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Impact of Potential Probiotic Lactobacillus Strains on Host Growth and Development in a [START]Drosophila melanogaster[END] Model. Lactobacillus paracasei SD1, Lactobacillus rhamnosus SD11, and Lactobacillus gasseri SD12 were proposed as potential probiotics for oral health. However, the effects of them on host physiology are still unknown. This study aimed to select strains that can promote host growth and development in monocolonized [START]Drosophila[END] model compared with axenic and the commercial Lactobacillus rhamnosus GG-treated flies. The morphogenesis and growth of axenic [START]Drosophila melanogaster[END] were assessed from embryo to adult stage when provided with each probiotic strain-supplemented food. The colonization and persistence of probiotic in fly gut were also evaluated. The results indicated that axenic condition caused the lowest adult weight and emergence rate. All probiotic groups had higher weight than axenic group. Lact. rhamnosus SD11 group presented high adult emergence rate equivalent to Lact. rhamnosus GG group, which is significantly higher than the others. However, Lact. gasseri SD12 group had significantly lower adult weight and emergence rate when compared with Lact. rhamnosus GG group. The gut probiotics levels were rapidly increased within the first day after receiving probiotics. After probiotic cessation, their number in gut decreased and was maintained at low level, except for Lact. gasseri SD12, which completely vanished since day one. In conclusion, Lact. paracasei SD1, Lact. rhamnosus SD11, and Lact. rhamnosus GG can affect morphogenesis and weight of flies when fed since immature stage and have short period gut colonization. The findings of this study could possibly imply comparable health-promoting effects between Lact. paracasei SD1 and Lact. rhamnosus SD11 to commercial strain in [START]Drosophila[END] model.

When neurobiology and genetics meet: the study of visual system development in [START]Drosophila melanogaster[END]. The visual system of [START]Drosophila melanogaster[END] is an exceptionally well suited model to study the molecular and genetic mechanisms underlying inter- and intracellular signalling during nervous system development. This review highlights some of the recent advances in this field, that show not only the power of the combined neurobiological and genetical approaches in shedding new light into long standing questions regarding nervous system development but also uncover a striking evolutionary conservation, at the molecular level, of the basic developmental pathways and signal transduction cascades underlying metazoan ontogenesis.

Can [START]Drosophila melanogaster[END] represent a model system for the detection of reproductive adverse drug reactions? Once a molecule is identified as a potential drug, the detection of adverse drug reactions is one of the key components of its development and the FDA approval process. We propose using [START]Drosophila melanogaster[END] to screen for reproductive adverse drug reactions in the early stages of drug development. Compared with other non-mammalian models, [START]D. melanogaster[END] has many similarities to the mammalian reproductive system, including putative sex hormones and conserved proteins involved in genitourinary development. Furthermore, the [START]D. melanogaster[END] model would present significant advantages in time efficiency and cost-effectiveness compared with mammalian models. We present data on methotrexate (MTX) reproductive adverse events in multiple animal models, including [START]fruit flies[END], as proof-of-concept for the use of the [START]D. melanogaster[END] model.

Evidences for circadian rhythmicity in the per0 mutant of [START]Drosophila melanogaster[END]. per0 Mutants of [START]Drosophila melanogaster[END] which are exposed to light-dark cycles (LD) with different Zeitgeber period (T) have a limited range of entrainment. Entrained flies show a characteristic phase relationship of activity to the LD which depends on the period of the driving cycle as expected by oscillator theory. Both facts are taken as evidence that per. possesses endogenous oscillators and that the per gene product is not concerned with central clock structures but rather might be responsible for the mutual coupling between the individual oscillators in a multioscillatory system controlling locomotor activity.

Infection of [START]Drosophila melanogaster[END] by Tubulinosema kingi: stage-specific susceptibility and within-host proliferation. Despite its importance as a model organism very little is known about the interaction between [START]Drosophila[END] and its microsporidian pathogens. Here we report on the relative susceptibility of [START]Drosophila melanogaster[END] life history stages to infection by Tubulinosema kingi, and on patterns of pathogen proliferation. We find that only larvae can be infected, and that this susceptibility decreases with larval age. Following infection, the pathogen shows little subsequent proliferation in larvae, a limited amount in pupae while it replicates greatly in adults. We present evidence that the host launches a cellular immune response after infection with the pathogen, although its effectiveness remains to be demonstrated.

Symbiotic bacteria are responsible for diet-induced mating preference in [START]Drosophila melanogaster[END], providing support for the hologenome concept of evolution. Diet-induced mating preference in [START]Drosophila melanogaster[END] results from amplification of the commensal bacterium Lactobacillus plantarum, providing a new role for gut microbiota and support for the hologenome concept of evolution. When the flies were treated with antibiotics prior to changing their diet, mating preference did not occur. These data also indicate that other potentially beneficial bacteria could be irreversibly lost by antibiotic treatment and that their replacement could provide a health benefit. We suggest that [START]D. melanogaster[END] can be a useful model organism to study the activities of gut microbiota and their interaction with the immune system.

