincent Wigglesworth who described them as compounds that inhibit the final stage of metamorphosis when applied to the insect exoskeleton. He also showed that JH was required in adult insects to promote egg maturation. JH-III is synthesized in a pair of glands in the insect head called the corpora allata and functions in much the same way as 20E in that it regulates gene expression through a receptor-mediated signaling process. In fact, both 20E and JH-III have been found to regulate the same developmental process, but often in an opposing way, with JH-III antagonizing, or modulating, 20E signaling functions. Peak levels of JH-III and 20E in the hemolymph of female mosquitoes are diametrically opposed depending on whether the mosquito is sugar fed or blood fed, respectively.
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Although much is known about the synthesis and biological function of JH-III in several insect species, it is still not exactly clear how JH-III regulates gene expression in target tissues. The search for a JH receptor in D. melanogaster has so far led to two candidate proteins, both of which are DNA-binding transcription factors. One is the USP protein that forms a complex with ECR and has been found to bind JH-III as a ligand with low affinity ( 10–7 M). More recent studies have reported that USP also binds the metabolic precursor to JH-III, called methyl farnesoate, with high affinity ( 10–9 M), which is closer to what would be expected for a physiological ligand of a typical nuclear receptor protein. The second candidate JH receptor is a basic helix–loop– helix protein encoded by the methoprene-tolerant gene (Met) that binds JH-III with affinities in the nanomolar range. Met contains a PAS domain that is 40% identical to the dioxin receptor translocator protein ARNT which binds to the dioxin receptor, another ligand-activated transcription factor. Based on studies showing modulation of 20E-mediated transcription by increased levels of JH-III, it has been proposed that ECR– USP heterodimers might form complexes with Met in a way that facilitates cross-talk modulation of gene expression by E20 and JH-III at specific promoters. In addition, several other DNA-binding proteins in D. melanogaster, called Chd64 and FKBP39, have been shown by yeast two-hybrid assays to be interaction partners with Met and to bind with high affinity to DNA sequences that function as JH response elements.
Prothoracicotropic Hormone The neuropeptide PTTH is synthesized in the corpora allata gland in the insect brain and functions in metamorphosis as a hormonal activator of ecdysone synthesis in the prothoracic gland. The existence of PTTH was first described in the 1920s by Stefan Kopec, a Polish biologist, who used decapitation experiments to show that the insect head is required for molting. Later, Vincent Wigglesworth confirmed the presence of a brain hormone distinct from JH that was required for insect metamorphosis. The active form of PTTH is homodimer of identical subunits, each containing a proteolytically processed peptide of 109 amino acids. Because of its similarity to other growth hormones, it was hypothesized that PTTH most likely binds to a growth hormone-like receptor present in prothoracic gland cells during the molting cycle. A bioinformatics approach was recently used to identify receptor tyrosine kinase transcripts that are expressed in the D. melanogaster prothoracic gland at times when PTTH activity is known to be required for metamorphosis. One of the candidate genes identified was the Torso receptor tyrosine kinase, which was subsequently shown by genetic and biochemical analyses to be the long sought PTTH receptor. It was found that PTTH binding to the D. melanogaster Torso protein on the surface prothoracic gland cells stimulates the Ras ! Raf ! ERK intracellular signaling pathway and leads to the activation of ecdysone biosynthesis (Figure 5). Although several molecular details of ecdysteroidogenesis in the insect prothoracic gland still need to be resolved, at least six different P450 enzymes have recently been identified as essential components of the pathway in D. melanogaster. The genes encoding these P450 enzymes are called the Halloween genes because of their lethal mutant
PTTH
PTTH receptor (Torso)
Prothoracic gland cell
Ras
Raf
ERK Activation of P450 enzymes encoded by the halloween genes Ecdysone synthesis Figure 5 The insect neuropeptide hormone PTTH induces ecdysone synthesis in prothoracic gland cells by binding to and activating the receptor tyrosine kinase protein Torso. Activation of the Ras ! Raf ! ERK signaling pathway by Torso leads to stimulation of P450 enzyme activity and initiation of the ecdysteroidogenesis pathway. The Halloween genes discovered in Drosophila encode six of these p450 enzymes.
phenotype that is characterized by a defective exoskeleton. The D. melanogaster Halloween genes were first described by Eric Wieschaus and Christiane Nusslein-Volhard using a genetic screen for developmental mutants and creatively named spook, spookier, phantom, disembodied, shadow, and shade. The biochemical function and enzymatic properties of these six P450 enzymes are currently being investigated.
