Physiological Systems in Insects

Physiological Systems in Insects

Third Edition

Marc J. Klowden

Division of Entomology, University of Idaho, Moscow, Idaho

Table of Contents

Cover image

Title page

Copyright

Dedication

Preface to the Third Edition

Bibliography

Chapter 1. Signaling Systems

Paracrine Signaling

Endocrine Signaling

Types of Hormone Release Sites in Insects

Early Experiments that Set the Stage for Our Current Understanding

Types of Hormones in Insects

Prothoracicotropic Hormone

Ecdysteroids

The Juvenile Hormones

Other Neuropeptides Found in Insects

Terms for Understanding Signaling Systems

Bibliography

Chapter 2. Integumentary Systems

Insect Growth and Development

Strategies for Growth

Instars, Stadia, and Hidden Phases

Structure of the Integument

Modified Features of the Integument

Chemistry of the Cuticle

The Molting Process

Imaginal Disc Development

Metamorphosis, Cell Reorganization, and Cell Death

Terms for Understanding Integumentary Systems

Bibliography

Chapter 3. Developmental Systems

Morphogens and Developmental Patterns

Insect Eggs

Embryonic Development

Terms for Understanding Developmental Systems

Bibliography

Chapter 4. Reproductive Systems

Female Reproductive Systems

Types of Ovarioles

Vitellogenesis

Endocrinology of Female Reproduction

Ovulation, Fertilization and Oviposition

Male Reproductive Systems

Spermatogenesis

Unconventional Methods of Insect Reproduction

Mating Systems

Terms for Understanding Reproductive Systems

Bibliography

Chapter 5. Behavioral Systems

Ways of Looking at Behavior

Physiology of Learning and Memory

Physiology of Behaviors Accompanying Metamorphosis

Behavioral Modification by Parasites

Terms for Understanding Behavioral Systems

Bibliography

Chapter 6. Metabolic Systems

The Insect Alimentary Tract

Metabolic Processes in Insects

Metabolism of Carbohydrates

Metabolism of Proteins

Metabolism of Lipids

Diapause as a Metabolic Process

Terms for Understanding Metabolic Systems

Bibliography

Chapter 7. Circulatory Systems

Structure of the Insect Circulatory System

Immune Mechanisms in Insects

The Circulatory System and Temperature Variations

Terms for Understanding Circulatory Systems

Bibliography

Chapter 8. Excretory Systems

Major Excretory Products in Insects

Excretory Organs

Hormonal Control of Excretion and Osmoregulation

Storage Excretion

Other Functions of the Malpighian Tubules

Terms for Understanding Excretory Systems

Bibliography

Chapter 9. Respiratory Systems

Supplying Cells with Oxygen

The Tracheal System

Modifications that Increase Oxygen Uptake

Discontinuous Gas Exchange

Aquatic Respiration

Terms for Understanding Respiratory Systems

Bibliography

Chapter 10. Locomotor Systems

Basic Structure of Insect Muscles

Actin, Myosin and Muscle Activation

Types of Skeletal Muscles

Neural Excitation and Modulation of Muscle Contraction

Evolution of Insect Wings

Basic Structure of the Insect Wing

Muscles Involved in Wing Movements

Wing Movements During Flight

Wing Coupling and Control Mechanisms

Flight Muscle Metabolism

Terrestrial Locomotion

Terms for Understanding Locomotor Systems

Bibliography

Chapter 11. Nervous Systems

The Nervous System as a Key to the Evolution of Arthropods

Basic Components of the Insect Nervous System

Maintenance of Electrical Potential and Nervous Transmission

Structure of the Nervous System

Sensing the Environment

Terms for Understanding Nervous Systems

Bibliography

Chapter 12. Communication Systems

Visual Communication

Acoustic Communication

Tactile Communication

Chemical Communication

The Multicomponent Nature of Bee Communication

Terms for Understanding Communication Systems

Bibliography

Glossary

Index

Copyright

Academic Press is an imprint of Elsevier

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First edition 2002

Second edition 2007

Third edition 2013

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Dedication

Dedicated to the memory of Arden O. Lea, who made me think, and to Alex, Avi, Keira, and Shea, who make me smile.

Preface to the Third Edition

Sir Vincent Wigglesworth, the prescient founder of the field of insect physiology, introduced his 1934 treatise, Insect Physiology, with the comment, ‘The fundamental processes of vital activity, the ordered series of physical and chemical changes which liberate energy and maintain the immanent movement of life, are probably the same wherever living matter exists.’ In the 134 pages that followed in this first insect physiology textbook, Wigglesworth described what was known about the systems of insects based on what he estimated to be 2000 publications. Despite his prophetic introduction, few related comparisons of insect systems followed in the little book, most likely because there were no comparisons to make, as few others considered there to be much in common between arthropods and vertebrates. The experiments by Kopeć (1922) demonstrating that the insect brain was a source of hormones were largely ignored until Wigglesworth rediscovered them. Who besides another insect physiologist would believe that these simple creatures had hormones, let alone hormones produced by an insect brain the size of a poppy seed? The pioneering work by Berta and Ernst Scharrer (1944) made a strong case for neurosecretion in both insects and vertebrates, but they too had difficulty with the scientific community accepting the concept that nerve cells in any animal could produce hormones. Insects were evolutionarily distant from humans, classified in a primitive phylum, with a strange basic body ground plan and physiological makeup. During the cold war with the policy of mutually assured destruction, we knew that insects were different enough to survive the inevitable nuclear holocaust and repopulate the planet without us. The major reason to study insects was to find new ways to kill them that did not kill us.

In 1948, Wigglesworth presented a well-documented justification for using insects as models for studying general animal physiology. However, insects were still largely relegated to the category of pests we aim to tolerate or eradicate. Through five editions of the classic Destructive and Useful Insects, the value of insects to humans was summarized in a short chapter, while their destructive side dominated the remainder. Ten examples of how insects were beneficial or useful to humans were discussed, with the last being the limited use of insects and insect products in medicine. This included maggot therapy for the treatment of wounds, the use of insect venoms for treating rheumatism and arthritis, and the use of insect extracts and products such as royal jelly as medicines. However, over the last 15 years there has been an unprecedented explosion of information as molecular techniques have yielded information unobtainable by conventional biochemistry and physiology alone. Now that the genomes of humans and many species of insects can be compared, the similarities are truly remarkable and emphasize the wisdom of Wigglesworth’s remarks in 1934. Given that the arthropod lineage diverged from that of vertebrates more than 600 million years ago, parallels between the physiological systems of insects and humans are enough to make your respiratory system inspire, give your integument chills, and cause your tarsi to twitch. It appears that as many as 75% of the genes that are associated with human genetic diseases have homologies in Drosophila. These similarities have altered the focus of insect science, with insects seen more as model systems for studying human physiology, and a better understanding of them able to be applied to ourselves to advance the pace of human disease research.

