Gene manipulation in parasitic helminths

In light of recent growth in available DNA sequence information for a number of parasitic helminths, it is crucial that suitable gene manipulation technologies are developed to facilitate functional genomic studies in these organisms. In this review we discuss recent progress in the development of these technologies in nematode and platyhelminth parasites of medical and veterinary importance. Specifically, the current status of transient transfection, double-stranded RNA interference and antisense RNA as viable techniques for the manipulation of parasitic helminth gene expression is presented. In addition, the potential for the development of stable, or germ-line, transformation methods in these organisms is also discussed.     

Hormonal control of reproduction and reproductive behavior in crickets

In Orthoptera, the endocrine control of reproduction has been investigated mainly in Acrididae. Gryllidae are also good models for the study of hormonal control of reproduction and reproductive behavior. In this review, special attention will be focused on the house cricket, Acheta domesticus. In the house cricket, vitellogenesis is controlled according to the classical model described for most insect orders. However, whereas allatectomy completely abolished oocyte growth in A. domesticus, it did not totally suppress ovarian development and egg-laying in Teleogryllus commodus, Gryllus bimaculatus, and G. campestris. In the Gryllidae studied thus far, juvenile hormone (JH) is not needed for mating behavior. In adult A. domesticus, the expression of oviposition movement, although independent of the presence of the ovaries, is strictly controlled by JH III. Recent findings suggest hormonal actions on the central nervous system of the house cricket, and provide a stimulating basis for further research on the respective involvement of hormones and nervous system in the control of reproductive behavior in adult crickets. Arch. Insect Biochem. Physiol. 35:393–404, 1997. © 1997 Wiley-Liss, Inc.     

Experience-based agonistic behavior in female crickets, Gryllus bimaculatus

Fighting behavior in male crickets is already well described, and some of the mechanisms underlying aggression and aggressive motivation have already been revealed. Much less is known about female/female interactions. Here, we report that adult female crickets that had been isolated for several days readily entered into agonistic interactions with conspecific individuals. Characteristic dyadic encounters between isolated females escalated in a stepwise manner and were concluded with the establishment of a dominant/subordinate relationship. For 15 to 30 minutes following an initial fight, former subordinate females showed a dramatic change in agonistic behavior. If they were paired with the former dominant opponent during this interval, a significant majority did not enter into any aggressive interaction but instead actively avoided the opponent. A similar experience-based and time-dependent increase in avoidance was observed when former subordinate females were paired with unfamiliar na?ve opponents. However, when faced with an unfamiliar subordinate individual in the second encounter, no such increase in avoidance behavior was observed. We propose that the observed changes in the behavior of former subordinate females are the consequence of a change in the general state of arousal and of the recognition of dominance status, but not of individual recognition. The fact that former dominant individuals did not show similar experience-based changes in agonistic behavior suggests that dominant/subordinate relationships between pairs of female crickets are maintained mainly by the behavior of subordinate individuals.     

蟋蟀对召唤声识别的趋声性定位行为

为了探究蟋蟀种内个体间的声音通讯原理,本研究通过回放利用Cool-edit2000声音采集软件采集的双斑蟋Gryllus bimachlatus召唤声,分别对雌、雄蟋蟀识别召唤声的趋声性定位行为进行了观测研究。实验结果表明:雌、雄双斑蟋对回放的召唤声均有较强的识别反应,但雌蟋蟀的趋声定位性较强,能在较短的时间内积极寻找到声源,路径成"s"型;雄蟋蟀反应相对较弱,路径较长,找到声源时间比雌蟋蟀长。本研究结果揭示召唤声在蟋蟀种内通讯中不仅对个体间的联系有作用,对异性求偶也有重要作用。     

Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus

Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.     

No evidence of sickness behavior in immune‐challenged field crickets

Sickness behavior is a taxonomically widespread coordinated set of behavioral changes that increases shelter‐seeking while reducing levels of general activity, as well as food (anorexia) and water (adipsia) consumption, when fighting infection by pathogens and disease. The leading hypothesis explaining such sickness‐related shifts in behavior is the energy conservation hypothesis. This hypothesis argues that sick (i.e., immune‐challenged) animals reduce energetic expenditure in order have more energy to fuel an immune response, which in some vertebrates, also includes producing an energetically expensive physiological fever. We experimentally tested the hypothesis that an immune challenge with lipopolysaccharide (LPS) will cause Gryllus firmus field crickets to reduce their activity, increase shelter use and avoid foods that interfere with an immune response (i.e., fat) while preferring a diet that fuels an immune response (i.e., protein). We found little evidence of sickness behavior in Gryllus firmus as immune‐challenged individuals did not reduce their activity or increase their shelter‐seeking. Neither did we observe changes in feeding or drinking behavior nor a preference for protein or avoidance of lipids. Males tended to use shelters less than females but no other behaviors differed between the sexes. The lack of sickness behavior in our study might reflect the fact that invertebrates do not possess energetically expensive physiological fever as part of their immune response. Therefore, there is little reason to conserve energy via reduced activity or increased shelter use when immune‐challenged.     