See also: Bioenergetics: Cytochrome P-450; Metabolism Vitamins and Hormones: Urea Cycle: Disease Aspects; Signaling: Vitamin D Receptor.
Further Reading Brown MR, Sieglaff DH, and Rees HH (2009) Gonadal ecdysteroidogenesis in arthropoda: Occurrence and regulation. Annual Review of Entomology 54: 105–125. Browning C, Martin E, Loch C, et al. (2007) Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor. Journal of Biological Chemistry 282: 32924–32934. Grimmelikhuijzen CJ, Cazzamali G, Williamson M, and Hauser F (2007) The promise of insect genomics. Pest Management Science 63: 413–416. Jakob M, Kolodziejczyk R, Orlowski M, et al. (2007) Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain. Nucleic Acids Research 35: 2705–2718. Kamleh MA, Hobani Y, Dow JA, and Watson DG (2008) Metabolomic profiling of Drosophila using liquid chromatography Fourier transform mass spectrometry. FEBS Letters 582: 2916–2922.
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
Law JH and Wells MA (1989) Insects as biochemical models. Journal of Biological Chemistry 264: 16335–16338. Li Y, Zhang Z, Robinson GE, and Palli SR (2007) Identification and characterization of a juvenile hormone response element and its binding proteins. Journal of Biological Chemistry 282: 37605–37617. Michaud RM, Benoit JB, Lopez-Martinez G, et al. (2008) Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica. Journal of Insect Physiology 54: 645–655. Rewitz KF, O’Connor MB, and Gilbert LI (2007) Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization and functional conservation. Insect Biochemistry and Molecular Biology 37: 741–753. Rewitz KF, Yamanaka N, Gilbert LI, and O’Connor MB (2009) The insect neuropeptide PTTH activates receptor tyrosine kinase Torso to initiate metamorphosis. Science 326: 1403–1405.
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Riddiford LM (2008) Juvenile hormone action: A 2007 perspective. Journal of Insect Physiology 54: 895–901. Scaraffia PY, Tan G, Isoe J, et al. (2008) Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes. Proceedings of the National Academy of Sciences of the United States of America 105: 518–523. Spindler KD, Honl C, Tremmel C, et al. (2009) Ecdysteroid hormone action. Cellular and Molecular Life Sciences 66: 3837–3850. Warren JT, O’Connor MB, and Gilbert LI (2009) Studies on the black box: Incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta. Insect Biochemistry and Molecular Biology 39: 677–687. Zhou G, Flowers M, Friedrich K, et al. (2004) Metabolic fate of [14C]-labeled meal protein amino acids in Aedes aegypti mosquitoes. Journal of Insect Physiology 50: 337–349.
Glossary Gonotrophic cycle Reproductive process of ovarian development and egg laying in blood-feeding insects.
