John Ewer

The genome of Tetranychus urticae reveals herbivorous pest adaptations

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant–herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

Identification of the Gene Encoding Bursicon, an Insect Neuropeptide Responsible for Cuticle Sclerotization and Wing Spreading

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon, but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon, we identified the Drosophila melanogaster gene CG13419 as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-beta (TGF-beta) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased bursicon bioactivity. In situ hybridization and immunocytochemistry revealed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lacked bursicon bioactivity. Our results indicate that CG13419 encodes bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to have a defined function in invertebrates. Mutants show that the spectrum of bursicon actions is broader than formerly demonstrated.

Increases in cyclic 3', 5'-guanosine monophosphate (cGMP) occur at ecdysis in an evolutionarily conserved crustacean cardioactive peptide-immunoreactive insect neuronal network.

At the end of each instar, insects shed their old cuticle by performing the stereotyped ecdysis behavior. In the moth, Manduca sexta, larval ecdysis is accompanied by increases in intracellular cyclic 3′,5′‐guanosine monophosphate (cGMP) in a small network of 50 peptidergic neurons within the ventral central nervous system (CNS). Studies on a variety of insects show that this cGMP response has been associated with ecdysis throughout most of insect evolution. In the mealbeetle (Tenebrio, Coleoptera) and the mosquito (Aedes, Diptera), all 50 neurons showed increases in cGMP immunoreactivity (‐IR) at ecdysis, and all were immunopositive for crustacean cardioactive peptide (CCAP). Other insects varied with respect to their cGMP response at ecdysis and their CCAP‐IR. In more primitive insects, such as the silverfish (Ctenolepisma, Zygentoma) and the grasshopper (Locusta, Orthoptera), an abdominal subset of these neurons did not show detectable cGMP‐IR at ecdysis, although the neurons were CCAP‐IR. Conversely, whereas CCAP‐IR was severely reduced in the thoracic and subesophageal neurons of Lepidoptera larvae and may be absent in a subset of the corresponding abdominal neurons in crickets (Gryllus, Orthoptera), the ecdysial cGMP response occurred in all 50 neurons. The most extreme case was found in cyclorrhaphous flies, in which most of the 50 neurons were CCAP‐IR, although none showed increases in cGMP at ecdysis. This situation in higher Diptera is discussed in terms of their highly modified ecdysis behaviors.

Requirement for Period Gene Expression in the Adult and Not During Development for Locomotor Activity Rhythms of Imaginal Drosophila Melanogaster

Mutations at the period (per) locus of Drosophila melanogaster disrupt the circadian rhythm of adult locomotor activity. Molecular studies have shown that this gene is expressed primarily at the embryonic, pupal and adult stages. We have used conditional per mutants to infer the stages of development during which per expression is required for adult rhythmicity. In experiments carried out with germline transformants in which the arrhythmic per01 allele has been transformed with a heat-shock protein 70 promoter-driven per gene (hsp-per transformants) we find that per expression in the adult is both necessary and sufficient for imaginal rhythms. Results obtained with existing per alleles and other per transformant strains that behave as conditional per mutants are consistent with those obtained with these molecularly engineered conditional mutants. Using hsp-per transformants we have found that the per gene product is apparently required only at the time of manifestation of rhythmicity, and can rescue the hosts arrhythmic phenotype even when supplied many days after transfer to constant darkness. We present evidence suggesting that it is necessary for pacemaker function itself, rather than being involved in a process that couples the activity of the pacemaker to the output pathway. The levels of per transcript and the abundance and tissue distribution of its protein product observed in hsp-per transformants exposed to different temperature regimes are described. An initial report of some of these results has been published previously (Ewer et al., 1988).

Neuroendocrine Control of Larval Ecdysis Behavior in Drosophila: Complex Regulation by Partially Redundant Neuropeptides

To complete each molting cycle, insects display a stereotyped sequence of behaviors to shed the remains of the old cuticle. These behavioral routines, as well as other related physiological events, are critical for proper development and are under the control of several neuropeptides. Their correct deployment and concatenation depends on the complex actions and interactions among several peptide hormones: ecdysis triggering hormone (ETH), eclosion hormone (EH), and crustacean cardioactive peptide (CCAP). Numerous theories, some in conflict, have been proposed to define the functional hierarchies by which these regulatory factors operate. Here we use wild-type Drosophila and transgenic flies bearing targeted ablations of either EH or CCAP neurons, or ablations of both together, to reevaluate their roles. Consistent with findings in moths, our results suggest that EH and ETH affect the release of each other via a positive feedback, although ETH can also be released in the absence of EH. We show that EH and ETH both contribute to the air filling of the air ducts (trachea) of the next stage but that EH may play a primary role in this process. We present evidence that EH, whose actions have always been placed upstream of CCAP, may also regulate ecdysis independently of CCAP. Finally, we confirm that flies lacking EH neurons do not ecdyse prematurely when injected with ETH peptides. These findings are surprising and not easily explained by currently available hypotheses. We propose that important additional neuropeptides, and additional interactions between known regulators, contribute to the mechanisms underlying insect ecdysis behaviors.

Neuropeptide Control of Molting in Insects

Publisher Summary This chapter focuses on the control by neuropeptides of behaviors used in preparation for ecdysis, during the shedding of the old cuticle, and during postecdysial events, such as wing inflation and cuticle tanning. Most recent advances have occurred in the area of the control of ecdysis itself. Recent findings have revealed that the timing of this behavior occurs via a positive-feedback endocrine system that produces an unambiguous hormonal signal that causes the all-or-nothing onset of ecdysis behavior. In addition, this endocrine system is such that it also ensures that ecdysis occurs at the end of the molt, when the old cuticle can be successfully shed. Insect growth and metamorphosis are punctuated by molts, during which the animal produces a new exoskeleton, or cuticle. The production of this new exoskeleton and the replacement of the old one are accomplished during the molt.

GENETIC AND HORMONAL REGULATION OF THE DEATH OF PEPTIDERGIC NEURONS IN THEDROSOPHILA CENTRAL NERVOUS SYSTEM

To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, we investigated the death of a set of peptidergic neurons in the CNS of the fruitfly Drosophila melanogaster. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). We provide genetic evidence that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, we demonstrate that these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. We show that the death of these neurons is dependent upon the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis, and demonstrate that the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis.

Behavioral actions of neuropeptides in invertebrates: insights from Drosophila.

This review discusses recent advances in our understanding of the hormonal control of ecdysis behavior in Drosophila, as well as methods that can more generally be used in this organism to investigate the in vivo function of neuropeptide hormones. Ecdysis is a dedicated, vital, behavior that is used by arthropods at the end of each molt to shed the remains of the old exoskeleton. It is under the control of several interacting neuropeptide hormones, and successful ecdysis requires that the behavior and accompanying peripheral events occur at a precise time and in the correct order. The tightly controlled timing and concatenation of these events are due to the complex hormonal control of ecdysis, with several neuropeptides contributing to a particular event, and, conversely, one neuropeptide effecting both central as well as peripheral actions. It is for the analyses of this type of behavior that Drosophila can provide unique insights, and some of these insights are summarized here. In addition, I discuss more generally approaches that are available in this organism, which make it especially useful for investigating the hormonal control of behavior.

The innervation of the pyloric region of the crab, Cancer borealis: homologous muscles in decapod species are differently innervated.

SummaryThe muscles of the pyloric region of the stomach of the crab,Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster,Panulirus interrruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons inP. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.