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Evolution of TRP channels inferred by their classification in diverse animal species. The functions of TRP channels have primarily been characterized in model organisms within a limited evolutionary context. We thus characterize the TRP channels in choanoflagellate, sponge, Cnidaria, Lophotrochozoa, and arthropods to understand how they emerged during early evolution of animals and have changed during diversification of various species. As previously reported, five metazoan TRP subfamily members (TRPA, TRPC, TRPM, TRPML, and TRPV) were identified in choanoflagellates, demonstrating that they evolved before the emergence of multicellular animals. TRPN was identified in Hydra magnipapillata, and therefore emerged in the last common ancestor of Cnidaria-Bilateria. A novel subfamily member (TRPVL) was identified in Cnidaria and Capitella teleta, indicating that it was present in the last common ancestor of Cnidaria-Bilateria but has since been lost in most bilaterians. The characterization of arthropod TRP channels revealed that Daphnia pulex and insects have specifically expanded the TRPA subfamily, which diverged from the ancient TRPA1 channel gene. The diversity of TRPA channels except TRPA1 was detectable even within a single insect family, namely the [START]ant[END] lineage. The present study demonstrates the evolutionary history of TRP channel genes, which may have diverged in conjunction with the specific habitats and life histories of individual species.

Opposite effect of capsaicin and capsazepine on behavioral thermoregulation in [START]insects[END]. Transient receptor potential channels are implicated in thermosensation both in mammals and [START]insects[END]. The aim of our study was to assess the effect of mammalian vanilloid receptor subtype 1 (TRPV1) agonist (capsaicin) and antagonist (capsazepine) on [START]insect[END] behavioral thermoregulation. We tested behavioral thermoregulation of mealworms larvae intoxicated with capsaicin and capsazepine in two concentrations (10(-7) and 10(-4) M) in a thermal gradient system for 3 days. Our results revealed that in low concentration, capsaicin induces seeking lower temperatures than the ones selected by the [START]insects[END] that were not intoxicated. After application of capsazepine in the same concentration, the mealworms prefer higher temperatures than the control group. The observed opposite effect of TRPV1 agonist and antagonist on [START]insect[END] behavioral thermoregulation, which is similar to the effect of these substances on thermoregulation in mammals, indicates indirectly that capsaicin may act on receptors in [START]insects[END] that are functionally similar to TRPV1.

Nociceptive Pathway in the Cockroach [START]Periplaneta americana[END]. Detecting and avoiding environmental threats such as those with a potential for injury is of crucial importance for an animal's survival. In this work, we examine the nociceptive pathway in an insect, the cockroach [START]Periplaneta americana[END], from detection of noxious stimuli to nocifensive behavior. We show that noxious stimuli applied to the cuticle of cockroaches evoke responses in sensory axons that are distinct from tactile sensory axons in the sensory afferent nerve. We also reveal differences in the evoked response of post-synaptic projection interneurons in the nerve cord to tactile versus noxious stimuli. Noxious stimuli are encoded in the cockroach nerve cord by fibers of diameter different from that of tactile and wind sensitive fibers with a slower conduction velocity of 2-3 m/s. Furthermore, recording from the neck-connectives show that the nociceptive information reaches the head ganglia. Removing the head ganglia results in a drastic decrease in the nocifensive response indicating that the head ganglia and the nerve cord are both involved in processing noxious stimuli.

Effect of Capsaicin and Other Thermo-TRP Agonists on Thermoregulatory Processes in the [START]American Cockroach[END]. Capsaicin is known to activate heat receptor TRPV1 and induce changes in thermoregulatory processes of mammals. However, the mechanism by which capsaicin induces thermoregulatory responses in invertebrates is unknown. Insect thermoreceptors belong to the TRP receptors family, and are known to be activated not only by temperature, but also by other stimuli. In the following study, we evaluated the effects of different ligands that have been shown to activate (allyl isothiocyanate) or inhibit (camphor) heat receptors, as well as, activate (camphor) or inhibit (menthol and thymol) cold receptors in insects. Moreover, we decided to determine the effect of agonist (capsaicin) and antagonist (capsazepine) of mammalian heat receptor on the [START]American cockroach[END]'s thermoregulatory processes. We observed that capsaicin induced the decrease of the head temperature of immobilized cockroaches. Moreover, the examined ligands induced preference for colder environments, when insects were allowed to choose the ambient temperature. Camphor exposure resulted in a preference for warm environments, but the changes in body temperature were not observed. The results suggest that capsaicin acts on the heat receptor in cockroaches and that TRP receptors are involved in cockroaches' thermosensation.

The roles of thermal transient receptor potential channels in thermotactic behavior and in thermal acclimation in the [START]red flour beetle[END], [START]Tribolium castaneum[END]. To survive in variable or fluctuating temperature, organisms should show appropriate behavioral and physiological responses which must be mediated through properly attuned thermal sensory mechanisms. Transient receptor potential channels (TRPs) are a family of cation channels a number of which, called thermo-TRPs, are known to function as thermosensors. We investigated the potential role of thermo-TPRs that have been previously identified in the fruit fly, Drosophila melanogaster, in thermotaxis and thermal acclimation in the [START]red flour beetle[END], [START]Tribolium castaneum[END]. Phylogenetic analysis of the trp genes showed generally one-to-one orthology between those in D. melanogaster and in [START]T. castaneum[END], although there are putative gene-losses in two TRP subfamilies of D. melanogaster. With RNA interference (RNAi) of [START]T. castaneum[END] thermo-TRP candidates painless, pyrexia and trpA1, we measured thermal avoidance behavior. RNAi of trpA1 resulted in reduced avoidance of high temperatures, 39 and 42 C. We also measured the effects of RNAi on heat-induced knockout and death under a short exposure to high temperature (1min at 52 C) either with or without a 10-min acclimation period at 42 C. Relatively short exposure to high temperature was enough to induce high temperature thermal acclimation. RNAi of trpA1 led to faster knockout at 52 C. RNAi of painless showed lower recovery rates from heat-induced knockout after thermal acclimation, and RNAi of pyrexia showed lower long-term survivorship without thermal acclimation. Therefore, we concluded that trpA1 is important in high temperature sensing and also in enhanced tolerance to high-temperature induced knockout; painless plays a role in rapid acclimation to high temperature; and pyrexia functions in protecting beetles from acute heat stress without acclimation.

The roles of thermal transient receptor potential channels in thermotactic behavior and in thermal acclimation in the red flour beetle, Tribolium castaneum. To survive in variable or fluctuating temperature, organisms should show appropriate behavioral and physiological responses which must be mediated through properly attuned thermal sensory mechanisms. Transient receptor potential channels (TRPs) are a family of cation channels a number of which, called thermo-TRPs, are known to function as thermosensors. We investigated the potential role of thermo-TPRs that have been previously identified in the [START]fruit fly[END], [START]Drosophila melanogaster[END], in thermotaxis and thermal acclimation in the red flour beetle, Tribolium castaneum. Phylogenetic analysis of the trp genes showed generally one-to-one orthology between those in [START]D. melanogaster[END] and in T. castaneum, although there are putative gene-losses in two TRP subfamilies of [START]D. melanogaster[END]. With RNA interference (RNAi) of T. castaneum thermo-TRP candidates painless, pyrexia and trpA1, we measured thermal avoidance behavior. RNAi of trpA1 resulted in reduced avoidance of high temperatures, 39 and 42 C. We also measured the effects of RNAi on heat-induced knockout and death under a short exposure to high temperature (1min at 52 C) either with or without a 10-min acclimation period at 42 C. Relatively short exposure to high temperature was enough to induce high temperature thermal acclimation. RNAi of trpA1 led to faster knockout at 52 C. RNAi of painless showed lower recovery rates from heat-induced knockout after thermal acclimation, and RNAi of pyrexia showed lower long-term survivorship without thermal acclimation. Therefore, we concluded that trpA1 is important in high temperature sensing and also in enhanced tolerance to high-temperature induced knockout; painless plays a role in rapid acclimation to high temperature; and pyrexia functions in protecting beetles from acute heat stress without acclimation.

Tiling of the body wall by multidendritic sensory neurons in [START]Manduca sexta[END]. A plexus of multidendritic sensory neurons, the dendritic arborization (da) neurons, innervates the epidermis of soft-bodied insects. Previous studies have indicated that the plexus may comprise distinct subtypes of da neurons, which utilize diverse cyclic 3',5'-guanosine monophosphate signaling pathways and could serve several functions. Here, we identify three distinct classes of da neurons in [START]Manduca[END], which we term the alpha, beta, and gamma classes. These three classes differ in their sensory responses, branch complexity, peripheral dendritic fields, and axonal projections. The two identified alpha neurons branch over defined regions of the body wall, which in some cases correspond to specific natural folds of the cuticle. These cells project to an intermediate region of the neuropil and appear to function as proprioceptors. Three beta neurons are characterized by long, sinuous dendritic branches and axons that terminate in the ventral neuropil. The function of this group of neurons is unknown. Four neurons belonging to the gamma class have the most complex peripheral dendrites. A representative gamma neuron responds to forceful touch of the cuticle. Although the dendrites of da neurons of different classes may overlap extensively, cells belonging to the same class show minimal dendritic overlap. As a result, the body wall is independently tiled by the beta and gamma da neurons and partially innervated by the alpha neurons. These properties of the da system likely allow insects to discriminate the quality and location of several types of stimuli acting on the cuticle.

Nociceptive neurons respond to multimodal stimuli in [START]Manduca sexta[END]. The caterpillar [START]Manduca sexta[END] produces a highly stereotyped strike behavior in response to noxious thermal or mechanical stimuli to the abdomen. This rapid movement is targeted to the site of the stimulus, but the identity of the nociceptive sensory neurons are currently unknown. It is also not known whether both mechanical and thermal stimuli are detected by the same neurons. Here, we show that the likelihood of a strike increases with the strength of the stimulus and that activity in nerves innervating the body wall increases rapidly in response to noxious stimuli. Mechanical and thermal stimuli to the dorsal body wall activate the same sensory unit, suggesting it represents a multimodal neuron. This is further supported by the effects of rapidly repeated thermal or mechanical stimuli, which cause a depression of neuronal responsiveness that is generalized across modalities. Mapping the receptive fields of neurons responding to strong thermal stimuli indicates that these multimodal, nociceptive units are produced by class gamma multidendritic neurons in the body wall.

Nociceptive neurons protect [START]Drosophila[END] larvae from parasitoid wasps. BACKGROUND: Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in [START]fruit fly[END] larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae "roll" with a motor pattern that is completely distinct from the style of locomotion that is used for foraging. RESULTS: We have precisely mapped the sensory neurons that are used by the [START]Drosophila[END] larvae to detect nociceptive stimuli. By using complementary optogenetic activation and targeted silencing of sensory neurons, we have demonstrated that a single class of neuron (class IV multidendritic neuron) is sufficient and necessary for triggering the unusual rolling behavior. In addition, we find that larvae have an innately encoded preference in the directionality of rolling. Surprisingly, the initial direction of rolling locomotion is toward the side of the body that has been stimulated. We propose that directional rolling might provide a selective advantage in escape from parasitoid wasps that are ubiquitously present in the natural environment of [START]Drosophila[END]. Consistent with this hypothesis, we have documented that larvae can escape the attack of Leptopilina boulardi parasitoid wasps by rolling, occasionally flipping the attacker onto its back. CONCLUSIONS: The class IV multidendritic neurons of [START]Drosophila[END] larvae are nociceptive. The nociception behavior of [START]Drosophila[END] melanagaster larvae includes an innately encoded directional preference. Nociception behavior is elicited by the ecologically relevant sensory stimulus of parasitoid wasp attack.

Thermal nociception in adult [START]Drosophila[END]: behavioral characterization and the role of the painless gene. Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult [START]Drosophila[END] avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in [START]Drosophila[END] larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult [START]Drosophila[END] as a useful model to study the genetic mechanisms of thermal nociception.

[START]Drosophila[END] NOMPC is a mechanotransduction channel subunit for gentle-touch sensation. Touch sensation is essential for behaviours ranging from environmental exploration to social interaction; however, the underlying mechanisms are largely unknown. In [START]Drosophila[END] larvae, two types of sensory neurons, class III and class IV dendritic arborization neurons, tile the body wall. The mechanotransduction channel PIEZO in class IV neurons is essential for sensing noxious mechanical stimuli but is not involved in gentle touch. On the basis of electrophysiological-recording, calcium-imaging and behavioural studies, here we report that class III dendritic arborization neurons are touch sensitive and contribute to gentle-touch sensation. We further identify NOMPC (No mechanoreceptor potential C), a member of the transient receptor potential (TRP) family of ion channels, as a mechanotransduction channel for gentle touch. NOMPC is highly expressed in class III neurons and is required for their mechanotransduction. Moreover, ectopic NOMPC expression confers touch sensitivity to the normally touch-insensitive class IV neurons. In addition to the critical role of NOMPC in eliciting gentle-touch-mediated behavioural responses, expression of this protein in the [START]Drosophila[END] S2 cell line also gives rise to mechanosensitive channels in which ion selectivity can be altered by NOMPC mutation, indicating that NOMPC is a pore-forming subunit of a mechanotransduction channel. Our study establishes NOMPC as a bona fide mechanotransduction channel that satisfies all four criteria proposed for a channel to qualify as a transducer of mechanical stimuli and mediates gentle-touch sensation. Our study also suggests that different mechanosensitive channels may be used to sense gentle touch versus noxious mechanical stimuli.