A reason often given for using insects in research is that they are simple. With few parts, moving or otherwise, an insect system can be studied without the complications of ancillary and redundant components, and is therefore much easier to dissect and manipulate. The Drosophila genome, on its four pairs of chromosomes, has been fully identified and encoded by fewer than 14 000 genes – about half that of humans. The other obvious aspects of short lifecycles, little space required for rearing, considerably less food needed than if one was working with elephants, and the advantage of being completely off the radar screens of university animal care and use committees, are additional points in their favor. Unfortunately, this view of simplicity often tends toward a pejorative label: insects are small, unsophisticated, and have little in common with us more complicated vertebrates. However, their size belies their complexity, because their incredible success has not been in spite of their simplicity but because of it. To downplay this simplicity is also to conclude that an IBM system 360 computer that once filled an entire air-conditioned room accompanied by a colossal 8 Mb of storage was more complex than a present-day iPhone with 64 Gb of memory that fits in your pocket.

Ignoring the possibilities of consciousness and personality, the far less complex insect nervous system shares numerous similarities with that of humans. Insects may not dream, but they certainly sleep. They meet the criteria established for sleep: a period of quiescence that is associated with a species-specific posture; a reduced responsiveness to external stimuli; a rapid reversibility to wakefulness; a homeostasis based on a longer recovery period following periods of sleep deprivation; and an appropriate expression of clock genes. Drosophila engage in periods of quiescence that are characterized by changes in brain activity and the specific expression of numerous genes. Dozing flies undergo sustained periods of quiescence during the night, but when prevented from sleeping are less adept at performing their usual tasks. Just as older humans sometimes have problems sleeping, old flies show disturbances in their sleep patterns. Although in older Drosophila total amounts of sleep do not decrease, their sleep–wake cycles tend to be more fragmented with age. The observation that the roundworm Caenorhabditis elegans also sleeps indicates that sleep is a basic biological phenomenon common to many living things, and that Drosophila can be an effective model for studying aging and sleep in humans, and even the phenomenon of sleep-related restless leg syndrome.

Several human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, muscular dystrophy, and Huntington’s disease, lack effective treatments and have undetermined causes. Given the identification of several homologous regulatory genes involved in brain development in both humans and Drosophila, the use of insects to examine the genetic dissection of the developing brain may expand our knowledge of how gene mis-expression or loss of function might be countered. The presence of the Drosophila homologue of the microtubule-associated protein that is related to Alzheimer’s disease could establish insects as suitable models for Alzheimer’s. The kynurenine pathway for the degradation of the amino acid tryptophan has been studied in transgenic flies as a model for the treatment of Huntington’s disease.

Because insects do not have vertebrate-type breathing organs and, except for the largest insects, appear to be free from having to take the deep breaths that we do, Aristotle, without the benefit of today’s scientific instrumentation in the ancient Greece of 350 BC, can be forgiven for characterizing them as animals that did not breathe. It is thus satisfying that the branching morphogenesis of the developing insect tracheal system is now recognized as a paradigm for the development of branching in mammalian lung and vascular systems. Drosophila has also been proposed as a model system for the study of asthma-susceptibility genes and the innate immune responses of airway epithelial cells – perhaps even more useful than the traditional mouse model.

Given the health concerns about our increasing waistlines, Drosophila metabolism and energy homeostasis may yield insights into human obesity and the related pathologies, such as diabetes. In mammals, insulin and leptin signaling to centers in the brain regulates our metabolism and food intake. Insulin has been recognized in insects for many years, but it is only recently that the role of insulin signaling has been shown to be phylogenetically conserved. Although a natural epidemic of obesity in the insect population has yet to be identified, transgenic flies with certain blocked neurons store more fat, and suggest that the insect brain apparently measures the level of fat stores by similar mechanisms as does our own. The physiological roles of insulin for both humans and insects in the sensing of nutritional state and triglyceride storage, controlling cell and organ size, and determining overall longevity make insects ideal model systems for understanding growth-related processes in vertebrates. The steroid insect hormone 20-hydroxyecdysone (20E) that initiates molting of the exoskeleton may also benefit the vertebrate skeleton as an anti-osteoporosis drug. Rats fed 20E over 3 months showed increases in bone mineral density over controls. Rats also benefited from increases in the cross-sectional area of muscles after ingesting 20E isolated from plant material. In neither case were any side effects noted.

An immunity to intestinal disease is essential, given the widespread exposure of insects to microbial organisms acquired while feeding on fermenting substrates. There are many similarities to the human intestinal mucosa which involve both physical and molecular mechanisms that maintain a resident flora and discourage pathogenic bacteria. Modeling human intestinal disease in Drosophila has been proposed, as many signaling pathways that regulate disease, as well as gut development and regeneration, have been conserved in human and insect systems.

It is odd that at the same time as insects are being utilized more and more as model systems for what we consider to be higher organisms, departments of entomology and the insect-specific courses associated with them are being eliminated. As insects become increasingly used as models for understanding human ailments, it is essential that we understand more about them. As Barbara McClintock, the winner of the 1983 Nobel Prize in Physiology or Medicine, so passionately stated, above all one must have ‘a feeling for the organism’ with which one is working. There is and will be an increasing large cadre of scientists who work with insects but do not have the opportunity to feel very much about them. I hope this text will provide a feeling for the beauty and complexity of physiological systems in insects for those scientists, as well as for the traditional entomologists who began their careers already so inspired.

Rather than this third edition evolving into a multivolume reference, I chose to maintain it as a textbook that is an introduction to physiological systems in insects. To meet the needs of readers who require more detail, there is an extensive bibliography at the end of each chapter to serve as a guide to further information from the primary literature.

Bibliography

1. Abdelsadik A, Roeder T. Chronic activation of the epithelial immune system of the fruit fly’s salivary glands has a negative effect on organismal growth and induces a peculiar set of target genes. BMC Genomics. 2010;11:265.

2. Apidianakis Y, Rahme LG. Drosophila melanogaster as a model for human intestinal infection and pathology. Dis Model Mech. 2011;4:21–30.

3. Al-Anzi B, Sapin V, Waters C, et al. Obesity-blocking neurons in Drosophila. Neuron. 2009;63:329–341.

4. Bolduc FV, Tully T. Fruit flies and intellectual disability. Fly (Austin). 2009;3:91–104.

5. Bushey D, Hughes KA, Tononi G, Cirelli C. Sleep, aging, and lifespan in Drosophila. BMC Neurosci. 2010;11:56.

6. Bushey D, Tononi G, Cirelli C. The Drosophila fragile X mental retardation gene regulates sleep need. J Neurosci. 2009;29:1948–1961.