Isolation and maintenance-free culture of contractile myotubes from [START]Manduca sexta[END] embryos. Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS) and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the [START]tobacco hornworm[END], [START]Manduca sexta[END], due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile [START]M. sexta[END] muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and [START]M. sexta[END] myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, [START]M. sexta[END] muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured [START]M. sexta[END] muscle to continuously function at ambient conditions without medium replenishment, combined with the interesting metabolic properties, suggests that this cell source is a promising candidate for further investigation toward bioactuator applications.

Developmental expression of [START]Manduca sexta[END] hemolin. Hemolin is hemolymph protein that is a member of the immunoglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the [START]Manduca sexta[END] hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wandering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the presence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and persisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same sequence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any detectable carbohydrate, but hemolin from the hemolymph of bacteria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and composition. These results suggest that a single hemolin gene is developmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch.

Localization of the Elastic Proteins in the Flight Muscle of [START]Manduca sexta[END]. The flight muscle of [START]Manduca sexta[END] (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of [START]Manduca[END] is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 microm and thick filament lengths were measured as ~2.5 microm. This structural information may help provide an interpretive framework for future studies using this muscle system.

Biomimicry of the Hawk Moth, [START]Manduca sexta[END] (L.), Produces an Improved Flapping-Wing Mechanism. Flapping-wing micro air vehicles (FWMAVs) that mimic the flight capabilities of insects have been sought for decades. Core to the vehicle's flight capabilities is the mechanism that drives the wings to produce thrust and lift. This article describes a newly designed flapping-wing mechanism (FWM) inspired by the North American hawk moth, [START]Manduca sexta[END]. Moreover, the hardware, software, and experimental testing methods developed to measure the efficiency of insect-scale flapping-wing systems (i.e., the lift produced per unit of input power) are detailed. The new FWM weighs 1.2 grams without an actuator and wings attached, and its maximum dimensions are 21 x 24 x 11 mm. This FWM requires 402 mW of power to operate, amounting to a 48% power reduction when compared to a previous version. In addition, it generates 1.3 gram-force of lift at a flapping frequency of 21.6 Hz. Results show progress, but they have not yet met the power efficiency of the naturally occurring [START]Manduca sexta[END]. Plans to improve the technique for measuring efficiency are discussed as well as strategies to more closely mimic the efficiency of the [START]Manduca sexta[END]-inspired FWM.

Enterococcus faecalis 6-phosphogluconolactonase is required for both commensal and pathogenic interactions with [START]Manduca sexta[END]. Enterococcus faecalis is a commensal and pathogen of humans and insects. In [START]Manduca sexta[END], E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of [START]M. sexta[END] using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in [START]M. sexta[END] and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with [START]M. sexta[END]. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections.

Gene structure and expression profile of [START]Manduca sexta[END] prophenoloxidase-activating proteinase-3 (PAP-3), an immune protein containing two clip domains. Prophenoloxidase-activating proteinase-3 (PAP-3) is a component of the defence system in [START]Manduca sexta[END]. We have isolated genomic clones and elucidated the organization of this gene. The 3' end of exon 2, the entire exon 3 and the 5' end of exon 4 encode the two amino-terminal clip domains. Southern blot analysis suggested a single copy of the PAP-3 gene in the genome. We identified several putative immune-responsive elements in the upstream region. The PAP-3 gene is not highly expressed in the fat body during larval development until the wandering stage begins. The mRNA level is high in the epithelium, fat body and haemocytes. Tissue-specific alternative splicing occurs in the fat body and trachea. A bacterial injection markedly induced the gene expression in the fat body and haemocytes.

Serine Protease Networks Mediate Immune Responses in Extra-Embryonic Tissues of Eggs in the [START]Tobacco Hornworm[END], [START]Manduca sexta[END]. The melanization and Toll pathways, regulated by a network of serine proteases and noncatalytic serine protease homologs (SPHs), have been investigated mostly in adult and larval insects. However, how these innate immune reactions are regulated in insect eggs remains unclear. Here we present evidence from transcriptome and proteome analyses that extra-embryonic tissues (yolk and serosa) of early-stage [START]Manduca sexta[END] eggs are immune competent, with expression of immune effector genes including prophenoloxidase and antimicrobial peptides. We identified gene products of the melanization and Toll pathways in [START]M. sexta[END] eggs. Through in vitro reconstitution experiments, we demonstrated that constitutive and infection-induced serine protease cascade modules that stimulate immune responses exist in the extra-embryonic tissues of [START]M. sexta[END] eggs. The constitutive module (HP14b-SP144-GP6) may promote rapid early immune signaling by forming a cascade activating the cytokine Spatzle and regulating melanization by activating prophenoloxidase (proPO). The inducible module (HP14a-HP21-HP5) may trigger enhanced activation of Spatzle and proPO at a later phase of infection. Crosstalk between the two modules may occur in transition from the constitutive to the induced response in eggs inoculated with bacteria. Examination of data from two other well-studied insect species, Tribolium castaneum and Drosophila melanogaster, supports a role for a serosa-dependent constitutive protease cascade in protecting early embryos against invading pathogens.

