Physiological Systems in Insects

Preface to the Fourth 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 since 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 groundplan 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 didn’t 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 past 20 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 melanogaster. These similarities have altered the focus of insect science, with insects viewed 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 D. melanogaster genome, on its four pairs of chromosomes, is encoded by about 14,000 genes, a bit more than half of the protein coding sequences identified in humans. Although each group has evolved its own distinctive genes, many of those related to major biological processes have been well conserved. The other obvious aspects of short life cycles, little space required for rearing, considerably less food needed than if one were 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, are 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 megabytes of storage was more complex than a present-day iPhone with 512 gigabytes 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. D. melanogaster engages 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 total sleep amounts do not decrease in older D. melanogaster, 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 D. melanogaster can be effective models 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 D. melanogaster, 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 D. melanogaster homolog 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 didn’t 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. D. melanogaster 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 increases in our waistlines, D. melanogaster 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 regulate 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 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 antiosteoporosis 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 that involve both physical and molecular mechanisms that maintain a resident flora and discourage pathogenic bacteria. Modeling human intestinal disease in D. melanogaster 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. Knowledge of the role of gut microbiota in the regulation of overall human health and multiple physiological processes, including the social dysfunction associated with autism-spectrum disorders and the etiologies of Parkinson’s and Alzheimer’s diseases, has increased dramatically in recent years. Neurophysiological effects of gut microbiota on the olfaction, communication, and behavior of social insects parallel and could model the gut microbiota–brain connection in humans.

The tools of molecular biology have provided incredible insights into the physiological systems that contribute to the success of insects, but an organismal focus is still essential to completely understand how these systems participate toward this success. 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 increasingly large cadre of scientists who work with insects but don’t have the opportunity to feel very much about them. We hope this text continues to 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.

As knowledge of insect physiology has grown, so has each edition of this book. Rather than evolving this fourth edition into a multivolume reference, we chose to maintain it as a textbook that is an introduction to the physiological systems in insects. To meet the needs of readers who require more detail, there is an extensive list of references at the end of each chapter to serve as a guide to further information from the primary literature.

In this fourth edition, we updated each chapter to include more current information. We also added information on insecticide target sites, insecticide mode of action, and resistance development. New references were also added to the lists at the end of each chapter. To provide basic information on genetics, genomics, and epigenetics and their applications in the field of insect physiology to the readers, we added a new chapter (Chapter 13) to this edition.

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Chapter 1: Signaling systems

Abstract

Cells in a multicellular animal can communicate to elicit responses during development and changing environmental conditions by the process of signal transduction. The various signaling pathways in insects, the origins of the signaling molecules, their targets, and the outcomes of the signaling, are described. Controlling insects by using hormone mimics may be an effective alternative to chemical pesticides. Some early experiments that set the stage for our modern understanding of molting and metamorphosis are discussed.

Keywords

Ecdysis; Ecdysteroids; Juvenile hormones; Metamorphosis; Neuropeptides; Prothoracicotropic hormone (PTTH)

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 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 (Fig. 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 (Fig. 1.2).

Fig. 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.

Fig. 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 to itself by producing a chemical that activates receptors within its own cytoplasm or on its surface (Fig. 1.3A). An example is the prothoracic gland, which during some developmental stages activates its own production of ecdysteroids.

Fig. 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 (Fig. 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, which gives 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 (Fig. 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 that 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 (Fig. 1.3D). Only those cells with receptors that recognize a given 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, while most others are peptides that must remain outside and bind to external receptors.

Paracrine signaling is a bit more intimate, with the signal molecules diffusing not through the blood but instead through the extracellular matrix (Fig. 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 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 even more beautiful parsimony are used at different times for different developmental events within the same organism. These are the morphogens that 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 (FGFR) protein 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 receptor tyrosine kinase (RTK) 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 (Fig. 1.4). This then recruits proteins functioning as adaptors that couple the receptor to other proteins that 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 (InR) is an RTK with an astonishing similarity to the human InR.

Fig. 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 Janus kinase-signal transducer and activator of transcription (JAK-STAT). The cytokine receptors involved activate their associated tyrosine kinases (JAKs) that 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, in 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. They bind and induce a conformational change in the transmembrane receptor Domeless (DOME) that 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 (Fig. 1.5).

Fig. 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 homolog, 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 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 (Fig. 1.6). In addition to signaling within the nucleus, an alternative Wnt pathway phosphorylates the cytoplasmic actin and microtubular cytoskeleton and consequently alters cell shape.

Fig. 1.6 The Wnt signaling pathway is activated when the Wnt ligand binds to its Fz 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.

The third group of paracrine factors consists of the Hedgehog (Hh) family of proteins. Hh was first identified in Drosophila and named after the embryonic cuticular denticles present in gene mutants that resembled those of hedgehogs. Hh 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 homologs have been identified in mammals, including Sonic Hh, Desert Hh, and Indian Hh, but only a single homolog has been found in Drosophila. During the development of the Drosophila imaginal disc, Hh binds to the membrane receptor, Patched (Ptc), that 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 (Fig. 1.7). Similar to the Wnt pathway, signal transduction to effect transcription occurs as a result of the inhibition of an inhibitor.

Fig. 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 is assigned to the transforming growth factor β (TGF-β) 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 utilized 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) that together recruit a third co-Smad. The phosphorylated Smad complex then enters the nucleus as a transcription factor (Fig. 1.8).

