Receptors for Neuronal or Endocrine Signalling Molecules as Potential Targets for the Control of Insect Pests

Abstract

In metazoans, neuronal and endocrine communication is based on the release of extracellular signalling molecules that are recognised in a physiological concentration range by specific receptor proteins present in the target cells. These receptors will elicit a cellular response upon activation by their physiological agonist. A highly diverse repertoire of naturally occurring receptor agonists has already been discovered. Peptides, proteins and biogenic amines constitute the most diverse agonist classes. Most of these interact with G protein-coupled receptors (GPCRs), the largest category of signal transducing receptors that controls virtually every physiological process in metazoans. For more than two decades, insect GPCRs have been hailed for their potentially excellent aptitude to serve as pharmacological targets for the development of novel products for insect pest control. In this review, we will address this issue and enumerate reasons why it would be worth investing more in these targets.

Introduction

As all metazoans, insects are heterotrophs, implicating the basic need for the intake and digestion of food, as well as for the intestinal absorption of nutrients. Important biological processes, such as growth, development and reproduction, strongly depend on this nutritional input, as well as on other environmental cues. Therefore, it is crucial for animals to rely on internal mechanisms for the physiological regulation and coordination of these fundamental processes. Nutrient-sensing, hormonal and neuronal signalling systems play an important role in this complex regulation.

This review starts with an introduction of general aspects peculiar to insects: their different life cycles, their development, reproduction, food uptake and metabolism. In addition, some important neurohormones are mentioned that play crucial roles in these processes. The introduction is followed by a thorough overview of GPCRs and their functional role in signal transduction. The vast majority of these biomolecular mechanistic analyses have been performed with vertebrate GPCRs, nonetheless, where possible, we also describe what is known about this receptor class in insects. After the general part about GPCRs, different neurohormones (neuropeptides and biogenic amines), their receptors and the possible suitability of these receptors as insecticide targets are described. Finally, the applicability of neurohormone receptors as targets for insecticides is discussed.

Three major insect life cycle strategies can be distinguished. Insects can be either ametabolous, hemimetabolous or holometabolous. A brief description of each type will be given and illustrated with the life cycle of some important pest insects that were subject of many studies described in the chapter.

Ametabolous insects, such as springtails (order Collembola) and silverfish (order Thysanura), hatch from their eggs nearly in the same form as they will retain throughout their entire life, only lacking mature gonads and functional genitals. After each moult, the genital primordia become larger, until the insect is able to reproduce. Ametabola, therefore, do not undergo metamorphosis.

The silverfish, Lepisma saccharina, is known as a cosmopolitan household pest insect. Silverfish are among the few types of insect that continue to moult after reaching adulthood, a condition that was probably more common in the ancestral arthropod groups (Fig. 3.1) (Houseman, 2007).

Hemimetabolous (also designated as ‘heterometabolous’) insects, such as the pea aphid, Acyrthosiphon pisum (order Hemiptera), grasshoppers and crickets (order Orthoptera), undergo an incomplete metamorphosis. After emerging from the egg, the nymphs actually appear as small and immature versions of the adult, lacking wings and a functional reproductive system. After a series of moults, the nymphs pass to the reproductive adult stage, often winged. This type of development generates adults that can combine reproduction with flight, a situation which is probably advantageous for finding a sexual partner and for spreading the resulting offspring, increasing the chances for survival at the population and species level.

A. pisum, as an example of a hemimetabolous insect, is sap-sucking and feeds on several species of legumes worldwide. The sap intake weakens the host plants, which consequently affects their yields. Additionally, pea aphids can be vectors of viral diseases of plants. Female pea aphids lay their fertilised eggs in autumn and these eggs overwinter and hatch in spring. The hatched nymphs undergo four moults before entering the adult stage. The nymphs that hatch from the overwintered eggs are all females. In spring and summer, the pea aphid reproduces by viviparous parthenogenesis. When the population density rises or when food quality drops winged females are produced. These females leave the colony to infest other plants. In the fall, there is a single generation of male and female aphids, which produce diapausing eggs through sexual reproduction (Fig. 3.2) (Shingleton et al., 2003, van Emden and Harrington, 2007).