Overview of Insects Insects are the most abundant multicellular organisms on planet Earth and are thought to account for >70% of all species. So far, over 1 million insect species have been identified, with estimates putting the total number of insect species worldwide at 6–10 million. Insects are invertebrates that represent a class of arthropods characterized by three pairs of jointed legs; a three-part body consisting of a head, thorax, and abdomen; compound eyes; a chitin exoskeleton; and two antennae. Holometabolous insects hatch from eggs and progress through three distinct life stages. Newly hatched eggs produce larvae that go through metamorphosis, which is separated by molts. Since the chitin exoskeleton cannot accommodate the growing insect, molting provides a way for insects to shed their exoskeleton multiple times until they reach the pupal stage in which they complete metamorphosis in a protected shell called a pupal case. In the final developmental stage, the adult insect emerges from the pupal case, and within a few hours or days, females and males mate to produce fertilized eggs. In some other insects (hemimetabolous), the adult form from the hatching can be reached gradually with successive molts during a process called incomplete metamorphosis (cockroaches and grasshoppers). In this type of development, the immature stages (nymphs, or when aquatic, naiads) are like the adults except in size, flight, and sexual maturity. Endocrine regulation in insects relies on a large number of peptide and nonpeptide hormones, several of which have been studied in great detail. Three of the best-characterized insect hormones are ecdysone, juvenile hormone (JH), and prothoracicotropic hormone (PTTH), all of which play important roles in insect metamorphosis and reproduction. Ecdysone, and most likely JH, bind to receptor proteins that function as transcription factors, which directly or indirectly regulate the expression of specific target genes. In contrast, PTTH binds to a membranebound receptor tyrosine kinase in the prothoracic gland of immature insects and initiates an intracellular phosphorylation cascade that activates ecdysone synthesis. Ecdysone synthesis in adult insects occurs primarily in the ovaries in response to the brain neuropeptide ovarian ecdysteroidogenic hormone.
Insect Biochemistry Insect Metabolomics Insects provide an ideal model system to investigate metabolic regulation in multicellular organisms because they can often
590
Hemolymph The circulatory fluid of insects, similar to blood or lymphatic fluid in other organisms. Holometabolous insects Insects that undergo complete metamorphosis from eggs to larvae to pupae to adults.
be easily manipulated for biochemical studies, and for some of the most important insect species, whole genome sequence information is readily available, which greatly facilitates molecular genetic approaches. Metabolomics is the systematic study of small molecules that are utilized or generated by metabolic processes in living cells. The most common type of metabolomic study involves collecting biochemical samples from cell or tissue cultures, or whole organisms, under various conditions that can be quantitatively and qualitatively analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. Based on bioinformatic analysis of genome sequences, it is often possible to predict what metabolic pathways in insects are responsible for changes in metabolite profiles as a consequence of environmental signals or genomic variations in metabolic enzymes. One example of how insect biochemistry can be investigated using a metabolomics approach is the mass spectrometrybased study of the rosy (ry) mutation in Drosophila melanogaster. The ry mutation maps to the xanthine oxidase gene that encodes an enzyme required to convert xanthine, a product of purine degradation, to uric acid. In D. melanogaster, loss of xanthine oxidase causes a reddish brown eye color due to a deficiency in red pigment, which is how this mutation was first identified. In humans, xanthine oxidase deficiency leads to a condition called xanthinuria that is characterized by xanthine accumulation in the urinary tract and kidney. Using wholebody extracts of D. melanogaster wild-type and ry mutant flies, mass spectrometric analysis revealed significant increases in xanthine and hypoxanthine, as well as higher levels of guanine metabolites and biopterins. Surprisingly, the ry mutant defect in xanthine oxidase was associated with a 15-fold increase in glycerophosphocholine levels, presumably due to osmotic stress caused by the inability to excrete uric acid. Another example of how metabolomics have been used to investigate biochemical processes in insects, was a recent study in which larvae of the Antarctic midge Belgica antarctica were exposed to high heat, freezing temperatures, or desiccation, and the levels of 75 metabolites were analyzed by mass spectrometry. It was found that the steady-state level of a variety of sugars, amino acids, and citric acid cycle intermediates were differentially affected by these extreme environmental conditions. Specifically, levels of alanine, aspartate, mannitol, and urea were elevated after 6 h of freezing conditions (–10 ºC), whereas glycine and serine levels were decreased. Similarly, it was found that levels of a-ketoglutarate and putrescine were elevated after 1 h at 30 ºC, but glycerol, serine, and glucose
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
levels were decreased after a 1 h heat shock. Since serine levels decreased under both conditions of extreme cold and heat, as well as when the larvae were subjected to desiccating conditions, it was proposed that decreased serine levels may be a marker of environmental stress in this insect species.