An assay for chemical nociception in [START]Drosophila[END] larvae. Chemically induced nociception has not yet been studied intensively in genetically tractable models. Hence, our goal was to establish a [START]Drosophila[END] assay that can be used to study the cellular and molecular/genetic bases of chemically induced nociception. [START]Drosophila[END] larvae exposed to increasing concentrations of hydrochloric acid (HCl) produced an increasingly intense aversive rolling response. HCl (0.5%) was subthreshold and provoked no response. All classes of peripheral multidendritic (md) sensory neurons (classes I-IV) are required for full responsiveness to acid, with class IV making the largest contribution. At the cellular level, classes IV, III and I showed increases in calcium following acid exposure. In the central nervous system, Basin-4 second-order neurons are the key regulators of chemically induced nociception, with a slight contribution from other types. Finally, chemical nociception can be sensitized by tissue damage. Subthreshold HCl provoked chemical allodynia in larvae 4 h after physical puncture wounding. Pinch wounding and UV irradiation, which do not compromise the cuticle, did not cause chemical allodynia. In sum, we developed a novel assay to study chemically induced nociception in [START]Drosophila[END] larvae. This assay, combined with the high genetic resolving power of [START]Drosophila[END], should improve our basic understanding of fundamental mechanisms of chemical nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

painless, a [START]Drosophila[END] gene essential for nociception. We describe a paradigm for nociception in [START]Drosophila[END]. In response to the touch of a probe heated above 38 degrees C, [START]Drosophila[END] larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. In a genetic screen for mutants defective in this noxious heat response, we identified the painless gene. Recordings from wild-type larval nerves identified neurons that initiated strong spiking above 38 degrees C, and this activity was absent in the painless mutant. The painless mRNA encodes a protein of the transient receptor potential ion channel family. Painless is required for both thermal and mechanical nociception, but not for sensing light touch. painless is expressed in peripheral neurons that extend multiple branched dendrites beneath the larval epidermis, similar to vertebrate pain receptors. An antibody to Painless binds to localized dendritic structures that we hypothesize are involved in nociceptive signaling.

Pickpocket is a DEG/ENaC protein required for mechanical nociception in [START]Drosophila[END] larvae. Highly branched class IV multidendritic sensory neurons of the [START]Drosophila[END] larva function as polymodal nociceptors that are necessary for behavioral responses to noxious heat (>39 degrees C) or noxious mechanical (>30 mN) stimuli. However, the molecular mechanisms that allow these cells to detect both heat and force are unknown. Here, we report that the pickpocket (ppk) gene, which encodes a Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunit, is required for mechanical nociception but not thermal nociception in these sensory cells. Larvae mutant for pickpocket show greatly reduced nociception behaviors in response to harsh mechanical stimuli. However, pickpocket mutants display normal behavioral responses to gentle touch. Tissue-specific knockdown of pickpocket in nociceptors phenocopies the mechanical nociception impairment without causing defects in thermal nociception behavior. Finally, optogenetically triggered nociception behavior is unaffected by pickpocket RNAi, which indicates that ppk is not generally required for the excitability of the nociceptors. Interestingly, DEG/ENaCs are known to play a critical role in detecting gentle touch stimuli in Caenorhabditis elegans and have also been implicated in some aspects of harsh touch sensation in mammals. Our results suggest that neurons that detect harsh touch in [START]Drosophila[END] utilize similar mechanosensory molecules.

Multidendritic sensory neurons in the adult [START]Drosophila[END] abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death. BACKGROUND: For the establishment of functional neural circuits that support a wide range of animal behaviors, initial circuits formed in early development have to be reorganized. One way to achieve this is local remodeling of the circuitry hardwiring. To genetically investigate the underlying mechanisms of this remodeling, one model system employs a major group of [START]Drosophila[END] multidendritic sensory neurons - the dendritic arborization (da) neurons - which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis. The 15 da neurons are identified in each larval abdominal hemisegment and are classified into four categories - classes I to IV - in order of increasing size of their receptive fields and/or arbor complexity at the mature larval stage. Our knowledge regarding the anatomy and developmental basis of adult da neurons is still fragmentary. RESULTS: We identified multidendritic neurons in the adult [START]Drosophila[END] abdomen, visualized the dendritic arbors of the individual neurons, and traced the origins of those cells back to the larval stage. There were six da neurons in abdominal hemisegment 3 or 4 (A3/4) of the pharate adult and the adult just after eclosion, five of which were persistent larval da neurons. We quantitatively analyzed dendritic arbors of three of the six adult neurons and examined expression in the pharate adult of key transcription factors that result in the larval class-selective dendritic morphologies. The 'baseline design' of A3/4 in the adult was further modified in a segment-dependent and age-dependent manner. One of our notable findings is that a larval class I neuron, ddaE, completed dendritic remodeling in A2 to A4 and then underwent caspase-dependent cell death within 1 week after eclosion, while homologous neurons in A5 and in more posterior segments degenerated at pupal stages. Another finding is that the dendritic arbor of a class IV neuron, v'ada, was immediately reshaped during post-eclosion growth. It exhibited prominent radial-to-lattice transformation in 1-day-old adults, and the resultant lattice-shaped arbor persisted throughout adult life. CONCLUSION: Our study provides the basis on which we can investigate the genetic programs controlling dendritic remodeling and programmed cell death of adult neurons, and the life-long maintenance of dendritic arbors.

The role of [START]Drosophila[END] Piezo in mechanical nociception. Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in [START]Drosophila melanogaster[END] and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in [START]D. melanogaster[END]; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in [START]D. melanogaster[END] (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.

Response of [START]Drosophila[END] to wasabi is mediated by painless, the fly homolog of mammalian TRPA1/ANKTM1. A number of repellent compounds produced by plants elicit a spicy or pungent sensation in mammals . In several cases, this has been found to occur through activation of ion channels in the transient receptor potential (TRP) family . We report that isothiocyanate (ITC), the pungent ingredient of wasabi, is a repellent to the insect [START]Drosophila melanogaster[END], and that the painless gene, previously known to be required for larval nociception, is required for this avoidance behavior. A painless reporter gene is expressed in gustatory receptor neurons of the labial palpus, tarsus, and wing anterior margin, but not in olfactory receptor neurons, suggesting a gustatory role. Indeed, painless expression overlaps with a variety of gustatory-receptor gene reporters. Some, such as Gr66a, are known to be expressed in neurons that mediate gustatory repulsion . painless mutants are not taste blind; they show normal aversive gustatory behavior with salt and quinine and attractive responses to sugars and capsaicin. The painless gene is an evolutionary homolog of the mammalian "wasabi receptor" TRPA1/ANKTM1 , also thought to be involved in nociception. Our results suggest that the stinging sensation of isothiocyanate is caused by activation of an evolutionarily conserved molecular pathway that is also used for nociception.

HsTRPA of the [START]Red Imported Fire Ant[END], [START]Solenopsis invicta[END], Functions as a Nocisensor and Uncovers the Evolutionary Plasticity of HsTRPA Channels. [START]Solenopsis invicta[END], the [START]red imported fire ant[END], represents one of the most devastating invasive species. To understand their sensory physiology, we identified and characterized their Hymenoptera-specific (Hs) TRPA channel, SiHsTRPA. Consistent with the sensory functions of SiHsTRPA, it is activated by heat, an electrophile, and an insect repellent. Nevertheless, SiHsTRPA does not respond to most of the honey bee ortholog (AmHsTRPA)-activating compounds. The jewel wasp ortholog (NvHsTRPA) is activated by these compounds even though it outgroups both AmHsTRPA and SiHsTRPA. Characterization of AmHsTRPA/SiHsTRPA chimeric channels revealed that the amino acids in the N terminus, as well as ankyrin repeat 2 (AR2) of AmHsTRPA, are essential for the response to camphor. Furthermore, amino acids in ARs 3 and 5-7 were specifically required for the response to diallyl disulfide. Thus, amino acid substitutions in the corresponding domains of SiHsTRPA during evolution would be responsible for the loss of chemical sensitivity. SiHsTRPA-activating compounds repel [START]red imported fire ants[END], suggesting that SiHsTRPA functions as a sensor for noxious compounds. SiHsTRPA represents an example of the species-specific modulation of orthologous TRPA channel properties by amino acid substitutions in multiple domains, and SiHsTRPA-activating compounds could be used to develop a method for controlling [START]red imported fire ants[END].

A TRPA1 channel that senses thermal stimulus and irritating chemicals in [START]Helicoverpa armigera[END]. Sensing and responding to changes in the external environment is important for insect survival. Transient receptor potential (TRP) channels are crucial for various sensory modalities including olfaction, vision, hearing, thermosensation and mechanosensation. Here, we identified and characterized a transient receptor potential gene named as HarmTRPA1 in [START]Helicoverpa armigera[END] antennae. HarmTRPA1 was abundantly expressed in the antennae and labial palps. Transcripts of HarmTRPA1 could also be detected in the head and proboscis. Furthermore, functional analyses of HarmTRPA1 were conducted in the Xenopus Oocyte system. The results showed that the HarmTRPA1 channel could be activated by increasing the temperature from 20 to 45 C. No significant adaptation was observed when the stimulus was repeated. In addition to thermal stimuli, pungent natural compounds including allyl isothiocyanate, cinnamaldehyde and citronellal also activated HarmTRPA1. Taken together, we infer that HarmTRPA1 may function as both a thermal sensor involved in peripheral temperature detection and as a chemical sensor detecting irritating chemicals in vivo. Our data provide valuable insight into the TRPA1 channel in this moth and lay the foundation for developing novel strategies for pest control.

Insights into the genomic evolution of insects from cricket genomes. Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket [START]Gryllus bimaculatus[END], and the annotation of the 1.60-Gb genome of the Hawaiian cricket Laupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

Insights into the genomic evolution of insects from cricket genomes. Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket Gryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricket [START]Laupala kohalensis[END]. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

A genome-wide [START]Drosophila[END] screen for heat nociception identifies alpha2delta3 as an evolutionarily conserved pain gene. Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in [START]Drosophila[END], we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the alpha2delta family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (alpha2delta3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, alpha2delta3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in alpha2delta3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in alpha2delta3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Evolutionary conservation and changes in insect TRP channels. BACKGROUND: TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and [START]fruit flies[END], and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in [START]Drosophila melanogaster[END], Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. RESULTS: All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and [START]D. melanogaster[END] contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in [START]Drosophila[END], is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. CONCLUSION: The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

Evolutionary conservation and changes in insect TRP channels. BACKGROUND: TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, [START]Bombyx mori[END], Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. RESULTS: All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in [START]B. mori[END], A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. CONCLUSION: The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

Evolutionary conservation and changes in insect TRP channels. BACKGROUND: TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, [START]Tribolium castaneum[END], Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. RESULTS: All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. [START]T. castaneum[END], A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. CONCLUSION: The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

Evolutionary conservation and changes in insect TRP channels. BACKGROUND: TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, [START]Apis mellifera[END], Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. RESULTS: All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, [START]A. mellifera[END], and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, [START]A. mellifera[END], and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in [START]A. mellifera[END] and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. CONCLUSION: The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

Evolutionary conservation and changes in insect TRP channels. BACKGROUND: TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, [START]Nasonia vitripennis[END], and Pediculus humanus genomes as part of genome sequencing efforts. RESULTS: All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and [START]N. vitripennis[END]. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and [START]N. vitripennis[END] contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and [START]N. vitripennis[END]; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. CONCLUSION: The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

[START]Honey bee[END] thermal/chemical sensor, AmHsTRPA, reveals neofunctionalization and loss of transient receptor potential channel genes. Insects are relatively small heterothermic animals, thus they are highly susceptible to changes in ambient temperature. However, a group of [START]honey bees[END] is able to maintain the brood nest temperature between 32 C and 36 C by either cooling or heating the nest. Nevertheless, how [START]honey bees[END] sense the ambient temperature is not known. We identified a [START]honey bee[END] Hymenoptera-specific transient receptor potential A (HsTRPA) channel (AmHsTRPA), which is activated by heat with an apparent threshold temperature of 34 C and insect antifeedants such as camphor in vitro. AmHsTRPA is expressed in the antennal flagellum, and ablation of the antennal flagella and injection of AmHsTRPA inhibitors impair warmth avoidance of [START]honey bees[END]. Gustatory responses of [START]honey bees[END] to sucrose are suppressed by noxious heat and insect antifeedants, but are relieved in the presence of AmHsTRPA inhibitors. These results suggest that AmHsTRPA may function as a thermal/chemical sensor in vivo. As shown previously, Hymenoptera has lost the ancient chemical sensor TRPA1; however, AmHsTRPA is able to complement the function of Drosophila melanogaster TRPA1. These results demonstrate that HsTRPA, originally arisen by the duplication of Water witch, has acquired thermal- and chemical-responsive properties, which has resulted in the loss of ancient TRPA1. Thus, this is an example of neofunctionalization of the duplicated ion channel gene followed by the loss of the functionally equivalent ancient gene.

Honey bee thermal/chemical sensor, AmHsTRPA, reveals neofunctionalization and loss of transient receptor potential channel genes. Insects are relatively small heterothermic animals, thus they are highly susceptible to changes in ambient temperature. However, a group of honey bees is able to maintain the brood nest temperature between 32 C and 36 C by either cooling or heating the nest. Nevertheless, how honey bees sense the ambient temperature is not known. We identified a honey bee Hymenoptera-specific transient receptor potential A (HsTRPA) channel (AmHsTRPA), which is activated by heat with an apparent threshold temperature of 34 C and insect antifeedants such as camphor in vitro. AmHsTRPA is expressed in the antennal flagellum, and ablation of the antennal flagella and injection of AmHsTRPA inhibitors impair warmth avoidance of honey bees. Gustatory responses of honey bees to sucrose are suppressed by noxious heat and insect antifeedants, but are relieved in the presence of AmHsTRPA inhibitors. These results suggest that AmHsTRPA may function as a thermal/chemical sensor in vivo. As shown previously, Hymenoptera has lost the ancient chemical sensor TRPA1; however, AmHsTRPA is able to complement the function of [START]Drosophila melanogaster[END] TRPA1. These results demonstrate that HsTRPA, originally arisen by the duplication of Water witch, has acquired thermal- and chemical-responsive properties, which has resulted in the loss of ancient TRPA1. Thus, this is an example of neofunctionalization of the duplicated ion channel gene followed by the loss of the functionally equivalent ancient gene.