Other examples of hemimetabolous insects are locusts in the order of Orthoptera that are well known as swarming agricultural pests. Depending on their population density, locusts have the ability to undergo a very remarkable transition between two extreme phenotypes (designated as ‘phases’). Most of the time, locusts appear in their solitary phase, which is not very harmful for agriculture. However, under specific environmental conditions, these solitarious animals undergo a transition to the swarming gregarious phase. This usually happens over a large area at the same time and all these hopper bands and swarms together, called a locust plague, can destroy entire crop harvests over vast areas (Pener and Simpson, 2009, Symmons and Cressman, 2009, Verlinden et al., 2009). One of the most notorious locust species is the desert locust, Schistocerca gregaria. This species has been a threat to agriculture in Africa, Southwest Asia and the Middle East since the early beginnings of human civilisation. The devastating effects of locust swarms were already documented in ancient texts, such as the Bible and the Quran. The life cycle of the desert locust consists of three components: egg (embryo), larva (hopper) and adult. Female locusts lay between 80 (gregarious females) and 160 (solitarious females) eggs per egg pod. After hatching, the desert locusts pass through five or six (some solitarious animals) larval stages. Only adults possess fully developed wings. Solitarious desert locusts become sexually mature more slowly and the reproductive cycles take longer, but on the other hand they generally live longer than gregarious locusts and produce more and larger egg pods (as reviewed by Pener and Simpson, 2009, Symmons and Cressman, 2009).

Holometabolous insects, such as flies (order Diptera), moths (order Lepidoptera), beetles (order Coleoptera) and bees (order Hymenoptera), undergo a complete metamorphosis. After hatching from the eggs, the larvae undergo several moults. After the final larval moult, they will enter a non-feeding pupal stage, in which an extreme anatomical makeover (complete metamorphosis) to a winged, hexapod adult takes place.

The life cycle of tsetse flies, Glossina spp, which are the biological vectors for the trypanosomes (protozoan parasite), that cause sleeping sickness in humans and cattle is characterised by adenotrophic vivipary. Tsetse flies are found only in sub-Saharan Africa, Yemen and Saudi Arabia and form a threat to millions of people and their cattle. During this process, the female fly produces one egg at the time, which hatches in the uterus (Fig. 3.3) (Leak, 2009).

Another example of a holometabolous insect is the tobacco hornworm, Manduca sexta, present throughout a large part of the American continent. As its name suggests, the caterpillars feed on foliage of the tobacco plant and other members of the nightshade family (Solanaceae). The tobacco hornworm has mechanisms to sequester the toxic nicotine present in tobacco. The moth has a typical holometabolic life cycle, containing four major stages: egg, larva (caterpillar), pupa and adult (moth). The caterpillars undergo four or five moults. Right before their final moult into the pupal stage the caterpillar stops feeding. This non-feeding stage is called the pre-pupal stage and the start of this stage is marked by wandering behaviour and the enzymatic breakdown of the cuticle (Dominick and Truman, 1984, Xu and Denlinger, 2003). The pupal stage lasts approximately 18 days under long-day photoperiod (17 h light/7 h dark), while it can enter diapause for several months under short-day photoperiod (12 h light/12 h dark). After eclosion, female moths are ready to mate, which they usually do only once, while males can mate several times (Fig. 3.4) (Bell et al., 1975, Madden and Chamberlin, 1945, Wink and Theile, 2002).

An additional example of a holometabolous insect is the red flour beetle, Tribolium castaneum, that is a global pest for stored grain products, such as pasta, cereal and flour. It lives in very dry environments and therefore is found in temperate areas, but will survive the winter in protected places, especially where there is central heating (Tripathi et al., 2001). The number of instars ranges from five to eleven and depends on external conditions such as temperature, humidity and food availability (Good, 1936, Weston and Rattlingourd, 2000).

The honeybee, Apis mellifera, is a beneficial insect that is often negatively influenced by insecticides against pest insects. This social insect has been introduced all over the world for plant pollination and for honey production. The queen of the colony is the only fertile female. In the mid to late winter, after mating with drones, she starts laying eggs. When the eggs hatch, the larvae are fed by the nurse bees, which are worker bees that maintain the colony. After about a week the pupal stage starts and after another week an adult bee emerges. Fertilised eggs develop into females, while unfertilised eggs develop into haploid males (drones). Depending on the food that the female larvae receive, they become a queen or a worker (Hutchins et al., 2003).

