Identification and characterization of uncoupling protein 4 in fat body and muscle mitochondria from the cockroach <Emphasis Type="Italic">Gromphadorhina cocquereliana</Emphasis>

We have identified and characterized an uncoupling protein in mitochondria isolated from leg muscle and from fat body, an insect analogue tissue of mammalian liver and adipose tissue, of the cockroach Gromphadorhina coquereliana (GcUCP). This is the first functional characterization of UCP activity in isolated insect mitochondria. Bioenergetic studies clearly indicate UCP function in both insect tissues. In resting (non-phosphorylating) mitochondria, cockroach GcUCP activity was stimulated by the addition of micromolar concentrations of palmitic acid and inhibited by the purine nucleotide GTP. Moreover, in phosphorylating mitochondria, GcUCP activity was able to divert energy from oxidative phosphorylation. Functional studies indicate a higher activity of GcUCP-mediated uncoupling in cockroach muscle mitochondria compared to fat body mitochondria. GcUCP activation by palmitic acid resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of UCPs in insects. GcUCP protein was immunodetected using antibodies raised against human UCP4 as a single band of around 36 kDa. GcUCP protein expression in cockroach muscle mitochondria was significantly higher compared to mitochondria isolated from fat body. LC-MS/MS analyses revealed 100% sequence identities for peptides obtained from GcUCP to UCP4 isoforms from D. melanogaster (the highest homology), human, rat or other insect mitochondria. Therefore, it can be proposed that cockroach GcUCP corresponds to the UCP4 isoforms of other animals.     

Conditioning of a glass surface for the outgrowth of insect epidermis (Leucophaea maderae,blattaria)

Summary A method has been developed by which the surface of cover slips can be conditioned for rapid outgrowth of the epidermis of cockroach legs. The method permits in vitro observation of wound-healing processes. The conditioning is brought about by materials given off by the hemocytes, possibly materials from the granules of plasmatocytes which are extruded during the flattening of the cells. Coating of the glass surface with other proteins (including cockroach hemolymph serum) and carbohydrates was completely ineffective. Dedicated to my teacher, Dr. Gerhard Krause, from whom I received the decisive stimulation to start tissue culture work, on the occasion of his 70th birthday. The work has been supported by the Deutsche Forschungsgemeinschaft (Grant Bo 453)     

Identification of resilin in the leg of cockroach, Periplaneta americana: confirmation by a simple method using pH dependence of UV fluorescence

We have examined the tarsus (foot) and tibial segments of the cockroach leg to identify structures that contain the elastic protein resilin. The presence of resilin was tested using the conventional criteria of fluorescent emission at 420 nm under UV illumination and histological staining of wholemount tissues by toluidine blue. We have also developed a simple method of confirming identification of resilin through changes in its fluorescence that occur with alteration of pH of the surrounding medium. Using a commonly available excitation filter that only passes light at >330 nm, we found that the emission was present at neutral pH but was eliminated at low pH. It then reversibly reappeared when medium of higher pH was restored. This effect is attributable to a known shift in the absorption maximum of amino acids of resilin that occurs in acidic media (from 330 to 285 nm). The accuracy of this method of identification was confirmed by examination of ligaments of the wing hinge, which has previously been shown to contain resilin in a number of insects. Using these techniques, we have identified resilin in association with ligaments at the tibio-tarsal joint and in the articulation between the fourth and fifth tarsal segments of the leg. The anatomical arrangement of these ligaments suggests that they could aid in the generation of leg movements during walking by functioning as elastic antagonists to the actions of leg muscles. The method of identification we have devised could readily be applied to aid in the localization of resilin in other animals.     

Wasp manipulates cockroach behavior by injecting venom cocktail into prey central nervous system

The parasitoid wasp Ampulex compressa induces behavioral changes in the cockroach prey by injecting venom into its central nervous system. In contrast to most other venomous predators, the wasp's sting does not induce paralysis. Rather, the two consecutive stings in the thoracic and head ganglia induce three stereotypic behavioral effects. The prey behavior is manipulated in a way beneficial to the wasp and its offspring by providing a living meal for its newborn larva. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis prevents the cockroach from fighting with its front legs, thereby facilitating the second sting in the head. A postsynaptic block of central synaptic transmission mediates this leg paralysis. Following the head sting, dopamine identified in the venom induces 30 minutes of intense grooming that appears to prevent the cockroach from straying until the last and third behavioral effect of hypokinesia commences. In this lethargic state that lasts about three weeks, the cockroach does not respond to various stimuli nor does it initiates movement. However, other specific behaviors of the prey are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to specific thoracic neurons.     