The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in [START]Drosophila[END]. The basic mechanisms underlying noxious cold perception are not well understood. We developed [START]Drosophila[END] assays for noxious cold responses. Larvae respond to near-freezing temperatures via a mutually exclusive set of singular behaviors-in particular, a full-body contraction (CT). Class III (CIII) multidendritic sensory neurons are specifically activated by cold and optogenetic activation of these neurons elicits CT. Blocking synaptic transmission in CIII neurons inhibits CT. Genetically, the transient receptor potential (TRP) channels Trpm, NompC, and Polycystic kidney disease 2 (Pkd2) are expressed in CIII neurons, where each is required for CT. Misexpression of Pkd2 is sufficient to confer cold responsiveness. The optogenetic activation level of multimodal CIII neurons determines behavioral output, and visualization of neuronal activity supports this conclusion. Coactivation of cold- and heat-responsive sensory neurons suggests that the cold-evoked response circuitry is dominant. Our [START]Drosophila[END] model will enable a sophisticated molecular genetic dissection of cold nociceptive genes and circuits.

Development and organization of a nitric-oxide-sensitive peripheral neural plexus in larvae of the moth, [START]Manduca sexta[END]. Each hemisegment of the [START]Manduca sexta[END] larva is supplied with a subepidermal plexus of approximately 350 multidendritic neurons. An initial set of neurons, the primary plexus neurons, arise at 35-45% of embryogenesis. These neurons comprise 12-16 uniquely identifiable neurons per hemisegment that have homologues in other insect larvae. Each spreads processes across a characteristic portion of the body wall and has an axon that projects into the central nervous system. Secondary plexus neurons are born in two waves: the first between 70% and 80% of embryogenesis and the second during the molt to the second larval stage. The secondary plexus neurons are multidendritic, spread uniformly across the body wall, and appear to make contacts with the primary plexus neurons. Each secondary plexus cell arises as part of a five-cell cluster; the other cells produce a sensory bristle and socket along with the bristle sensory neuron and a glial cell. Application of nitric oxide (NO) donors induces plexus neurons to produce cyclic 3',5' guanosine monophosphate (cGMP), suggesting the presence of soluble guanylate cyclase. With few exceptions, plexus neurons become sensitive to NO stimulation approximately 10 hours after their birth and remain so throughout larval life. Cyclic GMP is detected primarily in the cytoplasm of plexus neurons and extends into the finest peripheral dendrites. Our results suggest that cGMP participates in the development and/or physiology of this peripheral neural plexus.

Altered heat nociception in cockroach [START]Periplaneta americana[END] L. exposed to capsaicin. Some natural alkaloids, e.g. capsaicin and camphor, are known to induce a desensitization state, causing insensitivity to pain or noxious temperatures in mammals by acting on TRP receptors. Our research, for the first time, demonstrated that a phenomenon of pharmacological blockade of heat sensitivity may operate in [START]American cockroach[END], [START]Periplaneta americana[END] (L.). We studied the escape reaction time from 50 C for [START]American[END] cockroaches exposed to multiple doses of different drugs affecting thermo-TRP. Capsaicin, capsazepine, and camphor induced significant changes in time spent at noxious ambient temperatures. Moreover, we showed that behavioral thermoregulation in normal temperature ranges (10-40 C) is altered in treated cockroaches, which displayed a preference for warmer regions compared to non-treated insects. We also measured the levels of malondialdehyde (MDA) and catalase activity to exclude the secondary effects of the drugs on these processes. Our results demonstrated that increase in time spent at 50 C (five versus one trial at a heat plate) induced oxidative stress, but only in control and vehicle-treated groups. In capsaicin, capsazepine, menthol, camphor and AITC-treated cockroaches the number of exposures to heat had no effect on the levels of MDA. Additionally, none of the tested compounds affected catalase activity. Our results demonstrate suppression of the heat sensitivity by repeated capsazepine, camphor and capsaicin administration in the [START]American cockroach[END].

Ripped pocket and pickpocket, novel [START]Drosophila[END] DEG/ENaC subunits expressed in early development and in mechanosensory neurons. [START]Drosophila melanogaster[END] has proven to be a good model for understanding the physiology of ion channels. We identified two novel [START]Drosophila[END] DEG/ ENaC proteins, Pickpocket (PPK) and Ripped Pocket (RPK). Both appear to be ion channel subunits. Expression of RPK generated multimeric Na+ channels that were dominantly activated by a mutation associated with neurodegeneration. Amiloride and gadolinium, which block mechanosensation in vivo, inhibited RPK channels. Although PPK did not form channels on its own, it associated with and reduced the current generated by a related human brain Na+ channel. RPK transcripts were abundant in early stage embryos, suggesting a role in development. In contrast, PPK was found in sensory dendrites of a subset of peripheral neurons in late stage embryos and early larvae. In insects, such multiple dendritic neurons play key roles in touch sensation and proprioception and their morphology resembles human mechanosensory free nerve endings. These results suggest that PPK may be a channel subunit involved in mechanosensation.

Enhanced locomotion caused by loss of the [START]Drosophila[END] DEG/ENaC protein Pickpocket1. Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.

[START]Drosophila[END] painless is a Ca2+-requiring channel activated by noxious heat. Thermal changes activate some members of the transient receptor potential (TRP) ion channel super family. They are primary sensors for detecting environmental temperatures. The [START]Drosophila[END] TRP channel Painless is believed responsible for avoidance of noxious heat because painless mutant flies display defects in heat sensing. However, no studies have proven its heat responsiveness. We show that Painless expressed in human embryonic kidney-derived 293 (HEK293) cells is a noxious heat-activated, Ca(2+)-permeable channel, and the function is mostly dependent on Ca(2+). In Ca(2+)-imaging, Painless mediated a robust intracellular Ca(2+) (Ca(2+)(i)) increase during heating, and it showed heat-evoked inward currents in whole-cell patch-clamp mode. Ca(2+) permeability was much higher than that of other cations. Heat-evoked currents were negligible in the absence of extracellular Ca(2+) (Ca(2+)(o)) and Ca(2+)(i), whereas 200 nm Ca(2+)(i) enabled heat activation of Painless. Activation kinetics were significantly accelerated in the presence of Ca(2+)(i). The temperature threshold for Painless activation was 42.6 degrees C in the presence of Ca(2+)(i), whereas the threshold was significantly increased to 44.1 degrees C when only Ca(2+)(o) was present. Temperature thresholds were further reduced after repetitive heating in a Ca(2+)-dependent manner. Ca(2+)-dependent heat activation of Painless was observed at the single-channel level in excised membranes. We found that a Ca(2+)-regulatory site is located in the N-terminal region of Painless. Painless-expressing HEK293 cells were insensitive to various thermosensitive TRP channel activators including allyl isothiocyanate, whereas mammalian TRPA1 inhibitors, ruthenium red, and camphor, reversibly blocked heat activation of Painless. Our results demonstrate that Painless is a direct sensor for noxious heat in [START]Drosophila[END].

Analysis of [START]Drosophila[END] TRPA1 reveals an ancient origin for human chemical nociception. Chemical nociception, the detection of tissue-damaging chemicals, is important for animal survival and causes human pain and inflammation, but its evolutionary origins are largely unknown. Reactive electrophiles are a class of noxious compounds humans find pungent and irritating, such as allyl isothiocyanate (in wasabi) and acrolein (in cigarette smoke). Diverse animals, from insects to humans, find reactive electrophiles aversive, but whether this reflects conservation of an ancient sensory modality has been unclear. Here we identify the molecular basis of reactive electrophile detection in flies. We demonstrate that [START]Drosophila[END] TRPA1 (Transient receptor potential A1), the [START]Drosophila melanogaster[END] orthologue of the human irritant sensor, acts in gustatory chemosensors to inhibit reactive electrophile ingestion. We show that fly and mosquito TRPA1 orthologues are molecular sensors of electrophiles, using a mechanism conserved with vertebrate TRPA1s. Phylogenetic analyses indicate that invertebrate and vertebrate TRPA1s share a common ancestor that possessed critical characteristics required for electrophile detection. These findings support emergence of TRPA1-based electrophile detection in a common bilaterian ancestor, with widespread conservation throughout vertebrate and invertebrate evolution. Such conservation contrasts with the evolutionary divergence of canonical olfactory and gustatory receptors and may relate to electrophile toxicity. We propose that human pain perception relies on an ancient chemical sensor conserved across approximately 500 million years of animal evolution.

Identification, Characterization and Expression Analysis of TRP Channel Genes in the Vegetable Pest, [START]Pieris rapae[END]. Transient receptor potential (TRP) channels are critical for insects to detect environmental stimuli and regulate homeostasis. Moreover, this superfamily has become potential molecular targets for insecticides or repellents. [START]Pieris rapae[END] is one of the most common and widely spread pests of Brassicaceae plants. Therefore, it is necessary to study TRP channels (TRPs) in [START]P. rapae[END]. In this study, we identified 14 TRPs in [START]P. rapae[END], including two Water witch (Wtrw) genes. By contrast, only one Wtrw gene exists in Drosophila and functions in hygrosensation. We also found splice isoforms of Pyrexia (Pyx), TRPgamma (TRPgamma) and TRP-Melastatin (TRPM). These three genes are related to temperature and gravity sensation, fine motor control, homeostasis regulation of Mg2+ and Zn2+ in Drosophila, respectively. Evolutionary analysis showed that the TRPs of [START]P. rapae[END] were well clustered into their own subfamilies. Real-time quantitative PCR (qPCR) showed that PrTRPs were widely distributed in the external sensory organs, including antennae, mouthparts, legs, wings and in the internal physiological organs, including brains, fat bodies, guts, Malpighian tubules, ovaries, as well as testis. Our study established a solid foundation for functional studies of TRP channels in [START]P. rapae[END], and would be benefit to developing new approaches to control [START]P. rapae[END] targeting these important ion channels.

Identification, Characterization and Expression Analysis of TRP Channel Genes in the Vegetable Pest, Pieris rapae. Transient receptor potential (TRP) channels are critical for insects to detect environmental stimuli and regulate homeostasis. Moreover, this superfamily has become potential molecular targets for insecticides or repellents. Pieris rapae is one of the most common and widely spread pests of Brassicaceae plants. Therefore, it is necessary to study TRP channels (TRPs) in P. rapae. In this study, we identified 14 TRPs in P. rapae, including two Water witch (Wtrw) genes. By contrast, only one Wtrw gene exists in [START]Drosophila[END] and functions in hygrosensation. We also found splice isoforms of Pyrexia (Pyx), TRPgamma (TRPgamma) and TRP-Melastatin (TRPM). These three genes are related to temperature and gravity sensation, fine motor control, homeostasis regulation of Mg2+ and Zn2+ in [START]Drosophila[END], respectively. Evolutionary analysis showed that the TRPs of P. rapae were well clustered into their own subfamilies. Real-time quantitative PCR (qPCR) showed that PrTRPs were widely distributed in the external sensory organs, including antennae, mouthparts, legs, wings and in the internal physiological organs, including brains, fat bodies, guts, Malpighian tubules, ovaries, as well as testis. Our study established a solid foundation for functional studies of TRP channels in P. rapae, and would be benefit to developing new approaches to control P. rapae targeting these important ion channels.

Heat Perception and Aversive Learning in [START]Honey Bees[END]: Putative Involvement of the Thermal/Chemical Sensor AmHsTRPA. The recent development of the olfactory conditioning of the sting extension response (SER) has provided new insights into the mechanisms of aversive learning in [START]honeybees[END]. Until now, very little information has been gained concerning US detection and perception. In the initial version of SER conditioning, [START]bees[END] learned to associate an odor CS with an electric shock US. Recently, we proposed a modified version of SER conditioning, in which thermal stimulation with a heated probe is used as US. This procedure has the advantage of allowing topical US applications virtually everywhere on the [START]honeybee[END] body. In this study, we made use of this possibility and mapped thermal responsiveness on the [START]honeybee[END] body, by measuring workers' SER after applying heat on 41 different structures. We then show that [START]bees[END] can learn the CS-US association even when the heat US is applied on body structures that are not prominent sensory organs, here the vertex (back of the head) and the ventral abdomen. Next, we used a neuropharmalogical approach to evaluate the potential role of a recently described Transient Receptor Potential (TRP) channel, HsTRPA, on peripheral heat detection by [START]bees[END]. First, we applied HsTRPA activators to assess if such activation is sufficient for triggering SER. Second, we injected HsTRPA inhibitors to ask whether interfering with this TRP channel affects SER triggered by heat. These experiments suggest that HsTRPA may be involved in heat detection by [START]bees[END], and represent a potential peripheral detection system in thermal SER conditioning.

Mating behaviour in a slave-making [START]ant[END], Rossomyrmex minuchae (Hymenoptera, [START]Formicidae[END]). The mating behaviour of the [START]ant[END] Rossomyrmex minuchae, a rare, protected slave-making species in Spain, seems to be significantly affected by its particular life history and patchy habitat. The mating behaviour of the entire genus Rossomyrmex is virtually unknown. We present here the results of a 3-year study of mating behaviour in R. minuchae.Behavioural observations and limited nest excavations revealed that R. minuchae does not produce sexuals every year, the number of sexuals is low, and the sex ratio tends to be female biased. Females typically exhibit two distinct activity periods. The first, the mating period, takes place in early afternoon: the ants "call" near the natal nest, mate and then return to their nest. The second, the dispersal period takes place in late afternoon: the mated females exit their nest and fly in search of a new, non-parasitized Proformica longiseta host nest. Males are highly active during the mating period, but will remain inactive in the dispersal period even if experimentally presented with virgin females. It appears that females are monogamous, while males are polygamous. When males are late arriving at the female calling site, the females will frequently congregate presumably calling in chorus. The low reproductive efficiency exhibited by R. minuchae, coupled with the postulated low genetic variation in the population, as sisters may mate with the same male, could result in a low survival rate and risk of eventual extinction. The observed decrease in nest density we observed during the 2004 season may be indicative of such a process.