7. Campesan S, Green EW, Breda C, et al. The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington’s disease. Curr Biol. 2011;21:961–966.

8. Cirelli C, Bushey D. Sleep and wakefulness in Drosophila melanogaster. Ann NY Acad Sci. 2008;1129:323–329.

9. Chien S, Reiter LT, Bier E, Gribskov M. Homophila: human disease gene cognates in Drosophila. Nucleic Acids Res. 2002;30:149–151.

10. Cox LS, Clancy DJ, Boubriak I, Saunders RD. Modeling werner syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease. Ann NY Acad Sci. 2007;1119:274–288.

11. DiAngelo JR, Birnbaum MJ. Regulation of fat cell mass by insulin in Drosophila melanogaster. Mol Cell Biol. 2009;29:6341–6352.

12. DiAngelo JR, Bland ML, Bambina S, Cherry S, Birnbaum MJ. The immune response attenuates growth and nutrient storage in Drosophila by reducing insulin signaling. Proc Natl Acad Sci USA. 2009;106:20853–20858.

13. Dow JA, Romero MF. Drosophila provides rapid modeling of renal development, function, and disease. Am J Physiol Renal Physiol. 2010;299:F1237–F1244.

14. Freeman A, Pranski E, Miller RD, et al. Sleep fragmentation and motor restlessness in a Drosophila model of Restless Legs Syndrome. Curr Biol. 2012;22:1142–1148.

15. Gilbert LI. Drosophila is an inclusive model for human diseases, growth and development. Mol Cell Endocrinol. 2008;293:25–31.

16. Gohil VM, Offner N, Walker JA, et al. Meclizine is neuroprotective in models of Huntington’s disease. Hum Mol Genet. 2011;20:294–300.

17. Grice SJ, Sleigh JN, Liu JL, Sattelle DB. Invertebrate models of spinal muscular atrophy: insights into mechanisms and potential therapeutics. Bioessays. 2011;33:956–965.

18. Hendricks JC, Finn SM, Panckeri KA, et al. Rest in Drosophila is a sleep-like state. Neuron. 2000;25:129–138.

19. Hendricks JC, Sehgal A. Why a fly? Using Drosophila to understand the genetics of circadian rhythms and sleep. Sleep. 2004;27:334–342.

20. Hirth F. Drosophila melanogaster in the study of human neurodegeneration. CNS Neurol Disord Drug Targets. 2010;9:504–523.

21. Hirth F, Reichert H. Conserved genetic programs in insect and mammalian brain development. Bioessays. 1999;21:677–684.

22. Jumbo-Lucioni P, Ayroles JF, Chambers MM, et al. Systems genetics analysis of body weight and energy metabolism traits in Drosophila melanogaster. BMC Genomics. 2010;11:297.

23. Kapur P, Wuttke W, Jarry H, Seidlova-Wuttke D. Beneficial effects of β-ecdysone on the joint, epiphyseal cartilage tissue and trabecular bone in ovariectomized rats. Phytomedicine. 2010;17:350–355.

24. Keller EF. A feeling for the organism. The life and work of barbara McClintock San Francisco: W.H. Freeman; 1983.

25. Koh K, Evans JM, Hendricks JC, Sehgal A. A Drosophila model for age-associated changes in sleep: wake cycles. Proc Natl Acad Sci USA. 2006;103:13843–13847.

26. Kucherenko MM, Marrone AK, Rishko VM, Magliarelli HF, Shcherbata HR. Stress and muscular dystrophy: a genetic screen for Dystroglycan and Dystrophin interactors in Drosophila identifies cellular stress response components. Dev Biol. 2011;352:228–242.

27. Kuhnlein RP. Energy homeostasis regulation in Drosophila: a lipocentric perspective. Results Probl Cell Differ. 2010;52:159–173.

28. Kuhnlein RP. Drosophila as a lipotoxicity model organism--more than a promise? Biochim Biophys Acta. 2010;1801:215–221.

29. Kushner RF, Ryan EL, Sefton JM, et al. A Drosophila melanogaster model of classic galactosemia. Dis Model Mech. 2010;3:618–627.

30. Kopeć S. Studies on the necessity of the brain for the inception of insect metamorphosis. Biol Bull. 1922;42:323–342.

31. Lloyd TE, Taylor JP. Flightless flies: Drosophila models of neuromuscular disease. Ann N Y Acad Sci. 2011;1184:e1–20.

32. Loewen CA, Feany MB. The unfolded protein response protects from tau neurotoxicity in vivo. PLoS One. 2010;5:e13084.

33. Lu B, Vogel H. Drosophila models of neurodegenerative diseases. Annu Rev Pathol. 2009;4:315–342.

34. McClure KD, French RL, Heberlein U. A Drosophila model for fetal alcohol syndrome disorders: role for the insulin pathway. Dis Model Mech. 2011;4:335–346.

35. Medioni C, Senatore S, Salmand PA, et al. The fabulous destiny of the Drosophila heart. Curr Opin Genet Dev. 2009;19:518–525.

36. Metcalf RL, Metcalf RA. Destructive and useful insects ed 5 NY: McGraw Hill; 1993.

37. Naidoo N, Casiano V, Cater J, Zimmerman J, Pack AI. A role for the molecular chaperone protein BiP/GRP78 in Drosophila sleep homeostasis. Sleep. 2007;30:557–565.

38. Preus A: Aristotle’s parts of animals 2. 16. 659b13–19: is it Authentic? Classic Quart 18 270–278, 1968.

39. Ratcliffe NA, Mello CB, Garcia ES, Butt TM, Azambuja P. Insect natural products and processes: new treatments for human disease. Insect Biochem Mol Biol. 2011;41:747–769.

40. Reiter LT, Potocki L, Chien S, Gribskov M, Bier E. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res. 2001;11:1114–1125.

41. Roeder T, Isermann K, Kabesch M. Drosophila in asthma research. Am J Respir Crit Care Med. 2009;179:979–983.

42. Scharrer B, Scharrer E. Neurosecretion IV Comparison between the intercerebralis-cardiacum-allatum system of the insects and the hypothalamo-hypophyseal system of the vertebrates. Biol Bull. 1944;87:242–251.

43. Seidlova-Wuttke D, Christel D, Kapur P, et al. β-ecdysone has bone protective but no estrogenic effects in ovariectomized rats. Phytomedicine. 2010;17:884–889.

44. Seugnet L, Galvin JE, Suzuki Y, Gottschalk L, Shaw PJ. Persistent short-term memory defects following sleep deprivation in a Drosophila model of Parkinson disease. Sleep. 2009;32:984–992.

45. Seugnet L, Suzuki Y, Donlea JM, Gottschalk L, Shaw PJ. Sleep deprivation during early-adult development results in long-lasting learning deficits in adult Drosophila. Sleep. 2011;34:137–146.