Optogenetic long-term manipulation of behavior and animal development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.     

Aggregative group behavior in insect parasitic nematode dispersal

Movement behavior of foraging animals is critical to the determination of their spatial ecology and success in exploiting resources. Individuals sometimes gain advantages by foraging in groups to increase their efficiency in garnering these resources. Group movement behavior has been studied in various vertebrates. In this study we explored the propensity for innate group movement behavior among insect parasitic nematodes. Given that entomopathogenic nematodes benefit from group attack and infection, we hypothesised that the populations would tend to move in aggregate in the absence of extrinsic cues. Movement patterns of entomopathogenic nematodes in sand were investigated when nematodes were applied to a specific locus or when the nematodes emerged naturally from infected insect hosts; six nematode species in two genera were tested (Heterorhabditis bacteriophora, Heterorhabditis indica, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema riobrave). Nematodes were applied in aqueous suspension via filter paper discs or in infected insect host cadavers (to mimic emergence in nature). We discovered that nematode dispersal resulted in an aggregated pattern rather than a random or uniform distribution; the only exception was S. glaseri when emerging directly from infected hosts. The group movement may have been continuous from the point of origin, or it may have been triggered by a propensity to aggregate after a short period of random movement. To our knowledge, this is the first report of group movement behavior in parasitic nematodes in the absence of external stimuli (e.g., without an insect or other apparent biotic or abiotic cue). These findings have implications for nematode spatial distribution and suggest that group behavior is involved in nematode foraging.     

Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies--with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ~1 h per group for the behavioral assays.     

Motor program initiation and selection in crickets, with special reference to swimming and flying behavior

An air puff stimulus to the cerci of a cricket (Gryllus bimaculatus) evokes flying when it is suspended in air, while the same stimulus evokes swimming when it is placed on the water surface. After bilateral dissection of the connectives between the suboesophageal and the prothoracic ganglia or between the brain and the suboesophageal ganglion, the air puff stimulus evokes flying even when the operated cricket is placed on the water surface. A touch stimulus on the body surface of crickets placed on the water surface elicits only flying when the connectives between suboesophageal and prothoracic ganglia are dissected, while the same stimulus elicits either swimming or flying when the connectives between the brain and the suboesophageal ganglion are dissected. These results suggest that certain neurons running through the ventral nerve cords between the brain and the suboesophageal ganglion or between the suboesophageal and the prothoracic ganglia play important but different roles in the initiation and/or switching of swimming and flying. In the suboesophageal ganglion, we physiologically and morphologically identified four types of "swimming initiating neurons". Depolarization of any one of these neurons resulted in synchronized activities of paired legs with a similar temporal sequence to that observed during swimming.     

Sound localisation in crickets

Intracellular recordings were made in the brain of the cricket Gryllus bimaculatus from an ascending auditory interneuron (AN1). Acoustic stimuli with calling song temporal pattern were delivered via earphones in a preparation with the acoustic trachea cut (attenuation of crossing sound > 30 dB). The input-output function of this cell was then determined by recording its responses to stimulation of the ipsilateral ear alone, of the contralateral ear alone and to stimulation of both ears simultaneously with the same or different carrier frequencies and intensities.This interneuron was excited by the ear ipsilateral to its axon and dendritic field and unresponsive to stimuli presented to the axon-contralateral ear alone. However, in binaural stimulation experiments, the response to a constant ipsilateral stimulus was progressively reduced as the intensity of a simultaneous contralateral stimulus was increased, above a threshold intensity.Tuning curves for threshold of this inhibition, determined in binaural stimulation experiments, indicated significant inhibition in the range 3–20 kHz with lowest threshold at 4–5 kHz. The inhibition was unaffected by sectioning of the contralateral circumoesophageal or neck connective, indicating that the inhibitory influence crosses the midline at the level of the prothoracic ganglion. Intracellular recordings from AN1 in the prothoracic ganglion confirmed that it was indeed neurally inhibited by inputs from the contralateral ear.Tuning curves for excitation of an omega neuron (ON1) by the ear ipsilateral to its soma and also the tuning of inhibition of ON1 by its contralateral ON1 partner, closely match the tuning of inhibition of AN1 and to a lesser extent, of AN2. This was taken as evidence that each AN1 is inhibited by the contralateral ON1. The significance of this interaction for directional hearing and phonotaxis is discussed.Abbreviations AP/CHP action potentials per chirp - AN1, AN2 ascending auditory interneurons 1, 2 - ON1 omega neuron 1 - ipsi ipsilateral contra contralateral - PTG prothoracic ganglion loc lateral ocellar nerve - On optic nerve an antennal nerve - coc circum-oesophageal connective so sound off     