Effect of particulate contamination on adhesive ability and repellence in two species of ant (Hymenoptera; [START]Formicidae[END]). Tarsal adhesive pads are crucial for the ability of insects to traverse their natural environment. Previous studies have demonstrated that for both hairy and smooth adhesive pads, significant reduction in adhesion can occur because of contamination of these pads by wax crystals present on plant surfaces or synthetic microspheres. In this paper, we focus on the smooth adhesive pads of ants and study systematically how particulate contamination and the subsequent loss of adhesion depends on particle size, particle surface energy, humidity and species size. To this end, workers of ant species Polyrhachis dives and Myrmica scabrinodis (Hymenoptera; [START]Formicidae[END]) were presented with loose synthetic powder barriers with a range of powder diameters (1-500 mum) and surface energies (PTFE or glass), which they would have to cross in order to escape the experimental arena. The barrier experiments were conducted for a range of humidities (10-70%). Experimental results and scanning electron microscopy confirm that particulate powders adversely affect the adhesive ability of both species of ant on smooth substrates via contamination of the arolia. Specifically, the loss of adhesion was found to depend strongly on particle diameter, but only weakly on particle type, with the greatest loss occurring for particle diameters smaller than the claw dimensions of each species, and no effect of humidity was found. We also observed that ants were repelled by the powder barriers which led to a decrease of adhesion prior to their eventual crossing, suggesting that insect antennae may play a role in probing the mechanical fragility of substrates before crossing them.

Effect of gland extracts on digging and residing preferences of red imported fire ant workers (Hymenoptera: [START]Formicidae[END]). There is evidence that ant-derived chemical stimuli are involved in regulating the digging behavior in Solenopsis invicta Buren. However, the source gland(s) and chemistry of such stimuli have never been revealed. In this study, extracts of mandibular, Dufour's, postpharyngeal, and poison glands were evaluated for their effect on ant digging and residing preferences of S. invicta workers from three colonies. In the intracolonial bioassays, workers showed significant digging preferences to mandibular gland extracts in 2 of 3 colonies and significant residing preferences in 1 of 3 colonies; significant digging preferences to Dufour's gland extracts in 1 of 3 colonies and significant residing preferences in 2 of 3 colonies. No digging and residing preferences were found for postpharyngeal and poison gland extracts. In intercolonial bioassays, significant digging and residing preferences were found for mandibular gland extracts in 3 of 6 colony combinations. Significant digging preferences to Dufour's gland extracts were found in 4 of 6 colony combinations and significant residing preferences in all 6 colony combinations. For postpharyngeal gland extracts, significant digging preferences were found only in 1 of 6 colonial combinations and no significant residing preferences were found. For poison gland extracts, no significant digging preferences were found; significant residing preferences were found in 1 of 6 colony combinations. However, a significant residing deterrence (negative residing preference index) was found for 2 of 6 colony combinations. Statistical analyses using data pooled from all colonies showed that mandibular and Dufour's gland extracts caused significant digging and residing preferences in both intracolonial and intercolonial bioassays but not postpharyngeal and poison gland extracts. By analyzing the data pooled from the same three colonies used for gland extract bioassays, it was found that, in no cases, workers showed significant digging and residing preferences to 2-ethyl-3,6-dimethylpyrazine, an alarm pheromone component from mandibular gland.

Effects of a juvenile hormone analogue pyriproxyfen on monogynous and polygynous colonies of the Pharaoh ant Monomorium pharaonis (Hymenoptera: [START]Formicidae[END]). To evaluate the effects of the juvenile hormone analogue pyriproxyfen on colonies of the Pharaoh ant Monomorium pharaonis (L.), peanut oil containing different concentrations (0.3, 0.6, or 0.9%) of pyriproxyfen was fed to monogynous (1 queen, 500 workers, and 0.1 g of brood) and polygynous (8 queens, 50 workers, and 0.1 g of brood) laboratory colonies of M. pharaonis. Due to its delayed activity, pyriproxyfen at all concentrations resulted in colony elimination. Significant reductions in brood volume were recorded at weeks 3 - 6, and complete brood mortality was observed at week 8 in all treated colonies. Brood mortality was attributed to the disruption of brood development and cessation of egg production by queens. All polygynous colonies exhibited significant reduction in the number of queens present at week 10 compared to week 1. Number of workers was significantly lower in all treated colonies compared to control colonies at week 8 due to old-age attrition of the workers without replacement. At least 98.67 +- 1.33% of workers were dead at week 10 in all treated colonies. Thus, treatment with slow acting pyriproxyfen at concentrations of 0.3 - 0.9% is an effective strategy for eliminating Pharaoh ant colonies.

Microsatellite genotyping of red imported fire ant (Hymenoptera: [START]Formicidae[END]) colonies reveals that most colonies persist in plowed pastures. Our study focused on colony dynamics of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: [START]Formicidae[END]), in relation to the standard practice of planting rye grass (i.e., plowing) in the fall in Louisiana. Microsatellite molecular markers were used to determine genotypes of individuals from red imported fire ant colonies. These markers allowed us to monitor treatment effect by detecting changes in number and location of colonies in response to disking of pasture plots. Previous research on mound disturbance as a form of cultural control in pastures has produced mixed results. We found that the majority of colonies persisted on plots after plowing. Mound density and mound area, 5 mo after plowing, were not significantly different among treatments. In contrast, April measurements of mound volume were significantly smaller on plowed plots compared with control plots. A closer look at the rebuilding of mounds on plowed plots, during the 5 mo, showed that mound heights stayed below pretreatment measurements and they were significantly smaller than those of undisturbed mounds. Whether plowing has potential for use as a cultural control technique in reducing the impact of red imported fire ant mounds on agricultural practices in pastures remains to be seen. Conceivably, the best application of this technique will be in combination with other control measures in an integrated pest management approach to control red imported fire ants in pastures.

Incomplete homogenization of chemical recognition labels between Formica sanguinea and Formica rufa ants (Hymenoptera: [START]Formicidae[END]) living in a mixed colony. Formica sanguinea Latreille (Hymenoptera: [START]Formicidae[END]) is a slave-making species, i.e., it raids colonies of host species and pillages pupae, which are taken to develop into adult workers in a parasite colony. However, it has been unclear if the coexistence of F. sanguinea with slave workers requires uniformity of cuticular hydrocarbons (CHCs), among which those other than n-alkanes are believed to be the principal nestmate recognition cues utilized by ants. In this study, a mixed colony (MC) of F. sanguinea and Formica rufa L. as a slave species was used to test the hypothesis that CHCs are exchanged between the species. Chemical analysis of hexane extracts from ants' body surfaces provided evidence for interspecific exchange of alkenes and methyl-branched alkanes. This result was confirmed by behavioral tests during which ants exhibited hostility toward conspecific individuals from the MC but not toward ones from homospecific colonies of their own species. However, it seems that species-specific differences in chemical recognition labels were not eliminated completely because ants from the MC were treated differently depending on whether they were con- or allospecific to the individuals whose behavioral reactions were tested. These findings are discussed in the context of mechanisms of colony's odor formation and effective integration of slaves into parasite colony.

Rediscovery of the rare ant genus Bannapone (Hymenoptera: [START]Formicidae[END]: Amblyoponinae) and description of the worker caste. The genus Bannapone was described in 2000 on the basis of a single dealate queen specimen. Since its original collection in Yunnan, China, no other specimen has been reported, making it one of the rarest ant genera in the world. Here we report the collection of two workers of Bannapone also from Yunnan province. The description of the worker caste is presented. Furthermore, we found significant differences with the described B. mulanae Xu, 2000 which leads us to describe the workers as a new species, B. scrobiceps n. sp.. Finally, we briefly discuss the importance of leaf-litter collection methods to collect taxa considered as "rare".

A descriptive morphology of the ant genus Procryptocerus (Hymenoptera: [START]Formicidae[END]). Morphology is the most direct approach biologists have to recognize uniqueness of insect species as compared to close relatives. Ants of the genus Procryptocerus possess important morphologic characters yet have not been explored for use in a taxonomic revision. The genus is characterized by the protrusion of the clypeus forming a broad nasus and antennal scrobes over the eyes. The toruli are located right posterior to the flanks of the nasus opposite to each other. The vertex is deflexed posteriorly in most species. An in-group comparison of the external morphology is presented focusing on the workers. A general morphology for gynes and males is also presented. Previously mentioned characters as well as new ones are presented, and their character states in different species are clarified. For the metasoma a new system of ant metasomal somite nomenclature is presented that is applicable to Aculeata in general. Finally, a Glossary of morphological terms is offered for the genus (available online). Most of the terminology can be used in other members of the [START]Formicidae[END] and Aculeata.

The ant genus Polyrhachis F. Smith in sub-Saharan Africa, with descriptions of ten new species. (Hymenoptera: [START]Formicidae[END]). Ten new sub-Saharan species of the ant genus Polyrhachis are described: P. gibbula n. sp. and P. omissa n. sp. belonging in the viscosa-group; P. brevipilosa n. sp., P. dubia n. sp., P. longiseta n. sp., P. luteipes n. sp., P. submarginata n. sp., and P. terminata n. sp. in the revoili-group; P. doudou n. sp. and P. fisheri n. sp. in the militaris-group. Also, P. epinotalis Santschi and P. kohli Forel are revived from synonymy with P. militaris (Fabr.) and P. volkarti Forel respectively. The type of P. cubaensis Mayr is described, changing the interpretation of the taxon and revalidating two of its synonyms, P. gerstaeckeri Forel and P. wilmsi Forel stat. n.. The examination of the type of Polyrhachis revoili Andre has proved this taxon to be different from previous interpretations; its position is reviewed and all of its synonyms are transferred to P. weissi Santschi. In addition, the first description of the worker of P. andrei Emery is provided, as well as some new records and taxonomic and morphological notes concerning other species. Finally, a synonymic list of the 61 currently known sub-Saharan species, a key to species-groups and an updated key to the workers are given.

Morphophysiological differences between the metapleural glands of fungus-growing and non-fungus-growing ants (Hymenoptera, [START]Formicidae[END]). The metapleural gland is an organ exclusive to ants. Its main role is to produce secretions that inhibit the proliferation of different types of pathogens. The aim of the present study was to examine the morphophysiological differences between the metapleural gland of 3 non-fungus-growing ants of the tribes Ectatommini, Myrmicini, and Blepharidattini and that of 5 fungus-growing ants from 2 basal and 3 derived attine genera. The metapleural gland of the non-fungus-growing ants and the basal attine ants has fewer secretory cells than that of the derived attine ants (leaf-cutting ants). In addition, the metapleural gland of the latter had more clusters of secretory cells and sieve plates, indicating a greater storage capacity and demand for secretion in these more advanced farming ants. The glands of the derived attine ants also produced higher levels of polysaccharides and acidic lipids than those of Myrmicini, Blepharidattini, and basal attines. Our results confirm morphophysiological differences between the metapleural glands of the derived attines and those of the basal attines and non-fungus-growing ants, suggesting that the metapleural glands of the derived attines (leaf-cutting ants) are more developed in morphology and physiology, with enhanced secretion production (acidic lipids and protein) to protect against the proliferation of unwanted fungi and bacteria in the fungal garden, it is possible that leaf-cutting ants may have evolved more developed metapleural glands in response to stronger pressure from parasites.

[Nymphal feeding of Leptophlebiidae ([START]Insecta[END]: Ephemeroptera) in the Cano Paso del Diablo, Venezuela]. The food habits of aquatic nymphs of 12 species of the family Leptophlebiidae were determined in a tropical intermittent stream in Venezuela. Three monthly samples of aquatic [START]insects[END] were taken from January to June in 1989. Two hundred and twenty-five stomach contents were analyzed. The results obtained were compared by the functional feeding groups classification. Species of the Thraulodes and Farrodes genera show significant differences in their diets.

Feces production as a form of social immunity in an [START]insect[END] with facultative maternal care. BACKGROUND: Social animals have the unique capability of mounting social defenses against pathogens. Over the last decades, social immunity has been extensively studied in species with obligatory and permanent forms of social life. However, its occurrence in less derived social systems and thus its role in the early evolution of group-living remains unclear. Here, we investigated whether lining nests with feces is a form of social immunity against microbial growth in the European earwig Forficula auricularia, an [START]insect[END] with temporary family life and facultative maternal care. RESULTS: Using a total of 415 inhibition zone assays, we showed that earwig feces inhibit the growth of two GRAM+ bacteria, two fungi, but not of a GRAM- bacteria. These inhibitions did not result from the consumed food or the nesting environment. We then demonstrated that the antimicrobial activity against fungus was higher in offspring than maternal feces, but that this difference was absent against bacteria. Finally, we showed that family interactions inhibited the antibacterial activity of maternal feces against one of the two GRAM+ bacteria, whereas it had no effect on the one of nymphal feces. By contrast, antifungal activities of the feces were independent of mother-offspring interactions. CONCLUSION: These results demonstrate that social immunity occurs in a species with simple and facultative social life, and thus shed light on the general importance of this process in the evolution of group-living. These results also emphasize that defecation can be under selection for other life-history traits than simple waste disposal.