46. Seugnet L, Suzuki Y, Thimgan M, et al. Identifying sleep regulatory genes using a Drosophila model of insomnia. J Neurosci. 2009;29:7148–7157.

47. Toth N, Szabo A, Kacsala P, Heger J, Zador E. 20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat. Phytomedicine. 2008;15:691–698.

48. Wagner C, Isermann K, Roeder T. Infection induces a survival program and local remodeling in the airway epithelium of the fly. FASEB J. 2009;23:2045–2054.

49. Wigglesworth VB. Insect Physiology London: Methuen; 1934; 134 pp.

50. Wigglesworth VB. The insect as a medium for the study of physiology. Proc R Soc B. 1948;135:430–446.

51. Zimmerman JE, Naidoo N, Raizen DM, Pack AI. Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci. 2008;31:371–376.

Chapter 1

Signaling Systems

It is essential for cells to know where they are and what the environment around them is like. Single-celled organisms may have to detect changes in nutrients, temperature, mechanical pressure, electromagnetic fields, light, and the metabolic products of other cells of the same or different species, and make appropriate responses. This process becomes more critical for the cells of multicellular organisms, because beginning a few moments after the fertilized egg cell first divides, the individual cells must begin communicating with each other to coordinate their activities. As development proceeds, cells not only need to respond to changing conditions but also to exchange information that will determine their specialized identity and the positions they will assume in the mature organism. The information from the outside of the cell must be translated and converted to specific cellular responses in a series of steps referred to as signal transduction pathways.

In multicellular organisms these signals are most frequently chemical in nature, emanating from a signaling cell and detected by specific receptor proteins on a target cell. There are hundreds of different signal molecules, but each cell responds selectively depending on its particular function within the organism. Its response, or failure to respond, depends on whether or not it possesses a receptor for that signal. The receptor molecules are virtually always proteins that span the cell membrane. If the correct receptor receives this extracellular signal, the target cell converts it to an intracellular signal that affects cell physiology, such as changes in cell shape, metabolism, or gene expression. The external signal is thus internalized, amplified, and distributed to several internal targets (Figure 1.1). The chemical signals may consist of many types of molecules, categorized by their range and speed of activity. After the signal is transduced and arrives inside the cell, its target might be either already-synthesized proteins that become activated by the message, or changes in gene expression, either of which can alter cell physiology and behavior (Figure 1.2).

Figure 1.1 A signal molecule binds to specific receptor sites on the cell membrane. The activation of the receptor transduces the signal to the cell interior where it is amplified by parallel pathways and distributed to targets.

Figure 1.2 Targets of signal transduction in the cell. Cell physiology, metabolism, or behavior may be modified by altered gene expression or altered protein function.

Autocrine signaling is the most private of the signaling modes, with a cell signaling itself by producing a chemical that activates receptors within its own cytoplasm or on its surface (Figure 1.3A). An example is the prothoracic gland, which during some developmental stages activates its own production of ecdysteroids.

Figure 1.3 Types of cell signaling. A. Autocrine signaling. B. Contact-dependent signaling. C. Neuronal signaling. D. Endocrine signaling. E. Paracrine signaling. See the text for an explanation.

Contact-dependent signaling relies on direct contact between neighboring cells, with signaling molecules embedded in the cell membranes or passed directly through pores (Figure 1.3B). The signaling cell may produce a molecule that binds to a receptor in the adjacent target cell, or release ions that are transferred through gap junctions to trigger a response only in those cells that are in direct contact. Also called juxtacrine signaling, this is the fastest mode of communication and can be found in cardiac muscle cells whose contractions are coordinated, allowing these to occur simultaneously. Contact-dependent signaling also occurs during early development, giving adjacent cells information about their location relative to other cells, and can specify their developmental fate.

Neuronal signaling delivers messages across long distances within the organism to specific cells. As described in Chapter 11, a signaling neuron sends an electrical signal along its axon that triggers the release of a neurotransmitter at its synapse with a target cell (Figure 1.3C). The neurotransmitter binds to postsynaptic receptors in target cells, causing a physiological response in those cells. The speed of transmission is rapid, but depends on the physical distance over which the signal must travel.

Paracrine and endocrine signaling both involve the diffusion of signal molecules through an extracellular medium. Endocrine signaling is the most public, releasing hormones into the blood that are distributed to all cells of the body (Figure 1.3D). Only those cells with receptors that recognize the hormone are capable of responding. The signaling cells may consist of endocrine cells or more specialized neurosecretory cells. Some endocrine signaling molecules are hydrophobic and are able to cross the cell membrane and bind to internal receptor proteins, but most others are peptides that must remain outside and bind to external receptors.

Paracrine signaling is more intimate, with the signal molecules diffusing not through the blood but through the extracellular matrix instead (Figure 1.3E). The proteins secreted by the target cells are effective over only a short distance and induce changes only in neighboring cells that bear specific receptors localized in regions of cytoplasmic extensions called cytonemes. Because many of the signal molecules involved are proteins that are unable to enter cells through the plasma membrane, pathways of signal transduction operate within the target cells for their activation, with an extracellular receptor communicating the signal to the cell interior.

The remainder of this chapter is devoted to several important paracrine and endocrine systems in insects.

Paracrine Signaling

There is a remarkable conservation of protein messengers used for many diverse processes throughout the animal kingdom. Very similar factors operate in both mammals and insects, and, with an even more beautiful parsimony, are used at different times for different developmental events within the same organism. These are the morphogens, which diffuse short distances over fields of cells and regulate gene transcription. They have been grouped into four protein families on the basis of their structural similarities.

Fibroblast growth factors (FGF) are important during embryonic development, tissue construction, and regeneration after wounding. An example is the Breathless FGF protein that activates the Branchless FGF receptor protein (FGFR) to regulate terminal branching in the developing tracheal system of Drosophila. An FGF protein binds to an FGFR and activates a kinase that phosphorylates proteins within the responding cell. One transduction cascade initiated by FGFs is the RTK (receptor tyrosine kinase) transduction pathway, where a specific paracrine factor ligand binds to the extracellular portion of the receptor and induces a conformational change that activates the kinase activity of the cytoplasmic portion and autophosphorylates key tyrosine residues (Figure 1.4). This then recruits proteins functioning as adaptors that couple the receptor to other proteins, which can activate still other proteins to pass the message along. One of these important proteins is Ras, which exchanges its bound GDP for GTP and then stimulates subsequent molecules in the pathway, including the mitogen-activated protein kinases (MAPK), to initiate a series of phosphorylations that ultimately are transferred to the nucleus to phosphorylate certain transcription factors. The Drosophila insulin receptor is a receptor tyrosine kinase with an astonishing similarity to the human insulin receptor.