Hemolymph protease-5 links the melanization and Toll immune pathways in the [START]tobacco hornworm[END], [START]Manduca sexta[END]. Proteolytic activation of phenoloxidase (PO) and the cytokine Spatzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spatzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in [START]Manduca sexta[END] Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpatzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by [START]Manduca sexta[END] serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

Competing beetles attract egg laying in a hawkmoth. In nature, plant-insect interactions occur in complex settings involving multiple trophic levels, often with multiple species at each level.1 Herbivore attack of a host plant typically dramatically alters the plant's odor emission in terms of concentration and composition.2,3 Therefore, a well-adapted herbivore should be able to predict whether a plant is still suitable as a host by judging these changes in the emitted bouquet. Although studies have demonstrated that oviposition preferences of successive insects were affected by previous infestations,4,5 the underlying molecular and olfactory mechanisms remain unknown. Here, we report that [START]tobacco hawkmoths[END] ([START]Manduca sexta[END]) preferentially oviposit on Jimson weed (Datura wrightii) that is already infested by a specialist, the three-lined potato beetle (Lema daturaphila). Interestingly, the moths' offspring do not benefit directly, as larvae develop more slowly when feeding together with Lema beetles. However, one of [START]M. sexta[END]'s main enemies, the parasitoid wasp Cotesia congregata, prefers the headspace of [START]M. sexta[END]-infested plants to that of plants infested by both herbivores. Hence, we conclude that female [START]M. sexta[END] ignore the interspecific competition with beetles and oviposit deliberately on beetle-infested plants to provide their offspring with an enemy-reduced space, thus providing a trade-off that generates a net benefit to the survival and fitness of the subsequent generation. We identify that alpha-copaene, emitted by beetle-infested Datura, plays a role in this preference. By performing heterologous expression and single-sensillum recordings, we show that odorant receptor (Or35) is involved in alpha-copaene detection.

Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system. Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the [START]tobacco hornworm[END], [START]Manduca sexta[END]. In this study, we purified [START]M. sexta[END] PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). [START]M. sexta[END] PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naive larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglycans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the [START]M. sexta[END] prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.

Memory span for heterospecific individuals' odors in an ant, Cataglyphis cursor. Only recently have researchers studied the ability of ants to learn and remember individual heterospecific odors. Cataglyphis cursor adults have the capacity to learn these odors, but the duration of their memory and the factors that affect its formation remain unknown. We used a habituation/discrimination paradigm to study some of these issues. C. cursor adult workers were familiarized to an anesthetized [START]Camponotus aethiops[END] on four successive encounters. Then they were either isolated or placed with 20 nestmates for a certain length of time before undergoing a discrimination test that consisted of reintroducing the familiar C. aethiops, as well as introducing an unknown member of the same colony. The results showed that adult C. cursor ants can retain in memory a complex individual odor for at least 30 min, as well as differentiate it from the odor of another closely related individual. However, when ants were replaced in a rich social background between the habituation and the discrimination trials, we did not observe a significant discrimination between the known and unknown C. aethiops ants. Our study shows, for the first time, the existence of long-term memory for individual odors in mature ant workers.

Individual differences in exploratory activity relate to cognitive judgement bias in carpenter ants. Emotional state may influence cognitive processes such as attention and decision-making. A cognitive judgement bias is the propensity to anticipate either positive or negative consequences in response to ambiguous information. Recent work, mainly on vertebrates, showed that the response to ambiguous stimuli might change depending on an individual's affective state, which is influenced by e.g. the social and physical environment. However, the response to ambiguous stimuli could also be affected by the individual's behavioural type (personality), a question that has been under-investigated. We studied the link between individual differences in exploratory activity and the response to an ambiguous stimulus in the ant [START]Camponotus aethiops[END]. Exploratory behaviour, quantified with an open-field test, was variable among individuals but consistent over time within individuals. Individual ants learned to associate a spatial position to a reinforcement and another spatial position to a punishment. Once the ants had acquired this discrimination, cognitive judgement bias was tested with the stimulus in an intermediate position. Fast explorers in the open-field took significantly more time to approach the ambiguous stimulus compared to slow explorers, suggesting a negative judgement bias for fast explorers and a positive bias for slow explorers. This previously unknown link between individual difference in exploratory activity and cognitive bias in a social insect may help understanding the evolution and organization of social life.

Associative learning in ants: conditioning of the maxilla-labium extension response in [START]Camponotus aethiops[END]. Associative learning has been studied in many vertebrates and invertebrates. In social insects, the proboscis extension response conditioning of honey bees has been widely used for several decades. However, a similar paradigm has not been developed for ants, which are advanced social insects showing different morphological castes and a plethora of life histories. Here we present a novel conditioning protocol using [START]Camponotus aethiops[END]. When the antennae of a harnessed ant are stimulated with sucrose solution, the ant extends its maxilla-labium to absorb the sucrose. We term this the "maxilla-labium extension response" (MaLER). MaLER could be conditioned by forward pairing an odour (conditioned stimulus) with sucrose (unconditioned stimulus) in the course of six conditioning trials (absolute conditioning). In non-rewarded tests following conditioning, ants gave significantly higher specific responses to the conditioned stimulus than to a novel odour. When trained for differential conditioning, ants discriminated between the odour forward-paired with sucrose and an odour forward-paired with quinine (a putative aversive stimulus). In both absolute and differential conditioning, memory lasted for at least 1h. MaLER conditioning allows full control of the stimulation sequence, inter-stimulus and inter-trial intervals and satiety, which is crucial for any further study on associative learning in ants.