The development of insects from egg to adult proceeds through different larval stages. Where growth in weight is a more or less continuous process, growth in size is discontinuous because the rigid cuticle limits expansion. Since insects are characterised by a hard exoskeleton, they need to undergo several moults before reaching the adult reproductive state. The process of moulting is therefore a potential target for insect pest control. Moulting is the periodic shedding of the old cuticle and formation of a new cuticle of greater surface area. During the post-moulting period, increases in body dimensions are confined, before the cuticle stiffens and sklerotizes. The stages in between moults are called larval instars and the number of instars varies between different insect species. There is a tendency of decreasing number of moults in the course of insect evolution. In some insects, such as collembolans, diplurans and other apterygote insects, growth is indeterminate, meaning that they moult until they die. However, in most insects there is a limited number of moults, so their growth is determinate. Determinate insects become reproductively mature in their final, adult stage, which is often referred to as the ‘imago’. In some species, the number of instars is determined by environmental conditions, while in many others the number of instars is constant, regardless of their environment (Adams, 2009, Gullen and Cranston, 2005, Hutchins et al., 2003).

Larval development is characterised by an alternation between feeding and moulting periods. Moulting is a highly complex process accompanied with hormonal, behavioural and physiological changes. These changes will eventually lead to the formation of a new, larger cuticle and the shedding of the old cuticle. The cuticle or exoskeleton is a rigid structure which cannot expand and to which the muscles are attached. Under the cuticle, an epithelial monolayer is found, called the epidermis (also designated as ‘hypodermis’). Epidermal cells are responsible for both the partial breakdown of the old cuticle and the production of the new cuticle. Shortly before ecdysis, the process of shedding of the remains of the old cuticle, the epidermal cells secrete enzymes for digestion of the proteins and chitin in the old cuticle. After ecdysis the insect fills its trachea with air and retains more water in order to increase its body volume before the new cuticle starts to harden. The hardening of the cuticle, also called sclerotisation, is based on the cross-linking of cuticular proteins in the procuticle, which also contains chitin and phenol oxidases (Adams, 2009, Gullen and Cranston, 2005, Hutchins et al., 2003).

Despite the differences in the pattern of development between insects, the endocrine system that regulates growth, moulting and metamorphosis appears to be conserved to a large extent. Several endocrine centres are responsible for the control of moulting, namely the brain neurosecretory cells, the epitracheal glands, the prothoracic glands, the brain–corpora cardiaca (CC) complex, and the corpora allata (CA). The success of moulting is dependent on a coordinated gene expression, controlled by juvenile hormone (JH) and ecdysteroids. The critical signal for the switch from feeding to moulting is an elevated titer of ecdysteroids, including ecdysone (E), 20-hydroxyecdysone (20E) and other hydroxylated analogues. Which ecdysteroid is the most active compound can be species-dependent. The nature of the moult on the other hand, namely from larva to larva, larva to pupa or pupa to adult in holometabolous insects for example, depends on the concentration of JH at the time of moulting (Gullen and Cranston, 2005, Riddiford, 2009, Žitñan and Adams, 2012). Low JH concentration permits expression of adult characteristics. The precise mode of functional interaction of both hormones and their regulation still remains largely unclear. Although several regulatory factors are already known, the overall regulatory scheme is not yet entirely clear. During the intermoult phase, JH levels are high, while at the end of the larval stage these levels decline and ecdysteroid levels rise. In lepidopteran species, the elevation of ecdysteroid levels is due to stimulation of ecdysteroidogenesis in the prothoracic glands by prothoracicotropic hormone (PTTH) (Kawakami et al., 1990). This peptide hormone is synthesised in the brain in a pair of large neurosecretory cells and then stored in the CA, where its release will be triggered by several physiological factors, such as nutritional status, and environmental cues, like photoperiod (for a review, see: Gilbert et al., 2002). The sensory input, however, is species-specific. In the migratory locust, Locusta migratoria, for example, stretch receptors in the wall of the pharynx are stimulated when food passes. This stimulatory information is submitted via the stomatogastric nervous system to the brain–CC complex (Clarke and Langley, 1963). Anyway, PTTH is not the only ecdysiotropic factor responsible for the complex secretory pattern of ecdysteroids. The secretion of ecdysteroids is the result of a balanced interplay between ecdysiotropic and ecdysiostatic factors. For example, bombyxin and other insulin-like peptides (ILPs) show prothoracicotropic activities in some lepidopteran species and in the fruit fly Drosophila melanogaster (Kiriishi et al., 1992, Marchal et al., 2010). However, it is not known yet if this is a direct or an indirect effect. One hypothesis is that ILPs may control the release of PTTH, thereby acting indirectly on the moulting glands. Other peptides with reported prothoracicotropic effects are orcokinin, diapause hormone (FXPRL-amide), diapause hormone-like peptide and pyrokinin-II (FXPRL-amide). Known factors with reported prothoracicostatic activity are the prothoracicostatic peptides [also known as B-type allatostatins (AST-B) or myoinhibiting peptides (MIP)], myosupressin, FMRF-amide-related peptides and trypsin modulating oostatic factor (TMOF) (for more detailed reviews on the regulation of ecdysteroidogenesis, see: Marchal et al., 2010, Tanaka, 2011).