The earliest known holometabolous [START]insects[END]. The Eumetabola (Endopterygota (also known as Holometabola) plus Paraneoptera) have the highest number of species of any clade, and greatly contribute to animal species biodiversity. The palaeoecological circumstances that favoured their emergence and success remain an intriguing question. Recent molecular phylogenetic analyses have suggested a wide range of dates for the initial appearance of the Holometabola, from the Middle Devonian epoch (391 million years (Myr) ago) to the Late Pennsylvanian epoch (311 Myr ago), and Hemiptera (310 Myr ago). Palaeoenvironments greatly changed over these periods, with global cooling and increasing complexity of green forests. The Pennsylvanian-period crown-eumetabolan fossil record remains notably incomplete, particularly as several fossils have been erroneously considered to be stem Holometabola (Supplementary Information); the earliest definitive beetles are from the start of the Permian period. The emergence of the hymenopterids, sister group to other Holometabola, is dated between 350 and 309 Myr ago, incongruent with their current earliest record (Middle Triassic epoch). Here we describe five fossils--a Gzhelian-age stem coleopterid, a holometabolous larva of uncertain ordinal affinity, a stem hymenopterid, and early Hemiptera and Psocodea, all from the Moscovian age--and reveal a notable penecontemporaneous breadth of early eumetabolan [START]insects[END]. These discoveries are more congruent with current hypotheses of clade divergence. Eumetabola experienced episodes of diversification during the Bashkirian-Moscovian and the Kasimovian-Gzhelian ages. This cladogenetic activity is perhaps related to notable episodes of drying resulting from glaciations, leading to the eventual demise in Euramerica of coal-swamp ecosystems, evidenced by floral turnover during this interval. These ancient species were of very small size, living in the shadow of Palaeozoic-era 'giant' [START]insects[END]. Although these discoveries reveal unexpected Pennsylvanian eumetabolan diversity, the lineage radiated more successfully only after the mass extinctions at the end of the Permian period, giving rise to the familiar crown groups of their respective clades.

Metabolic scaling in [START]insects[END] supports the predictions of the WBE model. The functional association between body size and metabolic rate (BS-MR) is one of the most intriguing issues in ecological physiology. An average scaling exponent of 3/4 is broadly observed across animal and plant taxa. The numerical value of 3/4 is theoretically predicted under the optimized version of West, Brown, and Enquist's vascular resource supply network model. [START]Insects[END], however, have recently been proposed to express a numerically different scaling exponent and thus application of the WBE network model to [START]insects[END] has been rejected. Here, we re-analyze whether such variation is indeed supported by a global deviation across all [START]insect[END] taxa at the order and family levels to assess if specific taxa influence [START]insect[END] metabolic scaling. We show that a previous reported deviation is largely due to the effect of a single [START]insect[END] family (Termitidae). We conclude that the BS-MR relationship in [START]insects[END] broadly supports the core predictions of the WBE model. We suggest that the deviation observed within the termites warrants further investigation and may be due to either difficulty in accurately measuring termite metabolism and/or particularities of their life history. Future work on allometric scaling should assess the nature of variation around the central tendencies in scaling exponents in order to test if this variation is consistent with core assumptions and predictions of the WBE model that stem by relaxing its secondary optimizing assumptions that lead to the 3/4 exponent.

Developmental basis for vein pattern variations in [START]insect[END] wings. The venation patterns characteristics of different [START]insect[END] orders and of families belonging to the same order possess enormous variation in vein number, position and differentiation. Although the developmental basis of changes in vein patterns during evolution is entirely unknown, the identification of the genes and developmental processes involved in Drosophila vein pattern formation facilitates the elaboration of construction rules. It is thus possible to identify the likely changes which may constitute a source of pattern variation during evolution. In this review, we discuss how actual patterns of venation could be accounted for by modifications in different Pterygota of a common set of developmental operations. We argue that the individual specification of each vein and the modular structure of the regulatory regions of the key genes identified in Drosophila offer candidate entry points for pattern modifications affecting individual veins or interveins independently. Assuming a general conservation of the processes involved in different species, the transitions between different patterns may require few changes in the regulatory gene networks involved.

Embryonic development of Galloisiana yuasai Asahina, with special reference to external morphology ([START]insecta[END]: Grylloblattodea). The embryogenesis of Grylloblattodea, one of the most primitive of the polyneopteran orders, is described using Galloisiana yuasai with special reference to external morphology. The egg membranes are characterized by an endochorion crossed by numerous vertical aeropyles and a fairly thin vitelline membrane, features shared by Mantophasmatodea. The inner layer formation is of the fault type. Serosal elements in the amnioserosal fold differentiate into hydropylar cells, to function in water absorption together with specialized amniotic structures, i.e., an amniotic strand and a thickened amnion. The germ band is of the short germ type. The germ band immerses deep into the yolk after its full elongation along the egg surface, and in this respect blastokinesis closely resembles that of Mantophasmatodea. The embryological features, i.e., those on egg membranes and blastokinesis, may suggest a closer affinity of Grylloblattodea and Mantophasmatodea. Appendages, ectodermal invaginations, and sternal and pleural sclerites are discussed in the light of serial homology, to provide a new basis for elucidating the [START]insect[END] body plan. Appendages are divided into the proximal coxopodite and distal telopodite, the former being divided further into the subcoxa and coxa. Subcoxal and coxal elements are identified in the mandible as well as in the abdominal appendages. The subcoxa is divided into the epimeron and episternum by the pleural suture in thoracic segments. Likewise, in the abdominal segments the subcoxa is divided into two, although the homologs of the epimeron and episternum are not sclerotized, and in the labial segment the subcoxal derivative or the postmentum is divided into the submentum and mentum. Two coxal endites bulge out from the medial side of the gnathal appendages. The mandibular molar and incisor, maxillary lacinia and galea, and labial glossa and paraglossa are serially homologous with each other. In the thoracic segments the original embryonic sternum or "protosternum" is largely replaced by subcoxal elements, and merely remains as a small anterior presternum and a posterior spinasternum. A major part of the venter is represented by the derivatives of the episternum such as an extensive basisternum, katepisternum, and trochantin and the medial element of the epimeron. The pleuron is derived from the episternal elements or the anepisternum and preepisternum, which bears a spiracle in the mesothorax and metathorax, and the lateral element of the epimeron. The homolog of the preepisternum in the prothorax is the cervical sclerite, but with no spiracle developed. A median ventral invagination arises in the thoracic segments as a spina, and the homolog of the spina develops into the eversible sac in the first abdominal segment.

Revision of Chinese Dilaridae ([START]Insecta[END]: Neuroptera) (Part I): species of the genus Dilar Rambur from northern China. The pleasing lacewing genus Dilar Rambur is a dominant group of the family Dilaridae in Asia and is diverse in China with 18 described species. Herein we record five species of Dilar. Three species, i.e. Dilar hastatus sp.nov., Dilar spectabilis sp.nov. and Dilar taibaishanus sp.nov. are described as new to science. Dilar sinicus Nakahara and Dilar septentrionalis Navas are also redescribed. A key to the species of Dilar from northern China is given.

Evolution of [START]insect[END] wings and development - new details from Palaeozoic nymphs. The nymphal stages of Palaeozoic [START]insects[END] differ significantly in morphology from those of their modern counterparts. Morphological details for some previously reported species have recently been called into question. Palaeozoic [START]insect[END] nymphs are important, however - their study could provide key insights into the evolution of wings, and complete metamorphosis. Here we review past work on these topics and juvenile [START]insects[END] in the fossil record, and then present both novel and previously described nymphs, documented using new imaging methods. Our results demonstrate that some Carboniferous nymphs - those of Palaeodictyopteroidea - possessed movable wing pads and appear to have been able to perform simple flapping flight. It remains unclear whether this feature is ancestral for Pterygota or an autapomorphy of Palaeodictyopteroidea. Further characters of nymphal development which were probably in the ground pattern of Pterygota can be reconstructed. Wing development was very gradual (archimetaboly). Wing pads did not protrude from the tergum postero-laterally as in most modern nymphs, but laterally, and had well-developed venation. The modern orientation of wing pads and the delay of wing development into later developmental stages (condensation) appears to have evolved several times independently within Pterygota: in Ephemeroptera, Odonatoptera, Eumetabola, and probably several times within Polyneoptera. Selective pressure appears to have favoured a more pronounced metamorphosis between the last nymphal and adult stage, ultimately reducing exploitation competition between the two. We caution, however, that the results presented herein remain preliminary, and the reconstructed evolutionary scenario contains gaps and uncertainties. Additional comparative data need to be collected. The present study is thus seen as a starting point for this enterprise.

Three new caddisflies species of the fossil genus Archaeotinodes ([START]Insecta[END]: Trichoptera: Ecnomidae) from the Baltic Amber. Three new caddisflies species of the fossil genus Archaeotinodes: Archaeotinodes petropolitana sp. nov., Archaeotinodes regiomontana sp. nov., and Archaeotinodes rossica sp. nov. from the Baltic amber (Upper Eocene, 40 million years old), are described and illustrated.

Magnetic resonance imaging in entomology: a critical review. Magnetic resonance imaging (MRI) enables in vivo imaging of organisms. The recent development of the magnetic resonance microscope (MRM) has enabled organisms within the size range of many [START]insects[END] to be imaged. Here, we introduce the principles of MRI and MRM and review their use in entomology. We show that MRM has been successfully applied in studies of parasitology, development, metabolism, biomagnetism and morphology, and the advantages and disadvantages relative to other imaging techniques are discussed. In addition, we illustrate the images that can be obtained using MRM. We conclude that although MRM has significant potential, further improvements to the technique are still desirable if it is to become a mainstream imaging technology in entomology.

Myofilaments promote wing expansion and maintain genitalia morphology in the [START]American cockroach[END], [START]Periplaneta americana[END]. Insects are the most widely distributed and successful animals on the planet. A large number of insects are capable of flight with functional wings. Wing expansion is an important process for insects to achieve functional wings after eclosion and healthy genital morphology is crucial for adult reproduction. Myofilaments are functional units that constitute sarcomeres and trigger muscle contraction. Here, we identified four myofilament proteins, including Myosin, Paramyosin, Tropomyosin and Troponin T, from the wing pads of nymphs in the [START]American cockroach[END], [START]Periplaneta americana[END]. RNAi-mediated knockdown of Myosin, Paramyosin, Tropomyosin and Troponin T in the early stage of final instar nymphs caused a severely curly wing phenotype in the imaginal molt, especially in the Paramyosin and Troponin T knockdown groups, indicating that these myofilament proteins are involved in controlling wing expansion behaviors during the nymph-adult transition. In addition, the knockdown resulted in abnormal external genitalia, caused ovulation failure and affected male accessory gland development. Interestingly, the expression of myofilament genes was induced by methoprene, a juvenile hormone (JH) analog, and decreased by the depletion of the JH receptor gene Met. Altogether, we have determined that myofilament genes play an important role in promoting wing expansion and maintaining adult genitalia morphology, and their expression is induced by juvenile hormone signaling. Our data reveals a novel mechanism by which wing expansion is regulated by myofilaments and the functions of myofilaments are involved in maintaining genitalia morphology. This article is protected by copyright. All rights reserved.

Serotonin-immunoreactive sensory neurons in the antenna of the cockroach [START]Periplaneta americana[END]. The antennae of insects contain a vast array of sensory neurons that process olfactory, gustatory, mechanosensory, hygrosensory, and thermosensory information. Except those with multimodal functions, most sensory neurons use acetylcholine as a neurotransmitter. Using immunohistochemistry combined with retrograde staining of antennal sensory neurons in the cockroach [START]Periplaneta americana[END], we found serotonin-immunoreactive sensory neurons in the antenna. These were selectively distributed in chaetic and scolopidial sensilla and in the scape, the pedicel, and first 15 segments of the flagellum. In a chaetic sensillum, A single serotonin-immunoreactive sensory neuron cohabited with up to four serotonin-negative sensory neurons. Based on their morphological features, serotonin-immunopositive and -negative sensory neurons might process mechanosensory and contact chemosensory modalities, respectively. Scolopidial sensilla constitute the chordotonal and Johnston's organs within the pedicel and process antennal vibrations. Immunoelectron microscopy clearly revealed that serotonin-immunoreactivities selectively localize to a specific type of mechanosensory neuron, called type 1 sensory neuron. In a chordotonal scolopidial sensillum, a serotonin-immunoreactive type 1 neuron always paired with a serotonin-negative type 1 neuron. Conversely, serotonin-immunopositive and -negative type 1 neurons were randomly distributed in Johnston's organ. In the deutocerebrum, serotonin-immunoreactive sensory neuron axons formed three different sensory tracts and those from distinct types of sensilla terminated in distinct brain regions. Our findings indicate that a biogenic amine, serotonin, may act as a neurotransmitter in peripheral mechanosensory neurons.