Pheromone-Induced Accuracy of Nestmate Recognition in Carpenter Ants: Simultaneous Decrease in Type I and Type II Errors. The ecological and evolutionary success of social insects relies on their ability to efficiently discriminate between group members and aliens. Nestmate recognition occurs by phenotype matching, the comparison of the referent (colony) phenotype to the one of an encountered individual. Based on the level of dissimilarity between the two, the discriminator accepts or rejects the target. The tolerated degree of mismatch is predicted by the acceptance threshold model, which assumes adaptive threshold shifts depending on the costs of discrimination errors. Inherent in the model is that rejection (type I) and acceptance (type II) errors are reciprocally related: if one type decreases, the other increases. We studied whether alarm pheromones modulate the acceptance threshold. We exposed [START]Camponotus aethiops[END] ants to formic acid and subsequently measured aggression toward nestmates and nonnestmates. Formic acid induced both more nonnestmate rejection and more nestmate acceptance than a control treatment, thus uncovering an unexpected effect of an alarm pheromone on responses to nestmates. Nestmate discrimination accuracy was improved via a decrease in both types of errors, a result that cannot be explained by a shift in the acceptance threshold. We propose that formic acid increases the amount of information available to the ants, thus decreasing the perceived phenotypic overlap between nestmate and nonnestmate recognition cues. This mechanism for improved discrimination reveals a novel function of alarm pheromones in recognition processes and may have far-reaching implications in our understanding of the modus operandi of recognition systems in general.

Asymmetry in olfactory generalization and the inclusion criterion in ants. Animals constantly face the challenge of extracting important information out of their environment, and for many animals much of this information is chemical in nature. The ability to discriminate and generalize between chemical stimuli is extremely important and is commonly thought to depend mostly on the structural similarity between the different stimuli. However, we previously provided evidence that in the carpenter ant [START]Camponotus aethiops[END], generalization not only depends on structural similarity, but also on the animal's previous training experience. When individual ants were conditioned to substance A, they generalized toward a mixture of A and B. However, when trained to substance B, they did not generalize toward this mixture, resulting in asymmetrical generalization. This asymmetry followed an inclusion criterion, where the ants consistently generalized from a molecule with a long carbon chain to molecules with a shorter chain, but not the other way around. Here I will review the evidence for the inclusion criterion, describe possible proximate mechanisms underlying this phenomenon as well as discuss its potential adaptive significance.

Sick ants become unsociable. Parasites represent a severe threat to social insects, which form high-density colonies of related individuals, and selection should favour host traits that reduce infection risk. Here, using a carpenter ant ([START]Camponotus aethiops[END]) and a generalist insect pathogenic fungus (Metarhizium brunneum), we show that infected ants radically change their behaviour over time to reduce the risk of colony infection. Infected individuals (i) performed less social interactions than their uninfected counterparts, (ii) did not interact with brood anymore and (iii) spent most of their time outside the nest from day 3 post-infection until death. Furthermore, infected ants displayed an increased aggressiveness towards non-nestmates. Finally, infected ants did not alter their cuticular chemical profile, suggesting that infected individuals do not signal their physiological status to nestmates. Our results provide evidence for the evolution of unsociability following pathogen infection in a social animal and suggest an important role of inclusive fitness in driving such evolution.

Sucrose responsiveness, learning success, and task specialization in ants. Social insects possess remarkable learning capabilities, which are crucial for their ecological success. They also exhibit interindividual differences in responsiveness to environmental stimuli, which underlie task specialization and division of labor. Here we investigated for the first time the relationships between sucrose responsiveness, behavioral specialization, and appetitive olfactory learning in ants, including reproductive castes. We show that castes of the ant [START]Camponotus aethiops[END] differ in their responsiveness to sucrose and in their learning success in olfactory conditioning experiments in which sucrose is used as reward. Olfactory learning was better in foragers than in nurses, in agreement with their higher sucrose responsiveness. Interindividual variation in stimulus responsiveness and in learning may be, therefore, a crucial factor for division of labor in social insects.

Active explorers show low learning performance in a social insect. An intriguing question in behavioral biology is whether consistent individual differences (called animal personalities) relate to variation in cognitive performance because commonly measured personality traits may be associated with risk-reward trade-offs. Social insects, whose learning abilities have been extensively characterized, show consistent behavioral variability, both at colony and at individual level. We investigated the possible link between personality traits and learning performance in the carpenter ant [START]Camponotus aethiops[END]. Exploratory activity, sociability, and aggression were assessed twice in ant foragers. Behaviors differed among individuals, they were partly repeatable across time and exploratory activity correlated positively with aggression. Learning abilities were quantified by differential conditioning of the maxilla-labium extension response, a task that requires cue perception and information storage. We found that exploratory activity of individual ants significantly predicted learning performance: "active-explorers" were slower in learning the task than "inactive-explorers". The results suggest for the first time a link between a personality trait and cognitive performance in eusocial insects, and that the underlying individual variability could affect colony performance and success.