Ecdysteroids act via the Ecdysone receptor (EcR) subunit of a nuclear, heterodimer receptor complex, EcR/Ultraspiracle. Binding of the active moulting hormone to its receptor initiates transcription of ‘early’ genes, which in turn modulate the activity of ‘early-late’ and ‘late’ genes, which carry out the tissue specific processes of moulting (Riddiford et al., 2003). Just before moulting the ecdysteroid levels decline and a cascade of peptide hormones initiate ecdysis. Neuroendocrine signals, such as corazonin (Kim et al., 2004), ecdysis-triggering hormone (ETH) (Ewer et al., 1997, Žitñan et al., 1996, Žitñan et al., 2002), eclosion hormone (EH) (Ewer et al., 1997), crustacean cardioactive peptide (CCAP) and MIP (Kim et al., 2006a, Kim et al., 2006b), are crucial factors in the regulation of the ecdysis sequence (as reviewed by Ewer, 2005, Truman, 2005, Žitñan et al., 2007). Corazonin and EH act sequentially on the Inka cells of the epitracheal gland, in order to release pre-ETH and ETH. These two peptides act on the central nervous system and will form a positive feedback loop for the release of EH from the ventral median neurons. This positive feedback pathway will lead to the depletion of ETH from Inka cells, which is necessary for the ecdysis behaviour that leads to the shedding of the cuticle. The behavioural responses to ETH are enabled by exposure to high ecdysteroid levels, since ecdysteroids induce sensitivity of the central nervous system to ETH. Ecdysteroids are responsible for both increased rates of ETH production in the Inka cells, as well as elevated expression of the ETH receptor. However, ecdysteroids inhibit the release of ETH and a decline of ecdysteroids is necessary for the release of ETH from Inka cells. Ecdysteroid decline is also responsible for the increased excitability of ventral median neurons, which are primary targets of ETH in order to release EH. EH will not only induce the release of ETH, it will also induce an elevation of cyclic GMP in a subset of neurons to initiate ecdysis behaviour (Ewer and Truman, 1997). Another important factor in the ecdysis sequence is bursicon (Luo et al., 2005, Mendive et al., 2005). This protein hormone is released from neurosecretory cells in the brain and the central nerve cord after ecdysis and accelerates sclerotisation of the cuticle (as reviewed by Arakane et al., 2008, Gammie and Truman, 1997, Loveall and Deitcher, 2010, Truman, 2005, Žitñan and Adams, 2012, Žitñan et al., 1999). Most of the neuropeptides, regulating these critical processes, act via GPCRs; indicating that these receptors may be potential targets for insect pest management.