Selective actions of insecticides on desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach ([START]Periplaneta americana[END]) neurons. BACKGROUND: Insect desensitizing nicotinic acetylcholine (nAChD) receptors are desensitized by low concentrations of agonists, including neonicotinoid insecticides, but are essentially insensitive to spinosyns, while non-desensitizing nicotinic acetylcholine (nAChN) receptors are selectively activated by spinosyns and relatively insensitive to neonicotinoids. RESULTS: The single-electrode voltage-clamp technique was used to measure the actions of newer nicotinic insecticides dinotefuran, sulfoxaflor, triflumezopyrim, spinetoram and GS-omega/k-hexatoxin-Hv1a on cockroach neuronal nAChD and nAChN currents. Like imidacloprid and clothianidin, newer orthosteric nicotinic agonist insecticides dinotefuran and sulfoxaflor act by desensitizing nAChD receptors. The mesoionic insecticide triflumezopyrim selectively inhibited nAChD current with an half maximal inhibitory concentration (IC50 ) of 1.2 nmol L-1 , with no activation. Unlike other Group 4 insecticides, it did not activate nAChN current, but inhibited it with an IC50 of 3.8 mumol L-1 , indicating that the compound is a true antagonist. Spinosad and the spinosyn-derived insecticide spinetoram potently and selectively activated nAChN receptors. GS-omega/k-hexatoxin-Hv1a had no effect on nAChN currents and it had a complex action on nAChD currents, inhibiting at sub-nanomolar concentrations and causing some activation and enhancement of ACh-evoked currents at 30 nmol L-1 and above. Some cells express GS-omega/k-hexatoxin-Hv1a-resistant nAChD receptors. CONCLUSIONS: Nicotinic acetylcholine receptor competitive modulators (IRAC Group 4) and nicotinic acetylcholine receptor allosteric modulators, site II (hexatoxins, IRAC Group 32) are selective for nAChD receptors, while nicotinic acetylcholine receptor allosteric modulators, site I (spinosyns, IRAC Group 5) are selective for nAChN receptors. It is proposed that IRAC Groups 5 and 32 be re-named non-desensitizing nicotinic acetylcholine receptor allosteric modulators and desensitizing nicotinic acetylcholine receptor allosteric modulators, respectively. 2021 Society of Chemical Industry.

Characterization of a chemostable serine alkaline protease from [START]Periplaneta americana[END]. BACKGROUND: Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects' gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. RESULTS: In the present study, purification and properties of a serine alkaline protease from [START]Periplaneta americana[END] and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60 C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. CONCLUSION: Several important properties of a serine protease from [START]P. Americana[END] were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the [START]P. americana[END] protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource.

Periplanetols A-F, phenolic compounds from [START]Periplaneta americana[END] with potent COX-2 inhibitory activity. Six new compounds, periplanetols A - F (1-4, 6 and 7), a compound isolated from natural origin for the first time (5), and nine known ones (8-16) were isolated from the 70% ethanol extract of the whole bodies of [START]Periplaneta americana[END]. Their structures including absolute configurations were unambiguously identified by comprehensive spectroscopic analyses and computational methods. Biological evaluation toward COX-2 inhibition revealed that compounds 1, 2, and 10 could inhibit COX-2 activity with the IC50 values of 768.0 nM, 617.7 nM, and 599.5 nM respectively, indicating their potential in developping novel agents against inflammation related disorders.

The role of the [START]American cockroach[END] ([START]Periplaneta americana[END]) as transport host of Eimeria tenella to chickens. The role of the [START]American cockroach[END], [START]Periplaneta americana[END] as transport host for Eimeria tenella was evaluated. Twenty-four cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of the oocysts recovered from the digestive tract of infected cockroaches as well as from their feces was evaluated by orally inoculating them into Boris Brown chickens. E. tenella oocysts were found in the digestive tract and feces of infected cockroaches up to day 4 after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for 4 and 3 days after ingestion of oocysts, respectively. Presence of oocysts in the feces of chicken that had been inoculated with either digestive tract or feces of [START]P. americana[END] demonstrated the infectivity of E. tenella oocysts from digestive tract or feces, suggesting that [START]P. americana[END] may play a role in the transmission of E. tenella among chicken and between chicken flocks.

Polyphosphate polymers during early embryogenesis of [START]Periplaneta americana[END]. Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach [START]Periplaneta americana[END]. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca+2, Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca+2 levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca+2 and Pi homeostasis during early embryogenesis of [START]P. Americana[END].

Improved genome assembly provides new insights into the environmental adaptation of the [START]American cockroach[END], [START]Periplaneta americana[END]. The synanthropic pest and a model organism for entomological research, [START]American cockroach[END], [START]Periplaneta americana[END] (Linnaeus), can survive in unfavorable environments for humans. To investigate the genetic mechanisms of success in environmental adaptation of [START]P. americana[END], we de novo reassembled its whole genome based on next-generation sequencing and PacBio sequencing. The final genome reassembly consisted of approximately 3.34 Gb with scaffold N50 of 465.51 Kb. The completeness (95.4%) of the complete genome was evaluated with single-copy orthologous genes using BUSCO. We identified 18,618 protein-coding genes, 16,443 (88.32%) of which were well supported by public protein databases. We identified 482.04 Mb (approximately 14.45%) repeat elements, 1,385,093 perfect microsatellites simple sequence repeats in [START]P. americana[END] genome, which was higher than other four Blattaria insects. Comparative genomics analysis revealed obvious expansion in the gene families associated with chemoreception (olfactory receptors, gustatory receptors, ionotropic glutamate receptors, chemosensory protein, and sensory neuron membrane protein), which provided the necessary information for functional characterization of the chemosensory receptors of [START]P. americana[END], with potential for new or refined applications of semiochemicals-based control of this pest insect. Similarly, gene families (cytochrome P450s, carboxyl/choline esterases, and UDP-glycosyl-transferases) encoding receptors for bitter or toxic substances and detoxification enzymes were obviously expanded in [START]P. americana[END], enabling its ability to detect and detoxify many toxins. Enrichment analysis of positively selected genes in [START]P. americana[END] revealed items associated with metabolic process and catalytic activity, which possibly contributed to the pesticide resistance of [START]P. americana[END]. We also analyzed the homologs to antimicrobial peptide genes reported in the Drosophila genome, and identified two attacins and seven defensins in [START]P. americana[END]. Our data and findings will substantially facilitate molecular studies in [START]P. americana[END], including elucidation of detoxification mechanisms of xenobiotic, as well as development of new pest management strategies for the control of pests like [START]P. americana[END].

Assessment of dermal safety of oil extracted from [START]Periplaneta americana[END]: acute dermal toxicity, irritation, and sensitization. INTRODUCTION: The [START]American cockroach[END] ([START]Periplaneta americana[END]) is used in traditional Chinese medicine. [START]Periplaneta americana[END] ([START]P. americana[END]) is rich in oil that has shown potential antioxidant and antibacterial activities in vitro. OBJECTIVE: To evaluate the safety of oil extracted from [START]P. americana[END] by conducting acute dermal toxicity, irritation, and sensitization tests. MATERIALS AND METHODS: In an acute dermal toxicity study, adult Sprague-Dawley rats were exposed to [START]P. americana[END] oil (2000 mg/kg body weight) for 24 h. Clinical observations were conducted to evaluate the toxicity, behaviour, and health of the animals every day after dermal exposure for 14 days. For the dermal irritation test, the oil was applied to rabbits in single and multiple doses. Multi-dose treatment was administered once per day for 14 days. Each rabbit served as its own left- and right-side control and the rabbits' irritation reactions in local intact and damaged skin were recorded and scored. The skin sensitization study of guinea pigs with the oil was conducted for a period of 28 days. RESULTS: The acute dermal median lethal dose (LD50) of [START]P. americana[END] oil was > 2000 mg/kg body weight in adult rats. There was no significant difference in mean irritation scores between the negative control and oil groups. The oil caused very little or no irritation in the intact and damaged skin rabbits treated with either single or multiple doses and it was non-sensitizing to the skin of guinea pigs. CONCLUSIONS: These results suggest that [START]P. americana[END] oil does not produce any significant acute toxic effects and is safe for use in animal models with almost no dermal irritation or sensitization. Therefore, it presents a low risk of provoking skin reactions in humans.

Environmental decomposition of olefinic cuticular hydrocarbons of [START]Periplaneta americana[END] generates a volatile pheromone that guides social behaviour. Once emitted, semiochemicals are exposed to reactive environmental factors that may alter them, thus disrupting chemical communication. Some species, however, might have adapted to detect environmentally mediated breakdown products of their natural chemicals as semiochemicals. We demonstrate that air, water vapour and ultraviolet (UV) radiation break down unsaturated cuticular hydrocarbons (CHCs) of [START]Periplaneta americana[END] ([START]American cockroach[END]), resulting in the emission of volatile organic compounds (VOCs). In behavioural assays, nymphs strongly avoided aggregating in shelters exposed to the breakdown VOCs from cuticular alkenes. The three treatments (air, water vapour, UV) produced the same VOCs, but at different time-courses and ratios. Fourteen VOCs from UV-exposed CHCs elicited electrophysiological responses in nymph antennae; 10 were identified as 2-nonanone, 1-pentanol, 1-octanol, 1-nonanol, tetradecanal, acetic acid, propanoic acid, butanoic acid, pentanoic acid and hexanoic acid. When short-chain fatty acids were tested as a mix and a blend of the alcohols and aldehyde was tested as a second mix, nymphs exhibited no preference for control or treated shelters. However, nymphs avoided shelters that were exposed to VOCs from the complete 10-compound mix. Conditioned shelters (occupied by cockroaches with faeces and CHCs deposited on the shelters), which are normally highly attractive to nymphs, were also avoided after UV exposure, confirming that breakdown products from deposited metabolites, including CHCs, mediate this behaviour. Our results demonstrate that common environmental agents degrade CHCs into behaviourally active volatile compounds that potentially may serve as necromones or epideictic pheromones, mediating group dissolution.

A composite hydrogel loading natural polysaccharides derived from [START]Periplaneta americana[END] herbal residue for diabetic wound healing. In view of the long medicinal use history of [START]Periplaneta americana[END] for manifold ulcer or skin wounds treatment, the comprehensive utilization value of [START]P. americana[END] herbal residue was evaluated. In this study, we isolated a polysaccharide fraction from [START]P. americana[END] herbal residue with the potential wound healing effect, named as PAP faction, based on our previous study and provided the structural and monosaccharide composition characterization. To improve the topical wound dressing property, a novel composite hydrogel consisting of PAP, carbomer 940 (CBM), carboxymethyl cellulose (CMC) with different ratios were prepared and optimized. Mediated by the physical crosslinking effect among these polymers, the composite hydrogel exhibited good three-dimensional network structures, good swelling and water retention capacity, moderate mechanical property in rheological test. And then, the good cytocompatibility of hydrogel was corroborated by 3T3 fibroblast proliferation assay. Finally, the composite hydrogel loading PAP has been proved to accelerate wound healing in diabetic rat models, by promoting wound closure, collagen deposition, M2 macrophages polarization and angiogenesis. In summary, this study would provide an effective and promising wound dressing candidate for the prevention and treatment of diabetic wound, based on the ecological concept of the comprehensive utilization of natural herbal resources.

N-containing compounds from [START]Periplaneta americana[END] and their activities against wound healing. Three new compounds, periplanamides A (1) and B (2), periplanpyrazine A (3), a new naturally occurring compound salicyluric acid methyl ester (6), and seventeen known compounds were isolated from the medicinal insect [START]Periplaneta americana[END]. The structures of the new compounds were elucidated on the basis of spectroscopic methods. The absolute configurations of 2 were assigned by computational methods. Biological activities of these isolates except 1, 9, 11, and 13 toward nitric oxide (NO) production, cell proliferation in HDFs, cell migration and angiogenesis in HUVECs were evaluated.

Experimental infection of the cockroach [START]Periplaneta americana[END] with Toxocara canis and the establishment of patent infections in pups. The possible role of the cockroach [START]Periplaneta americana[END] in the transmission of Toxocara canis eggs and larvae via faeces and tissue migration was studied. Cockroaches fed with 3 x 105 and 5 x 105 embryonated eggs were found to harbour viable eggs and larvae from days 1 to 5 post-infection (DPI). At necropsy on 5 DPI, eggs and larvae were also recovered from the rectal contents but not from the tissues of cockroaches. In addition patent infections were established in pups fed on infected faeces of cockroaches, with eggs first appearing in the faeces of pups at 38 DPI. Adult worms of T. canis were also recovered at necropsy. Therefore the importance of cockroaches as good mechanical disseminators of ascarid eggs, especially T. canis, is discussed.

Actions of quinolizidine alkaloids on Periplaneta americana nicotinic acetylcholine receptors. BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [(3)H]imidacloprid (IMI) to nerve-cord membranes from [START]Periplaneta americana[END] L. Two alkaloids were also tested for their actions on nAChRs expressed by cockroach neurons using patch-clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [(3)H]IMI binding at 10 microM, although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration-inhibition curves was cytisine > sophocarpine >or= aloperine >or= matrine. Patch-clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in [START]P. americana[END] neurons, yet, at 10 microM, aloperine, but not sophocarpine, suppressed ACh-induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with [START]P. americana[END] nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose-response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands.

Efficacy of entomopathogenic hypocrealean fungi against [START]Periplaneta americana[END]. The [START]American cockroach[END] [START]Periplaneta americana[END], one of the worlds' most important urban insect pests was tested with entomopathogenic fungi. Most promising Metarhizium anisopliae, Metarhizium robertsii and Beauveria bassiana killed nymphs (>= 81.7% mortality, 25 days after treatment), and these fungi developed on all dead insects. Other fungi tested were less virulent (Metarhizium frigidum and Purpureocillium lilacinum) or avirulent (Isaria cateniobliqua, Isaria farinosa, Simplicillium lanosoniveum, Sporothrix insectorum and Tolypocladium cylindrosporum). Intrageneric and intraspecific variability of fungal activity was detected. Adults were highly susceptible, and oothecae proved to be more resistant than nymphs and adults to infection with M. anisopliae IP 46. Findings of the study underscore the potential of fungi as biocontrol agents against this pest.