Eye structure, activity rhythms, and visually-driven behavior are tuned to visual niche in ants. Insects have evolved physiological adaptations and behavioral strategies that allow them to cope with a broad spectrum of environmental challenges and contribute to their evolutionary success. Visual performance plays a key role in this success. Correlates between life style and eye organization have been reported in various insect species. Yet, if and how visual ecology translates effectively into different visual discrimination and learning capabilities has been less explored. Here we report results from optical and behavioral analyses performed in two sympatric ant species, Formica cunicularia and [START]Camponotus aethiops[END]. We show that the former are diurnal while the latter are cathemeral. Accordingly, F. cunicularia workers present compound eyes with higher resolution, while C. aethiops workers exhibit eyes with lower resolution but higher sensitivity. The discrimination and learning of visual stimuli differs significantly between these species in controlled dual-choice experiments: discrimination learning of small-field visual stimuli is achieved by F. cunicularia but not by C. aethiops, while both species master the discrimination of large-field visual stimuli. Our work thus provides a paradigmatic example about how timing of foraging activities and visual environment match the organization of compound eyes and visually-driven behavior. This correspondence underlines the relevance of an ecological/evolutionary framework for analyses in behavioral neuroscience.

Formic acid modulates latency and accuracy of nestmate recognition in carpenter ants. Decision-making processes face the dilemma of being accurate or faster, a phenomenon that has been described as speed-accuracy trade-off in numerous studies on animal behaviour. In social insects, discriminating between colony members and aliens is subject to this trade-off as rapid and accurate rejection of enemies is of primary importance for the maintenance and ecological success of insect societies. Recognition cues distinguishing aliens from nestmates are embedded in the cuticular hydrocarbon (CHC) layer and vary among colonies. In walking carpenter ants, exposure to formic acid (FA), an alarm pheromone, improves the accuracy of nestmate recognition by decreasing both alien acceptance and nestmate rejection. Here, we studied the effect of FA exposure on the spontaneous aggressive mandible opening response (MOR) of harnessed [START]Camponotus aethiops[END] ants presented with either nestmate or alien CHCs. FA modulated both MOR accuracy and the latency to respond to odours of conspecifics. In particular, FA decreased the MOR towards nestmates but increased it towards aliens. Furthermore, FA decreased MOR latency towards aliens but not towards nestmates. As response latency can be used as a proxy of response speed, we conclude that contrary to the prediction of the speed-accuracy trade-off theory, ants did not trade off speed against accuracy in the process of nestmate recognition.

Review of the [START]Camponotus[END] kiesenwetteri group (Hymenoptera, Formicidae) in the Aegean with the description of a new species. Based on recently collected material, the [START]Camponotus[END] kiesenwetteri group is redefined, and its members known from the Aegean region are diagnosed. [START]Camponotus[END] schulzi sp. nov. is described from Izmir Province, Turkey. [START]Camponotus[END] nadimi Tohme, 1969 syn. nov. is proposed as a junior synonym of [START]Camponotus[END] libanicus Andre, 1881 and [START]Camponotus[END] kiesenwetteri cyprius Emery, 1920 syn. nov. as a junior synonym of [START]Camponotus[END] kiesenwetteri (Roger, 1859). A key to workers of species of the C. kiesenwetteri group is provided. Niche modeling analyses are used to account for species habitat suitability across the Aegean region.

Impact of host demography and evolutionary history on endosymbiont molecular evolution: A test in [START]carpenter ants[END] (genus Camponotus) and their Blochmannia endosymbionts. Obligate endosymbioses are tight associations between symbionts and the hosts they live inside. Hosts and their associated obligate endosymbionts generally exhibit codiversification, which has been documented in taxonomically diverse insect lineages. Host demography (e.g., effective population sizes) may impact the demography of endosymbionts, which may lead to an association between host demography and the patterns and processes of endosymbiont molecular evolution. Here, we used whole-genome sequencing data for [START]carpenter ants[END] (Genus Camponotus; subgenera Camponotus and Tanaemyrmex) and their Blochmannia endosymbionts as our study system to address whether Camponotus demography shapes Blochmannia molecular evolution. Using whole-genome phylogenomics, we confirmed previous work identifying codiversification between [START]carpenter ants[END] and their Blochmannia endosymbionts. We found that Blochmannia genes have evolved at a pace ~30x faster than that of their hosts' molecular evolution and that these rates are positively associated with host rates of molecular evolution. Using multiple tests for selection in Blochmannia genes, we found signatures of positive selection and shifts in selection strength across the phylogeny. Host demography was associated with Blochmannia shifts toward increased selection strengths, but not associated with Blochmannia selection relaxation, positive selection, genetic drift rates, or genome size evolution. Mixed support for relationships between host effective population sizes and Blochmannia molecular evolution suggests weak or uncoupled relationships between host demography and Blochmannia population genomic processes. Finally, we found that Blochmannia genome size evolution was associated with genome-wide estimates of genetic drift and number of genes with relaxed selection pressures.