Blood Meal Metabolism in Mosquitoes Because of their role as biological vectors for many of the arthropod-borne infectious diseases in humans, mosquitoes have been extensively studied at the biochemical level. Mosquitoes are responsible for transmitting not only malarial parasites and a variety of arboviruses, but also nematode worms that cause lymphatic filariasis. It is estimated that 40% of the world’s population is at risk of being infected by malaria, with 300 million people being infected each year and over 1 million deaths. Malaria is primarily transmitted by the mosquito species Anopheles gambiae in Africa, Anopheles albimanus in South America, and by Anopheles stephensi in Asia. The other major mosquito-borne disease affecting humans is Dengue fever, which can develop into Dengue hemorrhagic fever and be fatal. Aedes aegypti mosquitoes transmit Dengue virus and are one of the most rapidly spreading human disease vectors in the world. It is estimated that up to 3 billion people in the Americas, Africa, and Asia are at risk of being infected by Dengue virus, and that of the nearly 50 million new Dengue infections each year, 20 000 deaths will occur from Dengue hemorrhagic fever. Newly emerged Ae. aegypti female mosquitoes feed on nectar for several days until they are able to take their first blood meal (males do not blood feed). The blood meal is required for Ae. aegypti egg development and results in the deposition of 100 fertilized eggs within 72 h of feeding. This process of blood digestion, oocyte maturation in the ovaries, and the laying of fertilized eggs is called the gonotrophic cycle. In the course of each gonotrophic cycle, blood meal metabolism requires efficient retrieval of nutrients, and rapid excretion of toxic products such as ammonia (defined here as NH3, NHþ 4, or combination of both), in order to produce viable eggs. This is an amazing accomplishment considering the mosquito’s size. A typical female mosquito weighs 2 mg and can consume a blood meal of 2ml in few minutes. Taking into account the mass of water ( 1.6mg), protein ( 400 mg), lipid ( 10 mg), and carbohydrate ( 1 mg) in this 2-ml blood meal, it can be
calculated that the female mosquito consumes her own body weight in a single feeding. This feat would be equivalent to a 125 lb woman drinking a 12 gallon smoothie that contains 25 lbs of hamburger meat, 0.5 lb of butter, and 2 tbls of sugar. As metabolically challenging as blood meal digestion must be, the female Ae. aegypti mosquito is highly capable and typically completes 4–7 gonotrophic cycles to produce up to 400 mosquito progeny over a 1-month period. The vast majority of nutrients, in a typical mosquito blood meal, are derived from blood meal proteins, of which three constitute 80% of the total protein. These are hemoglobin ( 330mg), serum albumin ( 50 mg), and Igg antibodies ( 15 mg). Biochemical and molecular genetic studies have shown that mosquito blood meal digestion is initiated by endoprotease and exopeptidase enzymes that are secreted into the lumen by midgut epithelial cells within 30 min of feeding. Although mosquitoes require 10 of the 20 naturally occurring amino acids in their diet (essential amino acids), metabolic labeling studies using 14C-protein revealed that only 14% of the ingested protein is recycled into newly synthesized maternal proteins or egg proteins (Figure 1). In contrast, 70% of the amino acids retrieved from the blood meal are deaminated to generate reduced carbon that is used for immediate energy needs or excreted as waste. Blood meal-derived amino acids are also used for lipogenesis to supply stored energy for the female mosquito (10%), and converted to egg lipids that are used to complete oocyte maturation ( 6%).