Fig. 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 thus allow a sustained message to be sent to all cells, but only those cells that possess 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, 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 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 classical 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 classical 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 (Fig. 1.9A). Examples of endocrine glands in insects are the prothoracic glands, which produce ecdysteroids, and the corpus allatum (CA), which produces juvenile hormones. Insects, like vertebrates, also have nerve cells that generate electrical impulses and translate these to chemical messages at the synapse and 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 thus be considered as a hormone that is acting locally within the synapse (Fig. 1.9B). The neurotransmitters may also be released directly at an endocrine cell (Fig. 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 (Fig. 1.9D). Thus, the utilization of endocrine messengers lies on a spectrum, with neurons at one end that provide 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 (Fig. 1.9E). Receptors present on the postsynaptic membrane and on target cells specifically bind the molecules and set into motion a biological effect, but nontarget cells that lack these receptors are unable to receive the message.

Fig. 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 nontarget cells results 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 the 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 (Fig. 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 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 (CC). 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.

Fig. 1.10 An experiment performed by Kopeć. 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, B., 1992. Insect Endocrinology. Elsevier Science Ltd. 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 the fourth-stage nymphs were decapitated within 4 days after their blood meal, they failed to molt. However, when decapitation occurred later than 5 days following blood ingestion, the nymphs did molt to the fifth stage (Fig. 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 nymphs that were decapitated early before the critical period, recipient nymphs 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.

Fig. 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.

Type of hormones in insects

Insects produce steroid hormones, such as ecdysteroids, sesquiterpenes that 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 (Fig. 1.12).

Fig. 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 (Fig. 1.13). 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.

Fig. 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 nonpolar 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 that, after binding to the hormones, translocate to the nucleus where they directly interact with DNA and its transcription (Fig. 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.

Fig. 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 (Fig. 1.15A). It 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 (Fig. 1.15B), one providing DNA specificity by a sequence of amino acids called the P-box, and the second zinc finger allowing dimerization with another DBD. Because these zinc fingers provide the principal interface between the DBD and specific nucleotides within the hormone response element (Fig. 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.

Fig. 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 coactivating 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 coactivators. 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 (Fig. 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 (Fig. 1.17A).

Fig. 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.

Fig. 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 that 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 (Fig. 1.17B). In both these pathways, preexisting enzymes are activated when the hormone binds, unlike the mechanism of steroid hormone action that 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, 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 (Fig. 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, influencing neural wiring, and controlling CA for the production of juvenile hormone (JH).

Fig. 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 coworkers 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 PTTH. The major action of PTTH is to regulate the synthesis of ecdysteroids by the prothoracic gland and their numerous consequent events.

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 (Fig. 1.19). Two pairs of neurosecretory cells were specifically identified in Manduca by the microdissection and implantation experiments of Agui and coworkers in 1979. PTTH is produced in the large lateral neurosecretory cells LNCs of the brain and is released in the CC that terminates in the wall of the aorta or, in some insects, is released by the CA. 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.

Fig. 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, C.M., 1952. Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Biol. Bull. 103, 120–138. 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 coworkers 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 nonstimulated gland is compared with 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 radioimmunoassay (Fig. 1.20). It is still not an entirely satisfactory bioassay because the prothoracic gland preparations that are required involve a sometimes difficult dissection and isolation.

Fig. 1.20 An assay for prothoracicotropic hormone (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) based on a rather serendipitous bioassay. Bombyx mori silkworm moths were plentiful because of rearing methods developed by the silk industry and were ideal sources of the hormone because so much biomass was required as a starting point for the isolation of the minute quantities of hormones. In contrast, surgery for brain removal is easier in the Samia cynthia moths, and they made ideal bioassay animals for testing the PTTH activity of introduced materials. The standard bioassay thus involved attempts to identify PTTH from Bombyx bioassayed in a debrained Samia, which then initiated metamorphosis if PTTH was present. The small PTTH isolated from Bombyx was indeed able to activate the prothoracic glands of the related moth Samia cynthia as well as those of the blood-sucking bug, Rhodnius prolixus, but curiously, it was unable to activate the glands of Bombyx. This small PTTH was renamed bombyxin and is no longer considered as a true PTTH, mainly because there is no relationship between its titer in the hemolymph of Bombyx and the levels of 20-hydroxyecdysone that result. Bombyxin is also capable of stimulating cultured prothoracic glands, but only at doses hundreds of times greater than normal physiological levels. The multiple molecular species of the bombyxins that have been identified appear to share some homology with vertebrate insulin, but their exact roles in insect systems have yet to be determined. Bombyxin receptors are present on the ovaries of some lepidopterans, and the hormone may be involved in ovarian development and the utilization of carbohydrate during egg maturation. Insulin-like molecules have also been implicated in the control of insect growth, and bovine insulin is able to stimulate incubated prothoracic glands of Bombyx mori.

It is the big PTTH that is acknowledged to act as the true PTTH: it stimulates the prothoracic glands to produce ecdysone. In Bombyx, big PTTH is synthesized as a large 224 amino acid precursor with three proteolytic cleavage signals. It is then cleaved to liberate a 109 amino acid subunit. The active molecule is a homodimer, consisting of two identical chains that are held together by three intramolecular disulfide bonds and one intermolecular cysteine-cysteine bond (Fig. 1.21). The folding of the molecule is largely controlled by these intra- and intermolecular disulfide bonds.

Fig. 1.21 The amino acid structure of prothoracicotropic hormone (PTTH). Only one of the two identical chains in the homodimer is shown. From Nagata, S., Kataoka, H., Suzuki, A., 2005. Silk moth neuropeptide hormones: prothoracicotropic hormone and others. Ann. N. Y. Acad. Sci. 1040, 38–52. Reprinted with permission.

Control of