Different neuroendocrine cell types in the pars intercerebralis of [START]Periplaneta americana[END] produce their own specific IGF-related peptides. Of the nine genes of the [START]American cockroach[END], [START]Periplaneta americana[END], coding for peptides related to insulin and insulin-like growth factor, seven show significant expression in the central nervous system as demonstrated by the polymerase chain reaction on reverse transcribed RNA. In situ hybridisation shows that five of those are expressed by cells in the pars intercerebralis. Antisera raised to the predicted peptides show that these cells are neuroendocrine in nature and project to the corpora cardiaca. Interestingly, there are at least three cell types that each express different genes. This contrasts with Drosophila where a single cell type expresses a number of genes expressing several such peptides. Whereas in Drosophila the neuroendocrine cells producing insulin-like peptides also express sulfakinins, the arthropod orthologs of gastrin and cholecystokinin, in [START]Periplaneta[END] the sulfakinins are produced by different cells. Other neuropeptides known to be produced by the pars intercerebralis in [START]Periplaneta[END] and other insect species, such as the CRF-like diuretic hormone, neuroparsin, leucokinin or myosuppressin, neither colocalize with an insulin-related peptide. The separate cellular localization of these peptides and the existence of multiple insulin receptors in this species implies a more complex regulation by insulin and IGF-related peptides in cockroaches than in the fruit fly.

Ovicidal and repellent activities of several plant essential oils against [START]Periplaneta americana[END] L. and enhanced activities from their combined formulation. Natural ovicidal and repellent agents against [START]Periplaneta americana[END] L. are urgently needed, and plant essential oils (EOs) can assume this role quite readily. In this study, ovicidal and repellent activities against [START]Periplaneta americana[END] of EOs from Cymbopogon citratus (Stapf.), Cinnamomum verum (J. Presl.), Eucalyptus globulus (Labill.), Illicium verum (Hook.f.), and Zanthoxylum limonella (Alston) in soybean oil and in ethyl alcohol were determined by topical and dual-choice assays, as well as 10% cypermethrin and a combined formulation of 5% C. verum EO + 5% I. verum EO. Cypermethrin at 10% provided the highest toxicity (100% inhibition rate) against the eggs, but only slightly higher than that (99.3%) provided by the combined EO formulation, while the highest repellent activity against the adults was provided by the combined formulation (89.5% repelled cockroaches at 48 h after treatment). In addition, all EO formulations in soybean oil provided higher ovicidal and repellent activities than those in ethyl alcohol. To conclude, the combined EO formulation in soybean oil can replace cypermethrin because their efficacy was nearly equivalent, but the combination should be much safer to use.

Function of the lipopolysaccharide-binding protein of [START]Periplaneta americana[END] as an opsonin. Previously, we reported the purification of an LPS-binding protein from the hemolymph of the [START]American cockroach[END] that was specific for E. coli LPS. In this study we found that this protein participated in the clearance of E. coli cells injected into the abdominal cavity of the cockroach, and that hemocytes ingested E. coli cells treated with this LPS-binding protein in vitro. These findings suggest that this LPS-binding protein acts as an opsonin.

Intraspecific Signals Inducing Aggregation in [START]Periplaneta americana[END] (Insecta: Dictyoptera). Chemical communication is necessary to induce aggregation and to maintain the cohesion of aggregates in [START]Periplaneta americana[END] (L.) cockroaches. We aimed to identify the chemical message inducing aggregation in this species. Two types of bioassays were used-binary choice tests in Petri dishes and tests in Y-olfactometer. Papers conditioned by direct contact of conspecifics induce aggregation when proposed in binary choice tests and were attractive in a Y-olfactometer. The identification of the molecules present on these conditioned papers indicated that dichloromethane extracts contained mainly cuticular hydrocarbons whereas methanol extracts contained more volatile molecules. Only a mixture of extracts in both solvents induced aggregation. High concentrations of cuticular hydrocarbons are necessary to induce aggregation when presented alone. When presented with volatile molecules present in methanol extracts, low concentrations of cuticular hydrocarbons are sufficient to induce aggregation if they are presented in contact. Among volatile molecules collected on filter paper, a mixture of three compounds-hexadecanoic acid, pentadecanoic acid, and pentaethylene glycol-induced aggregation. Our results provide evidence that aggregation processes in [START]P. americana[END] relies on a dual mechanism: attraction over long distances by three volatile molecules and maintenance on site by contact with cuticular hydrocarbons.

Comparative Transcriptomics Reveals the Expression Differences Between Four Developmental Stages of [START]American Cockroach[END] ([START]Periplaneta americana[END]). The globally distributed [START]American cockroach[END] ([START]Periplaneta americana[END]) is considered a pest, but it has been widely used in traditional Chinese medicine. In the past, the [START]American cockroach[END]'s genome and transcriptomes were sequenced, but the differential expression transcripts between developmental stages were unavailable. We performed the de novo assembly and analysis of [START]American cockroach[END] transcriptomes from four developmental stages. Approximately 200 million high-quality paired-end reads were generated by using Illumina Hiseq 2000 sequencer. The assembly produced 291,250 transcripts with an average length of 714 bp. In addition, 38,052 microsatellites and 11,060,020 transposable elements were identified. Based on sequence homology, 53,262 transcripts were annotated. After calculating the expression levels of all the transcripts, we found that 13 transcripts were highly expressed in all the samples and at least two, p10 and actin-related protein 1, played important roles during development. A total of 7954 differentially expressed transcripts (DETs) were identified. The adult had the largest number of DETs when compared to other samples (4818), while the 3rd and 8th larva had the least number of DETs (1332). We performed gene enrichment analysis with the DETs, and some interesting results were detected in the different groups. For example, chitin is the major component of the insect exoskeleton, and the chitin-related genes in larvae and new molted samples had higher expression levels than in adults. In addition, the enrichment analysis detected many chitin-related pathways. Our study performed the first large-scale comparative transcriptomics between the developmental stages of [START]American cockroach[END], which could provide useful gene expression data for future studies.

Characterization and diabetic wound healing benefits of protein-polysaccharide complexes isolated from an animal ethno-medicine [START]Periplaneta americana[END] L. [START]Periplaneta americana[END] L. (PA), a type of animal medicine, has been widely used for wound healing in clinical settings. In order to further investigate the bioactive wound healing substances in PA, crude PA protein-polysaccharide complexes were further purified by cellulose DE-52 and Sephadex G100 chromatography in succession. Among these isolated fractions, two fractions eluted by 0.3 M and 0.5 M NaCl with the higher yield, respectively named PaPPc2 and PaPPc3 respectively, were chosen for the wound healing experiments. Mediated by HPGPC, amino acid and monosaccharide composition analysis, circular dichroism spectrum, glycosylation type, FT-IR, and 1H NMR analysis, the characterization of PaPPc2 and PaPPc3 was implemented. And then, the benefits of PaPPcs to promote cell proliferation, migration, and tube formation of HUVECs were determined in vitro, indicated these fractions would facilitate angiogenesis. Finally, as proof of concept, PaPPc2 and PaPPc3 were employed to accelerate the acute wounds of diabetic mice, involving in increase blood vessels and the amounts of angiogenesis-related cytokines (alpha-SMA, VEGF, and CD31). In short, this study provides an experimental basis to demonstrate the protein-polysaccharide complexes of [START]Periplaneta americana[END] L. as its wound healing bioactive substances.

The molting gland of the cockroach [START]Periplaneta americana[END]: secretory activity and its regulation. 1. The prothoracic gland is the main source of ecdysteroids in larvae of the cockroach [START]Periplaneta americana[END]. 2. Besides ecdysone the molting gland of [START]Periplaneta[END] secretes 3-dehydroecdysone and proteins. 3. The molting gland of [START]Periplaneta[END] is regulated in different successive steps of cooperation of nervous and neuroendocrine activity. 4. Neurogenic effects on the molting gland via the prothoracic gland nerves are concentrated on the period of prepeak production of ecdysteroids. 5. Prior to the 17th day, the glands secretory activity is inhibited by GABA-ergic neuronal pathways from the subesophageal ganglion. 6. Neurogenic disinhibition by a peptidergic brain factor elicits the competence of the gland for prepeak activity, completed by the glandotropic effect of PTTH. 7. The 17th day of the larval stage is characterized as the head critical period, i.e., after this period the ecdysteroid secretion of the gland is independent of the prothoracicotropic hormone (PTTH) from the brain. 8. The main peak of ecdysteroid production is regulated by prothoracicotropic neuropeptids from the brain.

Experimental transmission of Toxocara canis from Blattella germanica and [START]Periplaneta americana[END] cockroaches to a paratenic host. The present study assessed the capacity of Blattella germanica and [START]Periplaneta americana[END] to disseminate and transmit infective phases of T. canis to rats, which were used as a model paratenic host. [START]P. americana[END] and B. germanica inoculated orally with T. canis larvated eggs shed eggs and larvae in their fecal matter during the first 6days post-inoculation. Larvae were recovered from the brain, lungs, kidneys and liver of rats that had been inoculated with either infected cockroaches or their feces. ELISAs of serum detected an increase of antibodies anti-T. canis excretion-secretion antigens, whereas Western Blot (WB) showed 4 bands (120, 50, 35 and 28kDa) that were similar to those found in positive control rats. Macroscopically, the liver and kidneys of infected rats had hemorrhagic areas with milk-spot-like lesions. The lungs showed diffuse grey protuberances. Histologically, hemorrhagic areas with leucocytic infiltrate were observed in the liver, lungs and kidneys. Some larvae were found within a granuloma that was surrounded by eosinophils and other leucocytic infiltrates. Larvae were found in the brain, but without inflammatory infiltrate. Both cockroach species that ingested larvated eggs of T. canis may shed viable larvae or eggs in their fecal matter. The induction of specific serum antibodies, presence of larvae in tissues and characteristic lesions associated with larval migration in the organs of rats that had ingested either whole adults or feces of B. germanica or [START]P. americana[END] demonstrate the capacity of these cockroaches to transmit toxocariosis to paratenic hosts.

Shock-induced Analgesia in the cockroach ([START]Periplaneta americana[END]). For 3 consecutive days cockroaches ([START]Periplaneta americana[END]) received escapable, inescapable, or no shock in an escape task. 24 hr. later minimum shock which initiated movement was identified. Reliably higher shocks were needed to initiate movement in the inescapably shocked roaches. In a second experiment the analgesia induced by inescapable shock was blocked by the opiate antagonist naloxone. The results are discussed in relation to the escape deficit and analgesia commonly seen following exposure to inescapable shock in a variety of species.

Antennal motor system of the cockroach, [START]Periplaneta americana[END]. The organization of the antennal muscles, nerves, and motor neurons has been investigated in the cockroach, [START]Periplaneta americana[END]. Antennal movements have been observed by video analysis, muscle actions have been determined by dissection and direct mechanical testing, and the motor neurons innervating each muscle have been defined with a recently developed selective backfill method. A model of the antennomotor system of [START]Periplaneta[END] has thus been established and compared with that of crickets. Five muscles located within the head capsule insert on the most proximal antennal segment, the scape. By their action, they allow the scape to move in essentially any direction within the dorsoventral and anteroposterior planes. An additional pair of muscles, one dorsal and one ventral, are found within the scape. They insert on the pedicel and move the pedicel in the dorsal-ventral plane. These seven muscles are controlled by at least 17 motor neurons with somata located in the deutocerebrum. By their action, these motor neurons enable cockroaches to move the long flagellum of each antenna through a wide range of positions in the frontal space, medio-laterally, and also allow depression toward the substrate and elevation well above the level of the head. The antennal motor neurons have been classified into five morphological types based on soma and axon location. Each morphological type has been correlated with a particular pattern of muscle innervation and control. The neurites of all motor neurons are located along the medial aspect of the dorsal lobe of the deutocerebrum.

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach, [START]Periplaneta americana[END]. Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar-binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, [START]Periplaneta americana[END] with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion-exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of [START]P. americana[END]. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from [START]P. americana[END]. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.

Presence of the [START]Periplaneta[END] lectin-related protein family in the [START]American cockroach[END] [START]Periplaneta americana[END]. We determined the partial amino acid sequences of [START]Periplaneta[END] lectin, which we had purified and characterized previously from the hemolymph of the [START]American cockroach[END] ([START]Periplaneta americana[END]) [Kubo T. and Natori S. (1987) Eur. J. Biochem. 168, 75-82]. Based on these sequences, we performed PCR and found that the cDNA library of the [START]Periplaneta[END] fat body contained many similar, but not identical, [START]Periplaneta[END] lectin-related cDNAs. Analysis of the cloned cDNAs suggested that [START]Periplaneta[END] has a protein family, of which the [START]periplaneta[END] lectin and LPS binding protein purified previously are members.

[Research status of [START]Periplaneta americana[END] with analyses and prospects of key issues]. [START]Periplaneta americana[END] is one of the common traditional Chinese medicines, which has a long application history. It can tonify spleen, promote blood circulation, induce diuresis to alleviate edema, and promote granulation. It is clinically used for the treatment of alimentary canal diseases, chronic heart failure, cutaneous lesion, periodontitis and other diseases. There are some representative prescriptions, such as Kangfu Xinye, Xinmailong injection, Ganlong capsule, and Xiaozheng Yigan tablet. This paper reviewed the chemical components, pharmacological effects and clinical applications of [START]P. americana[END], firstly summarized standards for the quality control of [START]P. americana[END], found out and analyzed the key problems in the research. The aim of this paper is to provide the references for the further development and application of [START]P. americana[END].

Learned helplessness in the cockroach ([START]Periplaneta americana[END]). For 3 consecutive days cockroaches ([START]Periplaneta americana[END]) were exposed to either escapable, inescapable, or no shock in an escape task. Twenty-four hours later they were tested in a shuttlebox escape task. There were reliable differences between escapable and inescapable animals and between inescapable and control animals in both escape latencies and the number of failures to escape.