A model of pattern generation of cockroach walking reconsidered

Cockroaches that have been decapitated or that have cut thoracic connectives can show rhythmic bursting in motoneurons to intrinsic leg muscles. These preparations have been studied as models for walking and to evaluate the functions of leg proprioceptors. The present study demonstrates that headless cockroaches walk extremely poorly and slowly with considerable discoordination of motoneuronal activity, these preparations show rhythmic motoneuron bursting that is similar to righting responses (attempts to turn upright) of intact animals when placed on their backs, and bursting is inhibited when a headless animal is turned or turns itself upright. Thus, rhythmic motoneuron activity of these preparations is most probably attempted righting rather than walking. It is concluded that the headless cockroach is useful for understanding the motor mechanisms underlying righting and walking but is not of value in assessing the functions of proprioceptive feedback.     

Colchicine reversibly inhibits electrical activity in arthropod mechanoreceptors

Colchicine and some other microtubule-active agents inhibit the electrical responses of cockroach tibial spine mechanoreceptors. Lumicolchicine, a colchicine analog which does not bind to microtubule protein, does not inhibit mechanoreceptive responses. Colchicine inhibition of peripheral mechanoreceptive responses is fully reversible and dose dependent, but colchicine has no effect on conduction in leg nerve axons. Colchicine inhibition is therefore an effect on the sensory dendrites or soma. The inhibition produced by colchicine could be produced by several effects. Colchicine may inhibit because it (1) disrupts the numerous intracellular microtubules which are a part of this sensory receptor's dendrite, (2) blocks axoplasmic transport of essential materials to the sensory dendrite, or (3) binds to tubulin or other proteins in the dendritic membrane.     

Learning genetics through a scientific inquiry game

In this paper we discuss an activity through which students learn basic concepts in genetics by taking part in a police investigation game. The activity, which we have called Recal, immerses students in a scientific-based scenario in which they play a role of a scientific assessor. Players have to develop and use scientific reasoning and evidence-based decision-making to solve the given enigmas along the game. The activity aims to improve students’ knowledge of genetics and show them how genetic evidence can be applied in forensic science. The activity (known as ‘the Recal case’) uses a problem-based learning educational methodology. It is learner-centred and students play an active collaborative role. The methodology requires students to structure their knowledge, and develop their reasoning processes and self-directed learning skills. The activity has been developed for a range of audiences, including high school students, undergraduates engaged in pre-service teaching and adults of all ages. A case study has also been carried out with a group of 120 pre-service student teachers from the Universitat Rovira i Virgili (Tarragona, Spain) to check whether the coherence in the running of the game, whether its effectiveness as a learning activity and whether its dynamics and motivational aspects are acceptable.     

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.     

Evaluation of specific activity of preparations of allergens from synanthropic insects

Physical, chemical and immunobiological characteristics of allergens from synanthropic insects were studied by tests for anaphylaxis, indirect degranulation of mast cells test and ELISA. Sera from 20 patients with severe and intermediate atopic asthma with sensiblization to common allergens have been studied. All extracts of allergens from synanthropic insects (german cockroach, oriental cockroach, american cockroach, speckled feeder cockroach, cricket, common house fly, brown house moth, confused flour beetle, rice weevil, grain weevil) have specific activity. Extracts of allergens from common house fly, brown house moth, german cockroach and oriental cockroach had the strongest allergenic activity as measured by ELISA. Obtained allergens can be used for insect allergy diagnostics.     

基于BOPPPS+翻转课堂的“蛋白质工程”线上线下混合式教学模式探索

“蛋白质工程”是山西省一流本科专业建设点生物技术专业的核心必修课程。为了解决“蛋白质工程”课堂教学模式单一、传授式教学学生参与度不够、听课不认真、授课时间缩短、实验价格昂贵等问题,课程团队进行了“蛋白质工程”课程教学模式改革与实践,提出了适合生物技术专业的“蛋白质工程”教学新策略:构建了基于BOPPPS+翻转课堂的教学模式,线下课堂讲授与学生在线自主学习与完成作业、章节测验和讨论等相结合,全面融入翻转课堂;特别是在“金课”两性一度标准的指导下,课程团队自建慕课(massive open online courses,MOOC)课程资源,利用超星泛雅网络教学平台开展BOPPPS+翻转课堂的“蛋白质工程”线上线下混合式教学工作,构建了全面、系统及动态的“蛋白质工程”课程新教学体系。经过3轮的教学实践表明,课程团队在“蛋白质工程”课程资源建设、探究性实验辅导、课堂研讨设计,以及课程成绩评定方式等方面形成了一套完整、可复制、科学、合理的线上线下混合式教学模式。基于BOPPPS+翻转课堂的“蛋白质工程”线上线下混合式教学模式有助于提高学生的自主学习能力,使学生深度参与到教学的全过程,对“蛋白质工程”有了全面深刻的认识,提高了“蛋白质工程”的教学质量,为后续学生学习其他专业课程奠定了基础,也为课程教学改革提供了参考。     