Figure 1.4 Receptor tyrosine kinase (RTK) transduction associated with a typical enzyme-linked receptor. The cytoplasmic domain of the receptor is switched on when a ligand binds to its extracellular domain. The cascade activates Ras when GTP is bound, activating a phosphorylation cascade that carries the signal to the nucleus via a MAP kinase cascade, affecting transcription factors and gene expression.

Another signal transduction cascade activated by FGFs to transmit extracellular signals to the nucleus is JAK-STAT (Janus kinase-signal transducer and activator of transcription). The cytokine receptors involved activate their associated tyrosine kinases (JAKs), which in turn activate gene regulatory proteins (STATs) that migrate to the nucleus to affect gene transcription. There are seven STAT genes in mammals, each of which binds to a different promoter, and four genes associated with the JAK family. These regulate cell proliferation, blood cell development, and the immune response, and are central to the proper growth and development of mammalian tissues. The cascade is responsible for the differentiation and renewal of both mammalian and Drosophila stem cells. In mammals, its disruption can cause leukemia and immunological disorders. The first identification of JAK-STAT signaling in insects was as a regulatory mechanism in embryonic segmentation, and it is now known to be involved in immune responses and hemocyte development, the formation of the eyes, wings, and legs, and in sex determination. It thus plays a major role in organ morphogenesis and cell rearrangement during development. Suppression of the JAK-STAT pathway in mosquitoes that are normally susceptible to dengue virus infection makes them even more susceptible to virus infection.

At least three ligands have been identified in Drosophila: Unpaired (UDP), UDP2, and UDP3. These bind and induce a conformational change in the transmembrane receptor Domeless (DOME), which phosphorylates and activates the JAK kinase Hopscotch (HOP) and transfers the phosphate group to a tyrosine residue on the transcription factor, dimerizing the STAT and allowing it to enter the nucleus where it binds to particular regions of DNA (Figure 1.5).

Figure 1.5 The JAK-STAT transduction cascade. The activation of JAK by the cytokine receptor subsequently activates the STAT that enters the nucleus and affects gene transcription.

A second family of paracrine factors consists of the cysteine-rich Wnt glycoproteins, found so far in mammals, insects, zebrafish, and nematodes. The name is derived from the combination of the wingless (wg) segment polarity gene in Drosophila and its vertebrate homologue, integrated (int). Wnt proteins are covalently bound to lipid molecules, responsible for their activity by making them hydrophobic. In both vertebrates and insects, Wnt signaling participates in the specification of cell fates and differentiation, and in cell adhesion and movement. When Wnt proteins are released from signaling cells, they bind to the Frizzled (Fz) receptor complex on target cells. The signal is transduced to several intracellular proteins and ultimately to β-catenin, a transcriptional cofactor that is normally degraded by cell metabolism. However, the Wnt signal inhibits this degradation so that levels of β-catenin increase and interact with nuclear transcription factors to influence transcription (Figure 1.6). In addition to signaling within the nucleus, there is an alternative Wnt pathway that phosphorylates the cytoplasmic actin and microtubular cytoskeleton and consequently alters cell shape.

Figure 1.6 The Wnt signaling pathway is activated when the Wnt ligand binds to its Frizzled receptor. The activated Disheveled protein inhibits the normal degradation of β-catenin that occurs through glycogen synthase kinase 3 (GSK3) and the APC protein. The β-catenin enters the nucleus and activates genes.

A third group of paracrine factors consists of the Hedgehog (Hh) family of proteins. Hedgehog was first identified in Drosophila and named after the embryonic cuticular denticles present in gene mutants, which resembled those of hedgehogs. Hedgehog signaling is involved in the growth of many organ systems as key organizers of patterning, most notably those regulating gene expression in Drosophila embryos and wing imaginal discs. It is responsible for the differences between anterior and posterior body segments. Three Hh homologues have been identified in mammals, including Sonic Hh, Desert Hh, and Indian Hh, but only a single homologue has been found in Drosophila. During development of the Drosophila imaginal disc, Hh binds to the membrane receptor Patched (Ptc), which is bound to a signal transducer, Smoothened (Smo). Ptc prevents the Smo protein from functioning, and without Hh bound to Ptc, the Cubitus interruptus (Ci) protein remains tethered to microtubules. On the microtubules, Ci is cleaved and a portion enters the nucleus to act as a transcriptional repressor. However, after Hh binding, Ptc no longer inhibits Smo and it becomes activated to release Ci from the microtubules to prevent it from becoming cleaved. The intact Ci enters the nucleus, where it serves as a transcriptional activator of Hh-associated genes (Figure 1.7). Similar to the Wnt pathway, signal transduction to effect transcription occurs as a result of the inhibition of an inhibitor.

Figure 1.7 The hedgehog signaling pathway. A. In the absence of the polypeptide ligand Hedgehog, the transmembrane receptor Patched prevents the expression of the Smoothened signal transducer, allowing the proteolytic cleavage of the Cubitus interruptus (Ci) protein to occur. B. When activated by Hedgehog, Patched is inhibited and allows Smoothened to accumulate and inhibit Ci cleavage. Ci accumulates and moves to the nucleus where it stimulates gene expression.

The fourth group of paracrine factors are assigned to the TGF-β (transforming growth factor) superfamily. There are more than 30 members of this superfamily, originally discovered for their involvement in stimulating vertebrate bone formation. They have since been implicated in many other signaling systems, including immunological processes, differentiation, cell division, and cell movement, and their disruption has been associated with the human diseases of cancer and hypertension. The Drosophila genome encodes at least seven members of the TGF-β family. The TGF-β ligands include TGF-β proteins, Bone morphogenic proteins (BMPs) and activins. In Drosophila, the BMP-type signal protein Decapentaplegic (Dpp) is required for patterning of the developing wing, along with Glass Bottom Boat (GBB), both closely related to vertebrate BMPs. The homology is so close that BMP can actually substitute for Dpp in otherwise deficient flies during their development. Activin, another TGF-β morphogen, is used for the establishment of synaptic connections in the Drosophila compound eye and is necessary for general cell proliferation during larval and pupal development.

TGF-β superfamily ligands bind to type II serine–threonine kinase receptors, which then bind and activate type I serine–threonine kinase receptors. The activated type I receptors then phosphorylate two Smad proteins (a name created by merging the names of the first family members to be identified, the nematode SMA protein and the Drosophila MAD protein) which together recruit a third co-Smad. The phosphorylated Smad complex then enters the nucleus as a transcription factor (Figure 1.8).

Figure 1.8 Ligands in the TGF-β superfamily bind to and activate serine-threonine kinase type I and II receptors. The activated receptor phosphorylates Smad proteins that then recruit a co-Smad. The complex enters the nucleus as a transcription factor.