Urea Synthesis in Ae. aegypti Excess nitrogen cannot be stored in cells, and therefore, nitrogen balance must be highly regulated to prevent ammonia toxicity, while at the same time, providing sufficient nitrogen for the biosynthesis of amino acids, nucleotide bases, and coenzymes. Aquatic organisms excrete most of their excess nitrogen directly into water as ammonia, whereas animals that spend all or part of their life cycle on land excrete nitrogen as uric acid or urea. Mammals synthesize urea via a metabolic pathway called the urea cycle that derives the two nitrogen atoms in urea from carbamoyl phosphate and aspartate. Certain amphibians and some fish do not express urea cycle enzymes; however, the urea they produce is the result of uric acid degradation by the uricolytic pathway (Figure 2). The
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Figure 1 Blood meal metabolism in female Aedes aegypti mosquitoes. The major nutrient in the mosquito blood meal is protein, which is converted to amino acids that are either deaminated and used as reduced carbon for energy conversion and lipid synthesis, or used directly for the synthesis maternal and egg proteins.
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Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
O H N
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Figure 2 The uricolytic pathway converts uric acid into urea. Feeding experiments in Aedes aegypti mosquitoes with 15NH4Cl have been used to quantitate uricolytic pathway intermediates by mass spectrometry (bolded N atoms were 15N-labeled in these experiments). RNAi-mediated knockdown of urate oxidase expression in blood-fed mosquitoes confirmed that the uricolytic pathway is functional under normal physiological conditions.
uricolytic pathway converts uric acid to urea and glyoxylic acid through a three-step process requiring the enzymes urate oxidase, allantoinase, and allantoicase. Early metabolic studies of nitrogen metabolism in blood-fed Ae. aegypti mosquitoes showed that excess nitrogen produced by blood meal digestion is excreted as ammonia, amino acids, uric acid, and urea. Based on studies in vertebrates, it was assumed that the urea present in mosquito feces was produced by converting excess dietary arginine into urea via the arginase reaction. However, by using a combination of 15 N-isotope labeling experiments, bioinformatic analysis of the Ae. aegypti genome sequence, and RNAi-mediated knockdown of urate oxidase expression, it was recently shown that a portion of this excreted urea is in fact derived from the uricolytic pathway. As illustrated in Figure 2, mass spectrometric analysis of uric acid containing 15N-labeled in two positions revealed that uric acid is metabolized to 2 mol of 15N-labeled urea, thereby providing biochemical evidence that the uricolytic pathway is functional in Ae. aegypti mosquitoes. In addition, RNAimediated knockdown of urate oxidase expression in 15NH4Clfed mosquitoes was found to significantly decrease the amount of 15N-urea in the feces. Similarly, knocking down urate oxidase expression in blood-fed mosquitoes resulted in a buildup of uric acid in mosquito tissues. Given the unusually high-protein content of the mosquito blood meal, it is likely that the uricolytic pathway provides a mechanism for the adult female mosquito to maximize nutrient uptake while minimizing the risk of ammonia toxicity.
Insect Hormones Endocrine regulation in insects is complex with many different types of hormones involved, including steroids (ecdysone), lipid compounds (JH), multiple peptide hormones (PTTH; ovarian ecdysteroidogenic hormone, OEH; adipokinetic hormone, AKH), and biogenic amines (serotonin, DOPA). The discussion here focuses on the two major gonadotropins, ecdysone and JH, and the peptide hormone PTTH, which
regulates ecdysteroidogenesis in the larval and pupal prothoracic gland. Insect endocrinology has been extensively studied in D. melanogaster, Manduca sexta, and Ae. aegypti, primarily with regard to the structure and function of 20-hydroxyecdysone (20E), the biologically active form of ecdysone, JH-III, the most abundant JH found in insects, and a variety of peptide hormones, of which PTTH is one of the best characterized at the biochemical level.