Myoinhibitory neuropeptides in the American cockroach

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.     

A proprioceptor with central cell bodies in the cockroach,Periplaneta americana (Insecta)

Summary A previously undescribed receptor in the coxo-trochantinal region of the metathoracic leg of the cockroach Periplaneta americana was found to have central cell bodies. This cockroach stretch receptor is the second sensory receptor in insects reported to possess somata in the CNS and its remarkable similarity to a locust proprioceptor suggests it to be homologous.     

PBL 结合案例教学法在病理生理学教学中的应用

以问题为基础(Problem-based learning,PBL)的教学法和案例教学法都是近年来我国医学教育改革中经常采用的两种新型教学方法。但二者从教学论方面来看,在教学材料的选择上、教学目的上都存在着差别。病理生理学是沟通基础和临床各学科间的桥梁学科,将"以问题为基础"的教学模式与案例教学法有机地结合引入病理生理学教学,符合病理生理学的学科特点,能够有效提高教学质量,在教育观念上更易于被医学生所接受,因而适应对高素质医学人才培养的需要。     

Modeling stress and strain in an insect leg for simulation of campaniform sensilla responses to external forces

Campaniform sensilla (cs) on insect legs are stimulated by strain in the cuticle produced by forces applied to the leg, and generate action potentials when the strain is sufficiently high. We modeled the tibia of the American cockroach, Periplaneta americana, as a hollow, circular tube and developed a simulation of cs responses to external forces by solving equations of mechanics as applied to the leg to obtain values for stress and strain, then converting strain to action potentials. Based on the close correspondence between our simulated responses and the 1981 physiological results of Zill and Moran, we suggest that our model may be useful in assessing the performance of experimentally inaccessible cs groups during walking. Received: 10 September 1998 / Accepted in revised form: 1 March 1999     

A neuromechanical simulation of insect walking and transition to turning of the cockroach Blaberus discoidalis

A neuromechanical simulation of the cockroach Blaberus discoidalis was developed to explore changes in locomotion when the animal transitions from walking straight to turning. The simulation was based upon the biological data taken from three sources. Neural circuitry was adapted from the extensive literature primarily obtained from the studies of neural connections within thoracic ganglia of stick insect and adapted to cockroach. The 3D joint kinematic data on straight, forward walking for cockroach were taken from a paper that describes these movements in all joints simultaneously as the cockroach walked on an oiled-plate tether (Bender et al. in PloS one 5(10):1–15, 2010b). Joint kinematics for turning were only available for some leg joints (Mu and Ritzmann in J Comp Physiol A Neuroethol Sens Neural Behav Physiol 191(11):1037–54, 2005) and thus had to be obtained using the methods that were applied for straight walking by Bender et al. (PloS one 5(10):1–15, 2010b). Once walking, inside turning, and outside turning were characterized, phase and amplitude changes for each joint of each leg were quantified. Apparent reflex reversals and joint activity changes were used to modify sensory coupling pathways between the CPG at each joint of the simulation. Oiled-plate experiments in simulation produced tarsus trajectories in stance similar to those seen in the animal. Simulations including forces that would be experienced if the insect was walking freely (i.e., weight support and friction) again produced similar results. These data were not considered during the design of the simulation, suggesting that the simulation captures some key underlying the principles of walking, turning, and transitioning in the cockroach. In addition, since the nervous system was modeled with realistic neuron models, biologically plausible reflex reversals are simulated, motivating future neurobiological research.     