Endocrine Signaling

Hormones are the chemical messengers of multicellular organisms that allow the cells to communicate to more distant and widespread targets and engage in coordinated responses. They are especially pervasive in insect systems, affecting a wide variety of physiological processes, including embryogenesis, postembryonic development, behavior, water balance, metabolism, caste determination, polymorphism, mating, reproduction, and diapause. Hormones work along with the nervous system to provide the necessary communications between the many distant cells that comprise a multicellular animal. As in other signaling systems, tissues process the message only if they have the proper receptors that enable them to recognize it. Hormones therefore allow a sustained message to be sent to all cells, but only those cells that posses the receptors are capable of responding.

The method by which insect epidermal cells communicate during molting is a good example of the difference between nervous and endocrine signaling. The molting process, regulated by several hormones, requires hours for its full completion in many insects. It could occur faster if it were coordinated by the nervous system, but that would mean that all the epidermal cells involved would have to receive a nervous message, thereby hopelessly complicating the internal environment with neurons and leaving little room in the body for other organs. There are some cells that may not participate in molting; these are oblivious to the hormonal conditions because they lack receptors. Other processes, such as feeding and escape, cannot rely on the slowness of the endocrine system and are regulated by the nervous system. If information regarding some threat in the environment, such as a predator, were to be relayed by the endocrine system in order to initiate escape behavior, the insect would probably be eaten well before the message arrived. By selecting hormones as a messenger for some systems, insects have made a trade-off between the speed of the response and the complexity of the system that would be required for its implementation.

The classic definition of a hormone, a word coined from the Greek for ‘I excite,’ includes those substances secreted by glands and transported by the circulatory system to other parts of the body, where they evoke physiological responses in target tissues in very minute quantities. Although the term ‘endocrine’ originally implied that multicellular glands were the sources of the chemical messengers, it is now recognized that hormones may also be produced by single cells that are not necessarily clustered into a distinct gland. In addition to these more discrete endocrine glands, there are a number of neurosecretory cells found throughout the body that also produce hormones.

Types of Hormone Release Sites in Insects

Insects have classic endocrine glands, which are tissues that specialize in the secretion of chemical messengers that are transported by the blood and act on receptor-bearing target tissues elsewhere in the body (Figure 1.9A). Examples of endocrine glands in insects are the prothoracic glands, which produce ecdysteroids, and the corpus allatum, which produces juvenile hormones. Like vertebrates, insects also have nerve cells that generate electrical impulses and translate these to chemical messages at the synapses, which then propagate the messages to other neurons to which they are connected. In this case, the messenger, a neurotransmitter, binds to receptors on the postsynaptic neuron, remaining compartmentalized within the synapse and not entering the bloodstream. The neurotransmitter can therefore be considered as a hormone that is acting locally within the synapse (Figure 1.9B). The neurotransmitters may also be released directly at an endocrine cell (Figure 1.9C). Insects also have functional hybrids of neurons and endocrine glands called neurosecretory cells. Neurosecretory cells are specialized neurons that produce chemical messengers that are released into the bloodstream and affect distant target tissues. Rather than doing this at the synapse between two neurons, the chemicals are released into circulation or delivered to cells at a specialized structure called a neurohemal organ (Figure 1.9D). Thus, the utilization of endocrine messengers lies on a spectrum, with neurons at one end providing a local release of neurotransmitter that affects other neurons only; neurosecretory cells in the middle, with their modified neurons releasing neurohormones into general circulation; and conventional endocrine glands at the other end, releasing hormones into general circulation. The chemical products released from these various sites are referred to as hormones if they are produced by endocrine glands, neurotransmitters if produced by neurons, and neurohormones if produced by neurosecretory cells. Neuromodulators may be released by neurons at the synapse and modify the conditions under which other nerve impulses are transmitted and received (Figure 1.9E). Receptors present on the postsynaptic membrane and on target cells specifically bind the molecules and set in motion a biological effect, but non-target cells that lack these receptors are unable to receive the message.

Figure 1.9 Some examples of neurotransmitter release. A. An endocrine cell releasing a hormone into the circulatory system. B. A neuron synapsing with a neurosecretory cell, releasing a neurotransmitter at the synapse. C. A neuron synapsing with an endocrine cell, releasing a neurotransmitter. D. A neurosecretory cell releasing a neurohormone into the circulatory system at a neurohemal organ. E. An inhibitory neuron synapsing with a neurosecretory cell, releasing a neuromodulator at the synapse. F. Receptors on target cells recognize specific neurohormones in circulation, resulting in a biological effect. The absence of receptors on non-target cells result in the cell not being able to respond to the circulating chemical messages, and any molecules taken up nonspecifically are degraded.

Early Experiments that Set the Stage for Our Current Understanding

The first evidence for the existence of hormones in insects is attributed to Bataillon in 1894, although at the time the actual involvement of chemical messengers was not recognized. When silkworm larvae were ligated, separating the anterior and posterior halves with a tightly knotted thread that restricted the flow of hemolymph between the two halves, only the anterior portions of the larvae successfully pupated. However, the result was attributed to differences in internal pressure and not to any hormones.

It was not until the experiments of Kopeć in 1917 that the presence of hormones in insects was confirmed. When Kopeć ligated last instar larvae of the gypsy moth just behind the head, the insects pupated normally except, of course, for abnormalities of the head. In contrast, if an earlier instar was ligated in the same way, pupation failed to occur at all. When the ligature was applied to the middle portion of the last instar larva before a critical period had passed, only the anterior half pupated, with the critical period believed to be the time at which hormone was released into circulation from the anterior portion. However, if the ligature was applied after the critical period, both halves pupated (Figure 1.10). Removal of the brain itself before the critical period prevented pupation, but if the removal occurred after the critical period it had no effect, indicating that the brain was the source of the hormone. This was the first demonstration of an endocrine function for nervous tissue in any animal. Unfortunately, this conclusion was not well accepted at the time because the brain was not believed to have the capacity to produce hormones. Not only did prevailing wisdom consider insects to be devoid of hormones, but the notion of the brain or any other nervous tissue as a source of hormones was unheard of. It was not even known at the time that neurons secrete chemicals at the synapse, so it is easy to understand how others were unwilling to accept that nerve cells could be secretory like an endocrine gland. The nervous and endocrine systems were viewed as functionally distinct in their roles of intercellular communication. It was not until the 1930s that Berta and Ernst Scharrer finally showed that the vertebrate brain had an endocrine function, and also used insects as a convenient model system. They demonstrated that neurosecretory material moved from the cell bodies in the pars intercerebralis of the cockroach brain to the corpus cardiacum. The presence of neurosecretory cells in relatively primitive invertebrates suggested to them that the neurosecretory system was the initial means of intracellular communication from which the more specialized endocrine and nervous systems ultimately evolved. Neurosecretory cells were not a late stage in evolution, but rather an evolutionarily ancient means of biological communication.