20-Hydroxyecdysone The steroid hormone ecdysone is major regulator of molting, metamorphosis, and reproduction in insects. The site of ecdysone synthesis in insect nymphs or larvae is the prothoracic gland in response to PTTH signaling, whereas in the adult the majority of ecdysone is synthesized in the follicle cells of the ovaries. Ecdysone is secreted into the hemolymph and absorbed by fat body cells where it is converted to 20E by the enzyme ecdysone 20 monooxygenase, a P450 enzyme expressed in fat body tissue. The 20E product of this reaction is released into the hemolymph where it travels to target tissues by classic endocrine mechanisms. Physiological studies of 20E function in insects have shown that it controls the molting process during metamorphosis, as well as a number of other physiological processes including the wandering behavior of larvae, development of complex neuronal networks, and oocyte maturation in adult female insects. In adult female mosquitoes, 20E is required for completion of the gonotrophic cycle and production of viable eggs. Shortly after taking a blood meal, the neuropeptide OEH is released from the brain and is transported by the hemolymph to the ovaries where it stimulates the synthesis of ecdysone and 20E via a cyclic-AMP second-messenger signaling cascade. The biochemical response to 20E production is yolk protein synthesis in the fat body, and egg maturation in the ovaries, a process known as vitellogenesis (the various yolk proteins are named vitellogenins). The yolk proteins are secreted by fat body cells into the hemolymph where they are recovered by receptormediated endocytosis in the developing oocytes. Within 48 h
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
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Figure 3 20-Hydroxyecdysone (20E) and juvenile hormone III (JH-III) control metamorphosis and reproduction in insects. (a) Chemical structure of 20E showing the four ring cholesterol core. (b) 20E binds to a hydrophobic pocked in the ligand-binding domain of the ecdysone receptor protein. Molecular structure based on PDB file 2R40. (c) Chemical structure of the terpenoid-derived hormone JH-III.
of blood feeding, 20E concentrations in the hemolymph decrease to previtellogenic levels, and JH titers rise sharply in preparation for the next blood meal and gonotrophic cycle. The chemical structure of 20E is shown in Figure 3(a) where it can be seen that it consists primarily of a central four-ring core derived from cholesterol, as is true of all steroidal hormones that have been characterized biochemically. Insects cannot synthesize cholesterol de novo, and therefore, ecdysone biosynthesis is dependent on dietary cholesterol uptake during the larval and adult stages. The mechanism of action of 20E is mediated through binding and activation of a heterodimeric transcription factor consisting of two DNAbinding nuclear receptor proteins called the ecdysone receptor (ECR) and ultraspiracle (USP). The ligand-binding domain of the ECR protein from Heliothis virescens (Tobacco budworm) contains numerous hydrophobic amino acids, such as phenylalanine, tyrosine, and tryptophan, that line the 20E-binding pocket (Figure 3(b)). Studies of the D. melanogaster ECR protein have shown that dimerization with USP occurs in the absence of 20E, but upon 20E binding, the ECR–USP complex is translocated to the nucleus where it interacts with specific DNA sequences that function as ecdysone response elements. The molecular structure of the DNA-binding domains of the D. melanogaster ECR and USP proteins bound to a pseudopalindromic ecdysone response element located upstream of the hsp27 gene promoter is shown in Figure 4. It can be seen that the USP-subunit binds within the major groove of DNA to the 50 half-site of the hsp27 response element and makes contacts through an a-helical region of the protein with nucleotides on both DNA strands. Similarly, the ECR subunit binds to the 30 halfsite sequence that also lies within the major groove of the DNA helix and makes several direct nucleotide contacts. The net result of this sequence-specific DNA-binding event is the recruitment of auxiliary transcription factors that bind to either adjacent DNA sequences, or through protein–protein interactions, to the ECR–USP complex. One of the most thoroughly characterized examples of 20E-mediated regulation of gene expression in insects is the induced expression of multiple vitellogenin genes in the fat body of blood-fed female Ae. aegypti mosquitoes. Using quantitative real-time polymerase chain reaction assays, it was shown that 20E signaling through
USP
ECR
5⬘-AGGGTTCAATGCACTTGT-3⬘ 3⬘-TCCCAAGTTACGTGAACA-5⬘ hsp27 Ecdysone response element Figure 4 20E signaling is mediated by a nuclear receptor heterodimer consisting of the ecdysone receptor (ECR) and the ultraspiracle (USP) proteins. The DNA-binding domains of ECR and USP make direct contacts to nucleotide bases contained in the two half-site sequences of the ecdysone response element in the Drosophila melanogaster hsp27 gene (bolded nucleotides). Molecular structure based on PDB file 2HAN.
the ECR–USP heterodimeric complex induces Ae. aegypti vitellogenin gene expression by as much as 10 000-fold within 24 h of blood feeding.