Trap-treat-release: horizontal transfer of fipronil in field colonies of black [START]carpenter ants[END], Camponotus pennsylvanicus. BACKGROUND: Horizontal insecticide transfer is thought to play an important role in controlling a wide range of urban pests including ants, bed bugs, cockroaches, and termites. Despite decades of research and numerous laboratory studies, horizontal transfer has never been demonstrated in the field. As a result, the importance of horizontal transfer (and the resulting secondary kill) for practical pest management remains unknown. The goal of this study was to provide the first experimental examination of horizontal transfer under field conditions. The specific objective was to investigate horizontal transfer of fipronil in field colonies of black [START]carpenter ants[END], Camponotus pennsylvanicus. RESULTS: Laboratory experiments demonstrated that fipronil is effectively transferred from treated donors to untreated recipients and causes significant secondary mortality. Fipronil was effectively vectored to untreated ants from donors exposed via residual and direct spray applications, and 100% mortality was achieved with both exposure routes. Furthermore, horizontal transfer continued beyond secondary mortality and resulted in significant tertiary mortality, which has not been previously demonstrated in ants. Field experiments utilized a novel, three-step control method consisting of trap-treat-release and demonstrated that fipronil is effectively transferred when foraging workers are trapped, treated, and subsequently released back into their colonies. CONCLUSION: The current study is the first field demonstration of the importance of horizontal transfer for the control of pest ants. The trap-treat-release method may be an effective alternative to broadcast spray applications and could help alleviate problems such as insecticide run-off, environmental contamination, and non-target effects. This method has the potential to provide effective management of invasive and pest ants and should be further tested across a wider range of ant species, habitats, and active ingredients. 2019 Society of Chemical Industry.

Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing. BACKGROUND: Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. [START]Carpenter ants[END] are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, [START]carpenter ants[END] carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus. RESULTS: The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8% of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of [START]carpenter ants[END] comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults. CONCLUSIONS: The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle.

Deinococcucins A-D, Aminoglycolipids from Deinococcus sp., a Gut Bacterium of the [START]Carpenter[END] Ant Camponotus japonicus. Four new aminoglycolipids, deinococcucins A-D (1-4), were discovered from a Deinococcus sp. strain isolated from the gut of queen [START]carpenter ants[END], Camponotus japonicus. The structures of deinococcucins A-D were elucidated as a combination of N-acetyl glucosamine, 2,3-dihydroxypropanoic acid, and an alkyl amine with a C16 or C17 hydrocarbon chain primarily based on 1D and 2D NMR and mass spectroscopic data. The exact location of the olefinic double bond in deinococcucins C and D (3 and 4) was assigned based on the liquid chromatography-mass spectroscopy data obtained after olefin metathesis. The absolute configurations of the N-acetyl glucosamine and 2,3-dihydroxy moieties were determined through gas chromatography-mass spectroscopy analysis of authentic samples and phenylglycine methyl ester-derivatized products, respectively. Deinococcucins A and C displayed significant induction of quinone reductase in murine Hepa-1c1c7 cells.

Taxonomic revision of imitating [START]carpenter ants[END], Camponotus subgenus Myrmopytia (Hymenoptera, Formicidae) of Madagascar, using morphometry and qualitative traits. The ant genus Camponotus (Mayr, 1861) is one of the most abundant and species rich ant genera in the Malagasy zoogeographical region. Although this group is commonly encountered, its taxonomy is far from complete. Here, we clarify the taxonomy of the Malagasy-endemic Camponotus subgenus Myrmopytia (Emery, 1920). Species delimitation was based on traditional morphological characters and multivariate morphometric analyses, including exploratory Nest Centroid clustering and confirmatory cross-validated Linear Discriminant Analysis. Four species are recognized: Camponotus imitator (Forel, 1891), Camponotus jodinasp. n., Camponotus karahasp. n., and Camponotus longicollissp. n. All four species appear to mimic co-occurring Aphaenogaster species. A diagnosis of the subgenus Myrmopytia, species descriptions, an identification key based on minor and major subcastes of workers, and the known geographical distribution of each species are provided.

L-proline feeding for augmented freeze tolerance of Camponotus japonicus Mayr. The successful cryopreservation of organs is a strong and widespread demand around the world but faces great challenges. The mechanisms of cold tolerance of organisms in nature inspirit researchers to find new solutions for these challenges. Especially, the thermal, mechanical, biological and biophysical changes during the regulation of freezing tolerance process should be studied and coordinated to improve the cryopreservation technique and quality of complex organs. Here the cold tolerance of the Japanese [START]carpenter ants[END], Camponotus japonicus Mayr, was greatly improved by using optimal protocols and feeding on L-proline-augmented diets for 5 days. When cooling to -27.66 C, the survival rate of frozen ants increased from 37.50% +- 1.73% to 83.88% +- 3.67%. Profiling of metabolites identified the concentration of whole-body L-proline of ants increased from 1.78 to 4.64 ng g-1 after 5-day feeding. High L-proline level, together with a low rate of osmotically active water and osmotically inactive water facilitated the prevention of cryoinjury. More importantly, gene analysis showed that the expression of ribosome genes was significantly up-regulated and played an important role in manipulating freezing tolerance. To the best of our knowledge, this is the first study to link genetic variation to the enhancement of ants' cold tolerance by feeding exogenous cryoprotective compound. It is worth noting that the findings provide the theoretical and technical foundation for the cryopreservation of more complex tissues, organs, and living organisms.