Figure 1.10 An experiment performed by ćKope. When a caterpillar was ligated early during the last larval instar, only the anterior half later pupated. However, when ligated late during the last larval instar, both halves pupated. After Cymborowski (1992). Reprinted with permission.

At about this same time, Wigglesworth repeated the experiments of Kopeć using the blood-sucking bug Rhodnius prolixus. Rhodnius has five larval instars, each of which requires a large meal of blood in order to molt. When fourth instar larvae were decapitated within 4 days after their blood meal, they failed to molt. However, when decapitation occurred later than 5 days after blood ingestion, the larvae did molt to the fifth instar (Figure 1.11). Because decapitation is far from precise and obviously removes a number of different structures located in the head, Wigglesworth next focused on the source of the endocrine effect by excising only a portion of the brain containing the neurosecretory cells. When these excised cells were implanted into the abdomens of other larvae that were decapitated early before the critical period, the recipient larvae molted, demonstrating that neurosecretory cells were indeed the source of the brain’s endocrine effect. The historical paths to additional insights into the existence of other insect hormones are discussed in the following sections that describe each hormone.

Figure 1.11 Wigglesworth’s decapitation experiments using Rhodnius larvae. Top: When fourth instar larvae were blood fed and decapitated within 4 days, they failed to molt. Bottom: When they were decapitated after 5 days, the body still molted even though the head was not attached at the time.

Types of Hormones in Insects

Insects produce steroid hormones, such as ecdysteroids, sesquiterpenes, which include all the juvenile hormones, and an abundance of peptide hormones produced by neurosecretory cells throughout the central nervous system and the midgut. There are also a number of biogenic amines, including octopamine, tyramine, and serotonin, that are primarily neurotransmitters derived from amino acids that also may have more widespread effects on the organism. Other hormones, such as prostaglandin, are derived from fatty acids. The circulating titers of a particular hormone and its ultimate effects on target cells are precisely modulated by the interplay between hormone synthesis, release, and degradation in the hemolymph once the hormone is released into circulation, and by the development and specificity of receptor sites on target tissues that allow the specific hormone to be recognized (Figure 1.12).

Figure 1.12 Factors that affect the activity of hormones. Hormonal activity in the circulatory system is regulated by its rate of synthesis by the endocrine glands, the rate of release into the blood, its degradation in the blood, and the development and presence of hormone receptors on target cells.

Peptide hormones are usually synthesized as larger precursor preprohormones and prohormones and then processed by proteolytic enzymes into the smaller final hormone (Figure 1.13). In order for this to occur, the peptide must first be inserted into the cisterna of the endoplasmic reticulum and a signal peptide portion attached. The pre and pro portions are cleaved and the peptide hormone is then released from the cell by exocytosis.

Figure 1.13 The synthesis and processing of peptide hormones.

Modes of Action

There are fundamental differences in the mechanisms by which different hormones act on target cells. Because of their hydrophobic non-polar nature, ecdysteroids and juvenile hormones are able to enter the cell and bypass the complex cascade of signal transduction prescribed for peptide hormones. Instead of bearing transmembrane receptors, cells that respond to steroid hormones have receptors that reside in the cytoplasm and which, after binding to the hormones, translocate to the nucleus where they directly interact with DNA and its transcription (Figure 1.14). These nuclear receptors are ligand-dependent intracellular transcription factors that stimulate or block the synthesis of mRNA, and their presence makes the cell a target for the hormone. The three families or classes of the nuclear receptor protein superfamily include the steroid receptor family (class I), the thyroid/retinoid family (class II), and the orphan receptor family (class III), based on differences in the way the receptors in each class recognize and bind to DNA.

Figure 1.14 The mode of action of steroid hormones. The cell membranes are permeable to steroid hormones, so they pass through both the cell and nuclear membranes. They bind to receptors that serve as transcription factors, and together they directly interact with DNA and regulate transcription of mRNA and the production of proteins.

All the members of the nuclear receptor superfamily have a common modular structure (Figure 1.15A). This consists of a highly conserved DNA-binding domain (DBD) that binds the receptor to nucleotide response elements that reside within gene promoters. The amino acid sequences of the DBD cause the proteins to fold around zinc ions and form projections of two fingers (Figure 1.15B), one providing DNA specificity by a sequence of amino acids called the P-box, and the second allowing dimerization with another DBD. Because these zinc fingers provide the principal interface between the DBD and specific nucleotides within the hormone response element (Figure 1.15C), small differences in the amino acid residues of the receptor protein can affect the nature of the projections and the activity as a transcription factor.

Figure 1.15 Characteristics of nuclear receptors. A. Modular structure of domains. B. Amino acids form fingers that fold around zinc ions. C. Binding of zinc finger transcription factors to hormone response elements.

The DBD is joined to the ligand-binding domain (LBD) by a flexible hinge region. The less-conserved LBD serves several important functions. It contains the binding pocket for its specific hormone as well as a ligand-regulated transcriptional activating function (AF-2) that can recruit other co-activating proteins. The LBD also mediates dimerization with other receptors to expand on the potential DNA sequences and regulatory functions it is able to target. At the N-terminal of the DBD the amino acid sequences create a transcriptional activation function, AF-1. Unlike the ligand-dependent AF-2, AF-1 is ligand independent. Upon ligand binding, the LBD is activated by rotating its structural helix to a configuration that allows AF-1 and AF-2 to recruit transcriptional co-activators. The cell responds to the hormone by activating or inactivating specific genes when these transcription factors bind to hormone response elements of the target gene promoter. Although nuclear receptors are built on a modular ground plan, the interaction between the modules is responsible for receptor activity. The various isoforms of each transcription factor may also account for the tissue and target gene specificity of the hormones.

In contrast to steroid hormones, peptide hormones are hydrophilic, much more polar, and cannot easily pass through the cell membrane. To affect cell function they must trigger the cellular response while remaining on the outside. The peptide hormones bind to protein receptors on the membrane’s outer surface, altering the conformation of the receptor and consequently initiating the synthesis of second messenger molecules that carry the message inside the cell. These second messengers then act through a cascade of phosphorylations, resulting in the activation or inactivation of specific enzymes. A small number of molecules of the first messenger, or hormone, can thus be amplified by the production of a larger number of these second messengers.

There are several different second-messenger signal transduction systems, many of which involve a membrane-bound G-protein that consists of three subunits (α, β, γ) and operates between the first and second messengers. For some hormone transduction systems the second messenger is cyclic AMP (Figure 1.16). When the membrane receptor for the hormone becomes bound, it changes its conformation and causes it to come in contact with the G-protein. This causes the G-protein subunits to dissociate, with the α subunit activating the membrane-bound enzyme adenylate cyclase and forming cyclic AMP from ATP. The cAMP that is formed then stimulates a protein kinase that phosphorylates and activates enzymes and ribosomal and nuclear proteins to elicit a biological response (Figure 1.17A).