Juvenile Hormone (JH-III) Insects have a class of terpenoid compounds, collectively called JHs that regulate metamorphosis. The best-characterized and most abundant of these hormones is JH-III, the structure of which is shown in Figure 3(c). JHs were first discovered in the kissing bug Rhodnius prolixus in 1934 by Vincent Wigglesworth who described them as compounds that inhibit the final stage of metamorphosis when applied to the insect exoskeleton. He also showed that JH was required in adult insects to promote egg maturation. JH-III is synthesized in a pair of glands in the insect head called the corpora allata and functions in much the same way as 20E in that it regulates gene expression through a receptor-mediated signaling process. In fact, both 20E and JH-III have been found to regulate the same developmental process, but often in an opposing way, with JH-III antagonizing, or modulating, 20E signaling functions. Peak levels of JH-III and 20E in the hemolymph of female mosquitoes are diametrically opposed depending on whether the mosquito is sugar fed or blood fed, respectively.
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Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
Although much is known about the synthesis and biological function of JH-III in several insect species, it is still not exactly clear how JH-III regulates gene expression in target tissues. The search for a JH receptor in D. melanogaster has so far led to two candidate proteins, both of which are DNA-binding transcription factors. One is the USP protein that forms a complex with ECR and has been found to bind JH-III as a ligand with low affinity ( 10–7 M). More recent studies have reported that USP also binds the metabolic precursor to JH-III, called methyl farnesoate, with high affinity ( 10–9 M), which is closer to what would be expected for a physiological ligand of a typical nuclear receptor protein. The second candidate JH receptor is a basic helix–loop– helix protein encoded by the methoprene-tolerant gene (Met) that binds JH-III with affinities in the nanomolar range. Met contains a PAS domain that is 40% identical to the dioxin receptor translocator protein ARNT which binds to the dioxin receptor, another ligand-activated transcription factor. Based on studies showing modulation of 20E-mediated transcription by increased levels of JH-III, it has been proposed that ECR– USP heterodimers might form complexes with Met in a way that facilitates cross-talk modulation of gene expression by E20 and JH-III at specific promoters. In addition, several other DNA-binding proteins in D. melanogaster, called Chd64 and FKBP39, have been shown by yeast two-hybrid assays to be interaction partners with Met and to bind with high affinity to DNA sequences that function as JH response elements.
Prothoracicotropic Hormone The neuropeptide PTTH is synthesized in the corpora allata gland in the insect brain and functions in metamorphosis as a hormonal activator of ecdysone synthesis in the prothoracic gland. The existence of PTTH was first described in the 1920s by Stefan Kopec, a Polish biologist, who used decapitation experiments to show that the insect head is required for molting. Later, Vincent Wigglesworth confirmed the presence of a brain hormone distinct from JH that was required for insect metamorphosis. The active form of PTTH is homodimer of identical subunits, each containing a proteolytically processed peptide of 109 amino acids. Because of its similarity to other growth hormones, it was hypothesized that PTTH most likely binds to a growth hormone-like receptor present in prothoracic gland cells during the molting cycle. A bioinformatics approach was recently used to identify receptor tyrosine kinase transcripts that are expressed in the D. melanogaster prothoracic gland at times when PTTH activity is known to be required for metamorphosis. One of the candidate genes identified was the Torso receptor tyrosine kinase, which was subsequently shown by genetic and biochemical analyses to be the long sought PTTH receptor. It was found that PTTH binding to the D. melanogaster Torso protein on the surface prothoracic gland cells stimulates the Ras ! Raf ! ERK intracellular signaling pathway and leads to the activation of ecdysone biosynthesis (Figure 5). Although several molecular details of ecdysteroidogenesis in the insect prothoracic gland still need to be resolved, at least six different P450 enzymes have recently been identified as essential components of the pathway in D. melanogaster. The genes encoding these P450 enzymes are called the Halloween genes because of their lethal mutant
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ERK Activation of P450 enzymes encoded by the halloween genes Ecdysone synthesis Figure 5 The insect neuropeptide hormone PTTH induces ecdysone synthesis in prothoracic gland cells by binding to and activating the receptor tyrosine kinase protein Torso. Activation of the Ras ! Raf ! ERK signaling pathway by Torso leads to stimulation of P450 enzyme activity and initiation of the ecdysteroidogenesis pathway. The Halloween genes discovered in Drosophila encode six of these p450 enzymes.