Complete Genome Sequence of the Blochmannia Endosymbiont of Camponotus nipponensis. Blochmannia endosymbionts, belonging to Gammaproteobacteria, live in bacteriocytes, which are specialized cells for these bacterial species in the Camponotus genus ([START]carpenter ants[END]). In this announcement, we describe the complete genome sequence of the Blochmannia endosymbiont of Camponotus nipponensis, which originated from a C. nipponensis colony collected in the Republic of Korea.

The balbyter ant [START]Camponotus[END] fulvopilosus combines several navigational strategies to support homing when foraging in the close vicinity of its nest. Many insects rely on path integration to define direct routes back to their nests. When shuttling hundreds of meters back and forth between a profitable foraging site and a nest, navigational errors accumulate unavoidably in this compass- and odometer-based system. In familiar terrain, terrestrial landmarks can be used to compensate for these errors and safely guide the insect back to its nest with pin-point precision. In this study, we investigated the homing strategies employed by [START]Camponotus[END] fulvopilosus ants when repeatedly foraging no more than 1.25 m away from their nest. Our results reveal that the return journeys of the ants, even when setting out from a feeder from which the ants could easily get home using landmark information alone, are initially guided by path integration. After a short run in the direction given by the home vector, the ants then switched strategies and started to steer according to the landmarks surrounding their nest. We conclude that even when foraging in the close vicinity of its nest, an ant still benefits from its path-integrated vector to direct the start of its return journey.

Blochmannia endosymbionts and their host, the ant Camponotus fellah: cuticular hydrocarbons and melanization. [START]Carpenter ants[END] (genus Camponotus) have mutualistic, endosymbiotic bacteria of the genus Blochmannia whose main contribution to their hosts is alimentary. It was also recently demonstrated that they play a role in improving immune function as well. In this study, we show that treatment with an antibiotic produces a physiological response inducing an increase in both the quantity of cuticular hydrocarbons and in the melanization of the cuticle probably due to a nutritive and immunological deficit. We suggest that this is because it enhances the protection the cuticle provides from desiccation and also from invasions by pathogens and parasites. Nevertheless, the cuticular hydrocarbon profile is not modified by the antibiotic treatment, which indicates that nestmate recognition is not modified.

Moderate Hyperglycemia-Preventive Effect and Mechanism of Action of [START]Periplaneta americana[END] Oligosaccharides in Streptozotocin-Induced Diabetic Mice. [START]Periplaneta americana[END] is a kind of medicinal and edible insect, and its oligosaccharides (PAOS) have been reported to exert anti-inflammatory effects by regulating immunity, reducing oxidative stress, and meliorating gut microbiota. We hypothesized PAOS might benefit experimental diabetes mellitus (DM), an inflammatory disease coordinated by both innate and adaptive immunity. This study aimed to evaluate the effect of PAOS on glycemia and its potential mechanisms. Mice model of diabetes was established, and then the potential effects of PAOS was tested in vivo. Here, we found that PAOS triggered a moderate hyperglycemia-preventive effect on DM mice, showing markedly alleviated symptoms of DM, reduced blood glucose, and meliorated functions of liver and pancreas beta cell. Deciphering the underlying mechanism of PAOS-improving diabetes, the results revealed that PAOS downregulated the blood glucose level by activating PI3K/AKT/mTOR and Keap/Nrf2/HO-1 pathways, meanwhile inhibiting TLR4/MAPK/NF-kappaB, Beclin1/LC3, and NLRP3/caspase1 pathways in vivo. Furthermore, analyses of the microbial community intriguingly exhibited that PAOS promoted the communities of bacteria producing short-chain fatty acids (SCFAs), whereas attenuating lipopolysaccharides (LPS)-producing ones that favored inflammatory tolerance. Collectively, balancing the intestinal bacterial communities by PAOS, which favored anabolism but suppressed inflammatory responses, contributed substantially to the glycemia improvement of PAOS in DM mice. Accordingly, PAOS might function as complementary and alternative medicine for DM.

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.

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.

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.