Figure 1.16 Two of the major roles of adenine in cells. As cyclic AMP, it acts as a second messenger in cells. As ATP, it serves as a form of energy storage and transfer.

Figure 1.17 Signal transduction via second messengers. A. A protein kinase is activated by the second messenger cAMP that is formed from the adenylate cyclase generated when the G-protein, shown as only a single component here, dissociates as the hormone binds to the membrane bound receptor. B. The two second messengers, triphosphoinositol (IP3) or diacylglycerol (DAG), release calcium or activate a protein kinase, respectively, that can then activate enzymes. The second messengers are formed when a phospholipase is activated from the binding of the hormone to the receptor-associated G-protein.

A second major signal transduction pathway coupled to G-proteins involves the activation of a phospholipase and a subsequent increase in intracellular calcium. The hormone-receptor complex acts through a G-protein to activate a membrane-bound phospholipase that hydrolyzes the complex membrane molecule phosphatidylinositol 4,5-diphosphate (PIP2) to form two second messengers, triphosphoinositol (IP3) and diacylglycerol (DAG). The IP3 causes the release of calcium from the endoplasmic reticulum, which can activate exocytosis in cell secretory mechanisms and cell enzyme cascades. The DAG activates a membrane-bound protein kinase that phosphorylates and activates other enzymes (Figure 1.17B). In both these pathways pre-existing enzymes are activated when the hormone binds, unlike the mechanism of steroid hormone action which directly involves the activation of gene transcription and the synthesis of new enzymes. G-proteins may also influence the opening of membrane channels that allow Ca²+ or K+ to enter the cell.

The gas nitric oxide (NO) can also serve as a second messenger in insect systems. Increases in intracellular calcium from intracellular stores or through Ca²+ membrane channels activate the enzyme nitric oxide synthase through the calcium-binding protein calmodulin, forming nitric oxide and citrulline from arginine (Figure 1.18). The nitric oxide is able to cross the cell membrane and activate a soluble guanylate cyclase in neighboring cells that increases levels of cyclic GMP. The cGMP has a wide variety of effects on the target cell, including the activation of cGMP-dependent enzymes and the permeability of membrane channels. NO can also bind to nuclear transcription factors and repress or induce gene transcription. NO signaling in insects is associated with the Malpighian tubules, salivary glands, the central nervous system, and the development of the compound eyes. As in vertebrates, NO signaling is important in chemosensory transduction, especially in olfactory receptors associated with the antennal lobes of insects. NO signaling is also involved in establishing patterns of neural outgrowth and migration, influence on neural wiring, and in the control of the corpus allatum for the production of juvenile hormone.

Figure 1.18 Nitric oxide as a second messenger. The enzyme nitric oxide synthase can be activated by several pathways. The nitric oxide formed in the cells diffuses easily through the cell membrane and is able to activate the enzyme guanylate cyclase, causing a rise in cGMP that then has cellular effects. It can also bind to transcription factors and affect gene expression.

Prothoracicotropic Hormone

Prothoracicotropic hormone (PTTH) was the first insect hormone to be discovered and the last of the major insect hormones to be structurally identified, spanning almost 70 years before its primary structure was reported by Kataoka and co-workers in 1991. PTTH was the brain hormone described in Kopeć’s early investigations, but because the brain was later found to produce so many hormones, the simple designation of ‘brain hormone’ was no longer descriptive. Although its current name indicates its ability to activate the prothoracic glands, there are several other structurally unrelated molecules that have PTTH-like activity and have been placed under the banner of prothoracicotropic hormones. The major action of PTTH is to regulate the synthesis of ecdysteroids by the prothoracic gland.

Wigglesworth’s work in the 1940s established that a region of the Rhodnius brain containing large neurosecretory cells was the source of PTTH activity. Williams demonstrated the relationship between the brain and prothoracic glands in the late 1940s and early 1950s. He implanted both the prothoracic glands and a brain from a chilled pupa into a diapausing pupa and showed that both the brain and prothoracic gland were required to terminate diapause, and that the brain activated the prothoracic glands. When he implanted a single chilled brain into a chain of brainless diapausing pupae connected by parabiosis, all the pupae successively underwent adult development (Figure 1.19). Two pairs of neurosecretory cells were specifically identified in Manduca by the microdissection and implantation experiments of Agui and co-workers in 1979. PTTH is produced in the large lateral neurosecretory cells of the brain and is released in the corpus cardiacum that terminates in the wall of the aorta, or in some insects, released by the corpus allatum. In a few lepidopterans that have been studied, hindgut extracts of last instar larvae were shown to stimulate the prothoracic glands and are also considered as prothoracicotropic sources.

Figure 1.19 An experiment by Williams where a chain of brainless parabiosed pupae (A) were activated to molt by the implantation of a single brain into the first pupa (B). From Williams (1952). Reprinted with permission.

The delay in its structural identification was largely due to the lack of a reliable bioassay. Early bioassays for PTTH consisted of debrained pupae, referred to as ‘dauer’ (German: a long time) pupae because they could survive for 2–3 years until all the nutrients within them had been exhausted. When extracts with PTTH activity were injected, the pupae initiated metamorphosis to the adult stage. There were several problems with this bioassay, including a low reproducibility due to physiological variations between pupae and the relatively long time it took to score a response. A more direct assay for PTTH was developed by Bollenbacher and co-workers in 1979 using the criterion of ecdysone production by a pair of prothoracic glands that were maintained in vitro. The basal rate of ecdysone synthesis by a non-stimulated gland is compared to the rate of ecdysone secretion by a gland that is incubated with a suspected source of PTTH. The activation of the gland is indicated by a significant increase in ecdysone synthesis, measured by a radioimmunoassay (Figure 1.20). It is still not a completely satisfactory bioassay because the prothoracic gland preparations that are required involve a sometimes difficult dissection and isolation.

Figure 1.20 An assay for PTTH developed by Bollenbacher et al. (1979). A pair of matched prothoracic glands are removed from the insect and placed in culture. If PTTH is added to the culture, the glands produce increased amounts of ecdysteroids into the medium.

The PTTHs from only a handful of insects have been identified, with most of the work centered on the lepidopterans Bombyx mori, Manduca sexta, Hyalophora cecropia, and Lymantria dispar, and the dipterans Drosophila melanogaster and Anopheles gambiae. Initial isolations characterized the PTTHs as multiple forms that fell into two groups: the ‘big’ PTTH (14–29 kDa) and the ‘small’ PTTH (3–7 kDa),