phenotype that is characterized by a defective exoskeleton. The D. melanogaster Halloween genes were first described by Eric Wieschaus and Christiane Nusslein-Volhard using a genetic screen for developmental mutants and creatively named spook, spookier, phantom, disembodied, shadow, and shade. The biochemical function and enzymatic properties of these six P450 enzymes are currently being investigated.
See also: Bioenergetics: Cytochrome P-450; Metabolism Vitamins and Hormones: Urea Cycle: Disease Aspects; Signaling: Vitamin D Receptor.
Further Reading Brown MR, Sieglaff DH, and Rees HH (2009) Gonadal ecdysteroidogenesis in arthropoda: Occurrence and regulation. Annual Review of Entomology 54: 105–125. Browning C, Martin E, Loch C, et al. (2007) Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor. Journal of Biological Chemistry 282: 32924–32934. Grimmelikhuijzen CJ, Cazzamali G, Williamson M, and Hauser F (2007) The promise of insect genomics. Pest Management Science 63: 413–416. Jakob M, Kolodziejczyk R, Orlowski M, et al. (2007) Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain. Nucleic Acids Research 35: 2705–2718. Kamleh MA, Hobani Y, Dow JA, and Watson DG (2008) Metabolomic profiling of Drosophila using liquid chromatography Fourier transform mass spectrometry. FEBS Letters 582: 2916–2922.
Metabolism Vitamins and Hormones | Insect Biochemistry/Hormones
Law JH and Wells MA (1989) Insects as biochemical models. Journal of Biological Chemistry 264: 16335–16338. Li Y, Zhang Z, Robinson GE, and Palli SR (2007) Identification and characterization of a juvenile hormone response element and its binding proteins. Journal of Biological Chemistry 282: 37605–37617. Michaud RM, Benoit JB, Lopez-Martinez G, et al. (2008) Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica. Journal of Insect Physiology 54: 645–655. Rewitz KF, O’Connor MB, and Gilbert LI (2007) Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization and functional conservation. Insect Biochemistry and Molecular Biology 37: 741–753. Rewitz KF, Yamanaka N, Gilbert LI, and O’Connor MB (2009) The insect neuropeptide PTTH activates receptor tyrosine kinase Torso to initiate metamorphosis. Science 326: 1403–1405.
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Riddiford LM (2008) Juvenile hormone action: A 2007 perspective. Journal of Insect Physiology 54: 895–901. Scaraffia PY, Tan G, Isoe J, et al. (2008) Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes. Proceedings of the National Academy of Sciences of the United States of America 105: 518–523. Spindler KD, Honl C, Tremmel C, et al. (2009) Ecdysteroid hormone action. Cellular and Molecular Life Sciences 66: 3837–3850. Warren JT, O’Connor MB, and Gilbert LI (2009) Studies on the black box: Incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta. Insect Biochemistry and Molecular Biology 39: 677–687. Zhou G, Flowers M, Friedrich K, et al. (2004) Metabolic fate of [14C]-labeled meal protein amino acids in Aedes aegypti mosquitoes. Journal of Insect Physiology 50: 337–349.