Cockroaches ([START]Periplaneta americana[END] and Blattella germanica) as potential vectors of the pathogenic bacteria found in nosocomial infections. Although it has been difficult to prove the direct involvement of cockroaches (i.e. insects of the order Blattaria) in the transmission of pathogenic agents to humans, such insects often carry microorganisms that are important in nosocomial infections, and their medical importance in the spread of bacteria cannot be ruled out. In houses and institutions with poor standards of hygiene, heavy infestations with cockroaches, such as the peridomestic [START]American cockroach[END] (Periplaneta americana L.) and the domestic German cockroach (Blattella germanica L.), can occur. In the present study, cockroaches (126 B. germanica and 69 [START]P. americana[END]) were collected from four buildings (three public training hospitals and one house) in central Tehran, Iran. Each insect was processed, under sterile conditions, so that the bacteria on its external surfaces and in its alimentary tract and faecal pellets could be isolated and identified. The oldest and largest of the three hospitals sampled (a 1400-bed unit built 80 years ago) appeared to be the one most heavily infested with cockroaches, and cockroaches from this hospital accounted for most (65.4%) of the isolates of medically important bacteria made during the study. No significant difference was found between the percentages of [START]P. americana[END] and B. germanica carrying medically important bacteria (96.8% v. 93.6%; P>0.05). At least 25 different species of medically important bacteria were isolated and identified, and at least 22 were Gramnegative. The genus of enteric bacteria most frequently isolated from both cockroach species, at all four collection sites, was Klebsiella. The cockroaches from each hospital were much more likely to be found contaminated with medically important bacteria than those from the house. The hospital cockroaches were also more likely to be carrying medically important bacteria internally than externally (84.3% v. 64.1%; P<0.05). The implications of these and other recent results, for the control of cockroaches and nosocomial infections, are discussed.

Developmental O-glycan profile analysis shows pentasaccharide mucin-type O-glycans are linked with pupation of [START]Tribolium castaneum[END]. Eukaryotic cells can decorate their proteins with carbohydrate structures or glycans, significantly affecting the properties and activities of these proteins. Despite the importance of protein glycosylation in numerous biological processes, our knowledge of this modification in insects is far from complete. While N-glycosylation is the most studied, the study of O-glycans in insects is still very fragmentary and these studies are limited to a specific developmental stage or a specific tissue. In this article, matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) technology was used to analyze the O-glycan profile for the different developmental stages of egg, larva, pupa, and adult of the [START]red flour beetle[END] [START]Tribolium castaneum[END], an important insect model and pest worldwide. The results on the O-glycan profile showed that the mucin-type glycans dominate the O-glycome of the [START]red flour beetle[END]. Interestingly, some of the more complex mucin-type O-glycans, such as a tetra- (O-GalNAcGalGlcAGalNAc) and pentasaccharide O-glycan (O-GalNAc(GalGlcA)GalNAcGlcA), were highly abundant during the pupa stage, the intermediate stage between larval and adult stage in holometabolous insects, demonstrating that insect metamorphosis is accompanied with a change in the insect O-glycan profile. Together with the N-glycan profile, the current data are a foundation to better understand the role of protein glycosylation in the development of insects.

[START]Tribolium castaneum[END]: A Model for Investigating the Mode of Action of Insecticides and Mechanisms of Resistance. The [START]red flour beetle[END], [START]Tribolium castaneum[END], is a worldwide insect pest of stored products, particularly food grains, and a powerful model organism for developmental, physiological and applied entomological research on coleopteran species. Among coleopterans, [START]T. castaneum[END] has the most fully sequenced and annotated genome and consequently provides the most advanced genetic model of a coleopteran pest. The beetle is also easy to culture and has a short generation time. Research on this beetle is further assisted by the availability of expressed sequence tags and transcriptomic data. Most importantly, it exhibits a very robust response to systemic RNA interference (RNAi), and a database of RNAi phenotypes (iBeetle) is available. Finally, classical transposonbased techniques together with CRISPR/Cas-mediated gene knockout and genome editing allow the creation of transgenic lines. As [START]T. castaneum[END] develops resistance rapidly to many classes of insecticides including organophosphates, methyl carbamates, pyrethroids, neonicotinoids and insect growth regulators such as chitin synthesis inhibitors, it is further a suitable test system for studying resistance mechanisms. In this review, we will summarize recent advances in research focusing on the mode of action of insecticides and mechanisms of resistance identified using [START]T. castaneum[END] as a pest model.

Benzoquinone synthesis-related genes of [START]Tribolium castaneum[END] confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface. Insects fight against invading microbial pathogens through various immune-related measures that comprise 'internal', 'external' as well as 'social' immunities. The defenses by external immunity associated with the cuticular integument are supposed to be of particular importance in repelling entomopathogenic fungi that infect host insects transcutaneously. Among such integument-related defenses, external secretions of benzoquinone derivatives typical of tenebrionid beetles have been suggested to play important roles in the antimicrobial defenses. In the present study, by utilizing the experimental infection system composed of the [START]red flour beetle[END] [START]Tribolium castaneum[END] and generalist ascomycete entomopathogens Beauveria bassiana and Metarhizium anisopliae, we performed the functional assays of the three [START]T. castaneum[END] genes whose involvement in benzoquinone synthesis in the adults has been reported, namely GT39, GT62 and GT63. Observations by scanning electron microcopy (SEM) revealed that the conidia of the two fungal species did not germinate on the wild-type adult body surface but did on the pupae. The expression analyses demonstrated that the levels of GT39 and GT62 mRNA increased from middle pupae and reached high in early adults while GT63 did not show a clear adult-biased expression pattern. The RNA interference-based knockdown of any of the three genes in pupae resulted in the adults compromised to the infection of the both fungal species. SEM observations revealed that the gene silencing allowed the conidial germination on the body surface of the knockdown beetles, thereby impairing the robust antifungal defense of adult beetles. Thus, we have provided direct experimental evidence for the functional importance in vivo of these benzoquinone synthesis-related genes that support the antifungal defense of tenebrionid beetles.

Functional diversification of three delta-class glutathione S-transferases involved in development and detoxification in [START]Tribolium castaneum[END]. Glutathione S-transferases (GSTs) are members of a multifunctional enzyme superfamily. Forty-one GSTs have been identified in [START]Tribolium castaneum[END]; however, none of the 41 GSTs has been functionally characterized. Here, three delta-class GSTs, TcGSTd1, TcGSTd2 and TcGSTd3, of [START]T. castaneum[END] were successfully cloned and expressed in Escherichia coli. All of the studied GSTs catalysed the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene. Insecticide treatment showed that the expression levels of TcGSTd3 and TcGSTd2 were significantly increased after exposure to phoxim and lambda-cyhalothrin, whereas TcGSTd1 was slightly upregulated only in response to phoxim. A disc diffusion assay showed that overexpression of TcGSTD3, but not TcGSTD1 or TcGSTD2, in E. coli increased resistance to paraquat-induced oxidative stress. RNA interference knockdown of TcGSTd1 caused metamorphosis deficiencies and reduced fecundity by regulating insulin/target-of-rapamycin signalling pathway-mediated ecdysteroid biosynthesis, and knockdown of TcGSTd3 led to reduced fertility and a decreased hatch rate of the offspring, probably caused by the reduced antioxidative activity in the reproductive organs. These results indicate that TcGSTd3 and TcGSTd2 may play vital roles in cellular detoxification, whereas TcGSTd1 may play essential roles in normal development of [START]T. castaneum[END]. These delta-class GSTs in [START]T. castaneum[END] have obtained different functions during the evolution.

Beetling around the genome. The [START]red flour beetle[END], [START]Tribolium castaneum[END], has been selected for whole genome shotgun sequencing in the next year. In this minireview, we discuss some of the genetic and genomic tools and biological properties of [START]Tribolium[END] that have established its importance as an organism for agricultural and biomedical research as well as for studies of development and evolution. A [START]Tribolium[END] genomic database, Beetlebase, is being constructed to integrate genetic, genomic and biological data as it becomes available.

[START]Tribolium castaneum[END] gene expression changes after Paranosema whitei infection. BACKGROUND: Microsporidia are obligate parasites that possess some of the smallest eukaryotic genomes. Several insect species are susceptible to infections by microsporidian parasites. Paranosema whitei frequently infects young larvae of [START]Tribolium castaneum[END] and obligately kills the host whereupon transmission to subsequent hosts is accomplished via spores. P. whitei infection results in developmental arrest of [START]T. castaneum[END], preventing larvae from pupation. The mechanisms underlying P. whitei virulence as well as the molecular underpinning of host defenses remain uncharacterized. In the present study, we evaluated gene expression differences of [START]T. castaneum[END] infected with the microsporidian parasite P. whitei. RESULTS: More than 1500 [START]T. castaneum[END] genes were differentially expressed after infection with P. whitei. Several important host pathways appeared to be differentially expressed after infection, where immune genes were among the highest differential expressed genes. Genes involved in the Toll pathway and its effectors were specifically upregulated. Furthermore, iron homeostasis processes and transmembrane transport appeared significantly altered after P. whitei infection. Kruppel homolog 1 (Kr-h1) and other genes of the juvenile hormone (JH) pathway appeared differentially expressed after parasite infection. In addition, a small number of long intergenic non-coding RNAs (lincRNAs) appeared differentially expressed after P. whitei infection. CONCLUSION: In this study we characterized for the first time using RNA-seq the immune response of [START]T. castaneum[END] to P. whitei. Other pathways (transmembrane transport, iron homeostasis, protein synthesis, JH) indicate possible alterations of the host by the parasite such as a possible developmental arrest caused by JH regulation. Furthermore we find evidence that some lincRNAs might be connected to defense as previously reported for other insect species.

Functional analysis of sense organ specification in the [START]Tribolium castaneum[END] larva reveals divergent mechanisms in insects. Insects and other arthropods utilise external sensory structures for mechanosensory, olfactory, and gustatory reception. These sense organs have characteristic shapes related to their function, and in many cases are distributed in a fixed pattern so that they are identifiable individually. In Drosophila melanogaster, the identity of sense organs is regulated by specific combinations of transcription factors. In other arthropods, however, sense organ subtypes cannot be linked to the same code of gene expression. This raises the questions of how sense organ diversity has evolved and whether the principles underlying subtype identity in D. melanogaster are representative of other insects. Here, we provide evidence that such principles cannot be generalised, and suggest that sensory organ diversification followed the recruitment of sensory genes to distinct sensory organ specification mechanism. RESULTS: We analysed sense organ development in a nondipteran insect, the flour beetle [START]Tribolium castaneum[END], by gene expression and RNA interference studies. We show that in contrast to D. melanogaster, [START]T. castaneum[END] sense organs cannot be categorised based on the expression or their requirement for individual or combinations of conserved sense organ transcription factors such as cut and pox neuro, or members of the Achaete-Scute (Tc ASH, Tc asense), Atonal (Tc atonal, Tc cato, Tc amos), and neurogenin families (Tc tap). Rather, our observations support an evolutionary scenario whereby these sensory genes are required for the specification of sense organ precursors and the development and differentiation of sensory cell types in diverse external sensilla which do not fall into specific morphological and functional classes. CONCLUSIONS: Based on our findings and past research, we present an evolutionary scenario suggesting that sense organ subtype identity has evolved by recruitment of a flexible sensory gene network to the different sense organ specification processes. A dominant role of these genes in subtype identity has evolved as a secondary effect of the function of these genes in individual or subsets of sense organs, probably modulated by positional cues.

Non-lethal genotyping of [START]Tribolium castaneum[END] adults using genomic DNA extracted from wing tissue. The [START]red flour beetle[END] [START]Tribolium castaneum[END] has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of [START]T. castaneum[END] adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.

Development of a new method for collecting hemolymph and measuring phenoloxidase activity in [START]Tribolium castaneum[END]. OBJECTIVE: Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of [START]Tribolium castaneum[END] and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from [START]T. castaneum[END]. RESULTS: By using this method, we collected approximately 5 microl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.

Abundant expression of somatic transposon-derived piRNAs throughout [START]Tribolium castaneum[END] embryogenesis. BACKGROUND: Piwi-interacting RNAs (piRNAs) are a class of short (~26-31-nucleotide) non-protein-coding RNAs expressed in the metazoan germline. The piRNA pathway in arthropods is best understood in the ovary of Drosophila melanogaster, where it acts to silence active transposable elements (TEs). Maternal loading of piRNAs in oocytes is further required for the inheritance of piRNA-mediated transposon defence. However, our understanding of the diversity, evolution and function of the piRNA complement beyond drosophilids is limited. The [START]red flour beetle[END], [START]Tribolium castaneum[END], is an emerging model organism separated from Drosophila by ~ 350 million years of evolution that displays a number of features ancestral to arthropods, including short germ embryogenesis. Here, we characterize the maternally deposited and zygotically expressed small RNA and mRNA complements throughout [START]T. castaneum[END] embryogenesis. RESULTS: We find that beetle oocytes and embryos of all stages are abundant in heterogeneous ~ 28-nucleotide RNAs. These small RNAs originate from discrete genomic loci enriched in TE sequences and display the molecular signatures of transposon-derived piRNAs. In addition to the maternally loaded primary piRNAs, [START]Tribolium[END] embryos produce secondary piRNAs by the cleavage of zygotically activated TE transcripts via the ping-pong mechanism. The two [START]Tribolium[END] piRNA pathway effector proteins, Tc-Piwi/Aub and Tc-Ago3, are also expressed throughout the soma of early embryos. CONCLUSIONS: Our results show that the piRNA pathway in [START]Tribolium[END] is not restricted to the germline, but also operates in the embryo and may act to antagonize zygotically activated transposons. Taken together, these data highlight a functional divergence of the piRNA pathway between insects.

UVB Radiation Delays [START]Tribolium castaneum[END] Metamorphosis by Influencing Ecdysteroid Metabolism. Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on [START]Tribolium castaneum[END] metamorphosis, assessed the function of the [START]T. castaneum[END] prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of [START]T. castaneum[END] larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.