Another major potential target for insecticide development is the insect's reproductive system. The act of reproducing comprises many different aspects and can be interfered with at multiple levels. The female reproductive system includes paired ovaries connected to a pair of lateral oviducts. The ovaries consist of ovarioles, in which the oocytes develop. The paired lateral oviducts join at the end and open into the gonopore. In addition, female insects possess a spermatheca, where sperm is stored until egg fertilisation. The accessory glands of the female can produce adhesive substances to attach the eggs to their substrate after oviposition. The male's reproductive tissues comprise two testes where spermatozoa are produced in follicles. The vas deferens with associated seminal vesicles ends in an ejaculatory duct. Accessory glands can secrete additional products needed to form a spermatophore and factors that are important for specific physiological activities within the reproductive system of the female. Oocyte and egg development occurs in the adult female and is controlled by multiple hormones. In the ovarioles, yolk accumulation occurs, a process often called vitellogenesis. However, this should not be confused with the vitellogenin synthesis itself, which mainly takes place in the fat body. In some lepidopteran, ephemeropteran and plecopteran species that do not feed as adults, yolk accumulation is completed already in the last larval stage or in the pupa. However, in most other insect species this process takes place in the adult stage. In many species, vitellogenin synthesis appears to be under the control of JH acting on the fat body. However, in D. melanogaster and the yellow fever mosquito Aedes aegypti, ecdysteroids synthesised by the ovaries are responsible for the induction of yolk protein synthesis in the fat body (Deitsch et al., 1995, Gilbert et al., 1998, Sun et al., 2002). After ovulation, the egg passes through the genital chamber, where it is fertilised by sperm that is released from the spermatheca as a response to mechanosensory signals.

Many insects have a short generation span and large reproductive capacity. It is obvious that failure to reproduce could greatly contribute to pest management and population control. In addition, most insects exhibit specific courtship behaviour involving approach, identification and copulation. Nevertheless, these reproductive behaviours can differ quite extensively amongst different insect species. For example, male fruit flies often have elaborate displays preceding copulation, while male houseflies and blowflies attempt to copulate with any object of the right size. Males of some butterflies and moths simply wait near the pupae and copulate with the female immediately after it emerges. In addition, pheromones play a very prominent role in insect reproduction as they are of crucial importance for mate localisation in many insect species. For instance, lepidopteran females release a species-specific plume of sex pheromones to attract males. The males then perceive these chemicals and thus can localise the female through its pheromone plume. Typical behaviours have been widely exploited in pest control techniques, such as pheromone traps and mating disruption (Reddy and Guerrero, 2010). The production of pheromones in lepidopteran females is controlled by a neuropeptide called pheromone biosynthesis-activating neuropeptide (PBAN). Interfering with this peptide signalling system could therefore be an interesting target allowing for the modulation of the behaviour of pest insects. Impairment of courtship and copulative behaviour could also greatly contribute to pest control.

Currently, novel control methods aim towards neuropeptidergic and hormonal systems as emerging targets. Apart from additional roles in insect physiology, such as feeding behaviour, growth and development, various neuropeptides and the classic insect hormones (e.g. 20E and JH) are known to influence reproduction and the associated behavioural aspects. For instance, the primary production sites of ecdysone during larval stages are the prothoracic glands. In the adult stage, however, these glands usually disappear and ecdysteroid production is taken over by the testes and ovaries of adult insects. In the adult, these hormones may elicit multiple actions, including oocyte maturation and play a paracrine role within developing ovaries. Depending on the species, JH and/or 20E are also known to induce the synthesis of vitellogenin in the fat body after their release into the haemolymph. In addition, ILPs are involved in the control of reproduction. Mutations in the Drosophila insulin receptor (InR) caused flaws in ecdysteroidogenesis and folliculogenesis, as well as JH deficiency, linking the ILP/InR pathway to the classical hormone pathways (as reviewed by Badisco et al., 2013, Simonet et al., 2004, Van Wielendaele et al., 2013a). Several neuropeptides also regulate insect reproductive physiology, for example by influencing JH production, ecdysteroidogenesis and folliculogenesis. Allatotropin (AT) and allatostatin (AST), respectively, can stimulate or inhibit JH biosynthesis by the CA. Interestingly, these neuropeptides also play important roles in other crucial physiological processes, such as feeding and digestion (Spit et al., 2012). Another neuropeptide known for its role in food uptake that appears to be involved in reproductive behaviour and physiology is neuropeptide F (NPF) (Schoofs et al., 2001, Van Wielendaele et al., 2013b, Van Wielendaele et al., 2013c, Van Wielendaele et al., 2013d). In mosquitoes, the ovary ecdysteroidogenic hormone seems to play a similar role as ILP in stimulating ovarian ecdysteroidogenesis. Ovary ecdysteroidogenic hormone is structurally related to locust neuroparsins, which have also been associated with reproductive physiology. However, neuroparsin, a small neuroprotein produced in the pars intercerebralis and secreted via the CC, was initially identified as an anti-gonadotropic factor that delays vitellogenesis in locusts (as reviewed by Badisco et al., 2007, Claeys et al., 2006, Van Wielendaele et al., 2013a).