Neonatal manipulation of oxytocin influences female reproductive behavior and success

During early neonatal development, oxytocin (OT) may influence the expression of adult behavior and physiology. Here we test the prediction that early postnatal exposure to OT or an oxytocin antagonist (OTA) can affect the subsequent expression of sexual receptivity and reproductive success of females. To test this hypothesis, female prairie voles (Microtus ochrogaster) received one of four treatments within 24 h of birth. Three groups received an intraperitoneal injection of OT, OTA, or isotonic saline. A fourth group was handled, but not injected. Around 75 days of age, females were paired with sexually experienced males for 72 h and sexual activity was recorded. Treatment had no effect on the probability of mating. Injection, regardless of treatment, reduced latency to mate compared with handled controls. OT and OTA treatment decreased mating bout frequency compared to saline and handled controls, while OTA treatment increased reproductive success, probability of successfully producing a litter. The results suggest that neonatally OT, both endogenous and exogenous, can affect the expression of adult female reproductive activity and that blocking the effects of endogenous OT during neonatal development can affect female reproductive success. Finally, the results suggest that a number of aspects of reproduction are regulated by OT during the postnatal period, but that the mechanism of action may differ depending upon the reproductive activity.     

Place memory in crickets

Certain insect species are known to relocate nest or food sites using landmarks, but the generality of this capability among insects, and whether insect place memory can be used in novel task settings, is not known. We tested the ability of crickets to use surrounding visual cues to relocate an invisible target in an analogue of the Morris water maze, a standard paradigm for spatial memory tests on rodents. Adult female Gryllus bimaculatus were released into an arena with a floor heated to an aversive temperature, with one hidden cool spot. Over 10 trials, the time taken to find the cool spot decreased significantly. The best performance was obtained when a natural scene was provided on the arena walls. Animals can relocate the position from novel starting points. When the scene is rotated, they preferentially approach the fictive target position corresponding to the rotation. We note that this navigational capability does not necessarily imply the animal has an internal spatial representation.     

Phonotaxis in flying crickets

The steering responses of three species of field crickets, Teleogryllus oceanicus, T. commodus, and Gryllus bimaculatus, were characterized during tethered flight using single tone-pulses (rather than model calling song) presented at carrier frequencies from 3-100 kHz. This range of frequencies encompasses the natural songs of crickets (4-20 kHz, Fig. 1) as well as the echolocation cries of insectivorous bats (12-100 kHz). The single-pulse stimulus paradigm was necessary to assess the aversive nature of high carrier frequencies without introducing complications due to the attractive properties of repeated pulse stimuli such as model calling songs. Unlike the natural calling song, single tone-pulses were not attractive and did not elicit positive phonotactic steering even when presented at the calling song carrier frequency (Figs. 2, 3, and 9). In addition to temporal pattern, phonotactic steering was sensitive to carrier frequency as well as sound intensity. Three discrete flight steering behaviors positive phonotaxis, negative phonotaxis and evasion, were elicited by appropriate combinations of frequency, temporal pattern and sound intensity (Fig. 12). Positive phonotactic steering required a model calling song temporal pattern, was tuned to 5 kHz and was restricted to frequencies below 9 kHz. Negative phonotactic steering, similar to the 'early warning' bat-avoidance behavior of moths, was produced by low intensity (55 dB SPL) tone-pulses at frequencies between 12 and 100 kHz (Figs. 2, 3, and 9). In contrast to model calling song, single tone-pulses of high intensity 5-10 kHz elicited negative phonotactic steering; low intensity ultrasound (20-100 kHz) produced only negative phonotactic steering, regardless of pulse repetition pattern. 'Evasive', side-to-side steering, similar to the 'last-chance' bat-evasion behavior of moths was produced in response to high intensity (greater than 90 dB) ultrasound (20-100 kHz). Since the demonstration of negative phonotactic steering did not require the use of a calling song temporal pattern, avoidance of ultrasound cannot be the result of systematic errors in localizing an inherently attractive stimulus when presented at high carrier frequencies. Unlike attraction to model calling song, the ultrasound-mediated steering responses were of short latency (25-35 ms) and were produced in an open loop manner (Fig. 4), both properties of escape behaviors.(ABSTRACT TRUNCATED AT 400 WORDS)