Improving horizontal plane locomotion via leg angle control

The lateral leg spring model has been shown to accurately represent horizontal plane locomotion characteristics of sprawled posture insects such as the cockroach Blaberus discoidalis. While passively stable periodic gaits result from employing a constant leg touch-down angle for this model, utilizing a similar protocol for a point mass model of locomotion in three dimensions produces only unstable periodic gaits. In this work, we return to the horizontal plane model and develop a simple control law that prescribes variations in the leg touch-down angle in response to external perturbations. The resulting control law applies control once per stance phase, at the instant of leg touch-down, and depends upon previous leg angles defined in the body reference frame. As a result, our control action is consistent with the neural activity evidenced by B. discoidalis during locomotion over flat and rough terrain, and utilizes variables easily sensed by insect mechanoreceptors. Application of control in the lateral leg spring model is shown to improve stability of periodic gaits, enable stabilization of previously unstable periodic gaits, and maintain or improve the basin of stability of periodic gaits. The magnitude of leg touch-down angle variations utilized during stabilization appear consistent with the natural variations evidenced by single legs during locomotion over flat terrain.     

Modeling population dynamics of two cockroach species: effects of the circadian clock, interspecific competition and pest control

The German cockroach Blattella germanica is obviously one of the most spread household pests in the world, and is now virtually impossible to sustain outside human constructions. The double-striped cockroach B. bisignata, on the other hand, is limited to Southeast Asia and mostly living in the open space, yet is able to establish in cockroach-free households, too. In this article, we develop a stage-structured population model of these two species to explore (i) whether their circadian clocks impact their long-term population dynamics, (ii) which of these species is a superior competitor, and (iii) how stringent potential pest control strategies have to be to significantly impact established populations of the German cockroach. The results of the model are as follows. Firstly, phase shifts in the light-to-dark cycle did not affect cockroach population dynamics unless males and females were out of phase and their mate finding abilities rather limited. In addition, for the hypothesized circadian clock genotypes, the shorter is the inactivity period relative to the activity one or the less arrhythmic is the population, the more viable the population is and the quicker it grows to large numbers. Secondly, the German cockroach was the superior competitor: it was able to invade and drive out established populations of the double-striped cockroach and prevent any invasion of the latter. Finally, only a significant and simultaneous reduction in a number of most sensitive German cockroach parameters resulted in species extirpation. Only carefully designed and data-based models of German (and double-striped) cockroach population dynamics can be helpful in our quest to win the fight over this unwelcome but very sturdy species.     

Re-evaluation of the absolute threshold and response mode of the most sensitive know “vibration” detector,the cockroach's subgenual organ: A cochlea-like displacement threshold and a direct response to sound

Earlier accounts claim from indirect measurements that the subgenual organ (SGO) in the proximal tibia of the cockroach leg can detect vibrational displacements down to 0.002 mm, two orders of magnitude below the threshold for vertebrate hair cells in the cochlea. The SGO vibration threshold is redetermined here more directly by a new method on a cantilever beam, while controlling for particular acoustic and vibrational artifacts that might have compromised earlier efforts. The threshold is revised upwards to about 0.2 mm in the most sensitive preparation, about the same as cochlea. Recently, it has been determined that the cockroach SGO also has an auditory response, and the data here on subthreshold summation and response-intensity relationships provide further evidence that sound and contact vibration are both sensed by the same receptor neurons. Direct measurements rule out the prevailing hypothesis that sound is detected indirectly as induced vibration of the ground, and also weigh strongly against any significant involvement of generalized leg resonance in acoustic pick-up. The results fit with a recent proposal that the auditory response is direct, and that acoustic fluctuations inside the tracheae may be the primary response mode in the transduction of both vibration and sound. 1994 John Wiley & Sons, Inc.     

Cockroach tissueIn Vitro: A system for the study of insect cell biology

Summary Anin vitro technique of culturing insect tissue is described in which isolated tissues and cells of the cockroach,Leucophaea maderae (F.), are maintained in a chemically defined nutrient medium. The system can be used for three types of cultures: organ cultures of nymphal leg regenerates for endocrine studies; drop cultures of embryo leg tissue for screening biologically active chemicals; and dispersed embryo cell cultures for physiological studies at the cellular and subcellular levels. The dispersed cultures show measurable mitotic rates and can be maintained as long as 170 days without adding undefined substances to the medium.     

A classical conditioning paradigm for the study of learning in a ganglion of the cockroach (Periplaneta Americana)

It has been demonstrated that a headless cockroach can be conditioned to hold up its leg if it receives a shock upon lowering it (Horridge 1962). This was demonstrated as a significant change in a large group of animals. The work here develops further the original preparation reducing the variability so that the change can be demonstrated in a single animal. A single leg can then be used as its own control. For electrophysiological study of conditioning a classical conditioning paradigm has advantages over an operant one and this is discussed. A method for applying the classical conditioning procedure is described.