The digestion of insects consists of a combination of physical, chemical and nutritional elements. When an insect starts to feed, the digestive system and its associated glands begin to store, grind and digest the food and facilitate the absorption of nutrients. Unwanted compounds will also be excreted and eliminated via the gut. The gut and the mouthparts of insects are innervated with both the central and the stomatogastric nervous system (Fig. 3.5) (Spit et al., 2012). In general, the stomatogastric nervous system in Arthropoda consists of a peripheral complex of ganglia and nerves which innervate the visceral organs (Ayali, 2009). This system is closely connected with the brain and the endocrine system. In order to start feeding, the insect first has to respond to the presence of food and has to begin to move towards the food (Griss et al., 1991). A part of the stomatogastric nervous system is the frontal ganglion, situated on the dorsal surface of the oesophagus. This frontal ganglion is linked with the tritocerebrum of the brain (the interface between the central and enteric nervous system) by paired frontal connectives (Robertson and Lange, 2010). A recurrent nerve stretches from the frontal ganglion to the hypocerebral ganglion, which is linked by two oesophageal nerves to a pair of ingluvial ganglia, located in the wall of the foregut (Hartenstein, 1997, Stern et al., 2007). The stomatogastric nervous system is known to regulate food uptake and transport in the visceral organs (Audsley and Weaver, 2009).

Food localisation and ingestion comprises different steps, such as attraction to food, arrest of movement, tasting, biting, ingestion and termination of feeding. The sensitivity to these factors depends on the physiological state of the insect. When an insect is starved, it becomes more sensitive to odours and tastes. On the other hand, female insects that carry eggs will not be interested in the uptake of food. Also, insects do not feed just before and after moulting. Some stages can survive without the uptake of food, such as embryos, pupae, diapausing insects and even some adult stages of ephemeropteran, lepidopteran and dipteran species (Gillott, 2005). These animals rely on the presence of stored, nutrient-rich molecules resulting from food uptake during the previous stages.

After contact with a food source, the insect will choose to start eating it or not. In the case of herbivorous insects, this choice is linked to host-plant selection. At this first contact, the insect can monitor the surface of the plant for chemicals or can detect the odour of the plant (Chapman, 1998, Greenwood and Chapman, 1984). This may lead to a continuation of the search for food or, if the food is accepted, to eating the plant. Substances in the sap of plants regulate the continuation or arrest of food uptake. These substances can have nutritional value for the insects or may be harmful. The presence of nutritional factors in the sap, such as for example sugars, will lead to stimulation of feeding because these are important substances for insects. Other substances in the sap can lead to inhibition of food uptake. The presence of alkoloids, terpenoids, glycosides and other secondary plant metabolites in plants will deter the insects (Gillott, 2005).

In insects, there is a wide range of peptides and biogenic amines which affect the control of food uptake and digestion and are thus of interest in possible control strategies of insect pests. A variety of different peptides will either stimulate or inhibit the muscle movements in the gut. Sulfakinins (SKs), tachykinins (TKs), ATs and proctolin are known to stimulate the muscles in the wall of the foregut of some insects. On the other hand, ASTs, myosuppressins and other myoinhibitory peptides induce inhibition of the contractions of these muscles and thus also food uptake and/or food progression within the gut. In addition, NPF, diuretic hormones, FMRF-amide-related peptides and several biogenic amines can also influence food uptake and digestion (as reviewed by Audsley and Weaver, 2009, Spit et al., 2012).