Behavioral responses to visual overstimulation in the cockroach [START]Periplaneta americana[END] L. In the visual systems of insects, different types of photoreceptors contribute to specialized visual channels that mediate distinct functions and behaviors. Large compound eyes of [START]Periplaneta americana[END] contain photoreceptors of two spectral classes, broadband green-sensitive photoreceptors and narrow-band UV-sensitive photoreceptors. Here, we investigated how visual stimulation by UV and green light affects locomotor, resting, and grooming behaviors in [START]P. americana[END] under conditions when light avoidance is not possible. We show that green but not UV light stimulates locomotor activity, inducing paradoxical positive masking. Duration of resting and grooming decreased with increasing light intensity, consistent with development of behavioral stress in response to visual overstimulation. A reaction of full immobility is described under UV light and at higher intensities of green light, with relative periods of immobility and grooming strongly negatively correlated. Low-intensity UV was more effective than low-intensity green light in suppressing grooming and inducing immobility. Our results suggest that locomotor activity in [START]P. americana[END] is mainly regulated by green-sensitive photoreceptors, and that dim UV light can trigger behavioral immobility, whereas both wavelengths induce stress-like reactions at high intensities. Considering the intrinsic UV sensitivity of green-sensitive photoreceptors, the contrasting behavioral responses indicate antagonistic interactions between UV and green visual channels.

Effect of natural toxins and adipokinetic hormones on the activity of digestive enzymes in the midgut of the cockroach [START]Periplaneta americana[END]. This study examined the effect of two natural toxins (a venom from the parasitic wasp Habrobracon hebetor and destruxin A from the entomopathogenic fungus Metarhizium anisopliae), and one pathogen (the entomopathogenic fungus Isaria fumosorosea) on the activity of basic digestive enzymes in the midgut of the cockroach [START]Periplaneta americana[END]. Simultaneously, the role of adipokinetic hormones (AKH) in the digestive processes was evaluated. The results showed that all tested toxins/pathogens elicited stress responses when applied into the cockroach body, as documented by an increase of AKH level in the central nervous system. The venom from H. hebetor showed no effect on digestive enzyme activities in the ceca and midgut in vitro. In addition, infection by I. fumosorosea caused a decrease in activity of all enzymes in the midgut and a variable decrease in activity in the ceca; application of AKHs did not reverse the inhibition. Destruxin A inhibited the activity of all enzymes in the midgut but none in the ceca in vitro; application of AKHs did reverse this inhibition, and no differences between both cockroach AKHs were found. Overall, the results demonstrated the variable effect of the tested toxins/pathogens on the digestive processes of cockroaches as well as the variable ability of AKH to counteract these effects.

RNA interference supports a role for Nanchung-Inactive in mechanotransduction by the cockroach, [START]Periplaneta americana[END], tactile spine. Proteins encoded by nanchung, inactive, nompC and piezo genes have been shown to play crucial roles in the initial detection of mechanical force by various insect auditory neurons, nociceptors and touch receptors. Most of this previous research has been performed on the larval and adult fruit fly, Drosophila melanogaster. We identified and assembled all four homologous genes in transcriptomes from the cockroach, [START]Periplaneta americana[END]. Injection of long double-stranded RNA (dsRNA) into the adult cockroach abdomen successfully reduced the expression of each gene, as measured by quantitative PCR (RT-qPCR). A simple electrophysiological assay was used to record action potential firing in afferent nerves of cockroach femoral tactile spines in response to a standardized mechanical step displacement. Responses of nanchung knockdown animals were significantly reduced compared to matched sham-injected animals at 14 and 21 days after injection, and inactive knockdowns similarly at 21 days. In contrast, responses of nompC and piezo knockdowns were unchanged. Our results support a model in which Nanchung and Inactive proteins combine to form a part of the mechanotransduction mechanism in the cockroach tactile spine.

Analyses of the mouthpart kinematics in [START]Periplaneta americana[END] (Blattodea, Blattidae) using synchrotron-based X-ray cineradiography. The kinematics of the biting and chewing mouthparts of insects is a complex interaction of various components forming multiple jointed chains. The non-invasive technique of in vivo cineradiography by means of synchrotron radiation was employed to elucidate the motion cycles of the mouthparts in the cockroach [START]Periplaneta americana[END]. Digital X-ray footage sequences were used in order to calculate pre-defined angles and distances, each representing characteristic aspects of the movement pattern. We were able to analyze the interactions of the mouthpart components and to generate a functional model of maxillary movement by integrating kinematic results, morphological dissections and fluorescence microscopy. During the opening and closing cycles, which take about 450-500 ms on average, we found strong correlations between the measured maxillary and mandibular angles, indicating a strong neural coordination of these movements. This is manifested by strong antiphasic courses of the maxillae and the mandibles, antiphasic patterns of the rotation of the cardo about its basic articulation at the head and by the deflection between the cardo and stipes. In our functional model of the maxilla, its movement pattern is explained by the antagonistic activity of four adductor-promotor muscles and two abductor-remotor muscles. However, beyond the observed intersegmental and bilateral stereotypy, certain amounts of variation across subsequent cycles within a sequence were observed with respect to the degree of correlation between the various mouthparts, the maximum, minimum and time course of the angular movements. Although generally correlated with the movement pattern of the mandibles and the maxillary cardo-stipes complex, such plastic behaviour was especially observed in the maxillary palpi and the labium.

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.

Establishment and Maintenance of Gnotobiotic American Cockroaches ([START]Periplaneta americana[END]). Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches ([START]Periplaneta americana[END]). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 C for approximately 4-6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.