Transneuronal induction of muscle atrophy in grasshoppers

Autotomy is a process in grasshoppers whereby one or both hindlimbs can be shed to escape a predator or can be abandoned if damaged. It occurs between the trochanter and the femur (second and third leg segments) and once lost, the legs never regenerate. Autotomy severs branches of the leg nerve (N5) but damages no muscles since none span the autotomy plane. We find, however, that undamaged muscles intrinsic to the thorax of grasshoppers, Barytettix psolus, atrophy to less than 15% of their normal mass after autotomy of a hindlimb. These muscles operate the coxa and trochanter (first and second leg segments) and are innervated by branches of nerves 3 and 4; nerve branches that are not damaged by autotomy. Atrophy is localized to the side and body segment where autotomy occurs. Atrophy is evident 7-10 days after loss of a limb, is complete by about 30 days, and follows a similar time course whether induced in young adult, or sexually mature grasshoppers. During autotomy, leg nerve 5 is served distal to the trochanter, the thoracic muscles lose their normal static and dynamic load, and these muscles are subsequently no longer used to support the weight of the insect during posture and locomotion. Experimental loading and unloading of the affected muscles, and cutting of nerves indicated that it is the severing of leg nerve 5 during autotomy that transneuronally induces muscle atrophy.     

Interactions Between Limb Regeneration and Molting in Decapod Crustaceans

Molting and regeneration of lost appendages are tightly-coupled,hormonally-regulated processes in decapod crustaceans. Precociousmolts are induced by eyestalk ablation, which reduces circulatingmolt-inhibiting hormone (MIH) and results in an immediate risein hemolymph ecdysteroids. Precocious molts are also inducedby autotomy of 5–8 walking legs; adult land crabs (Gecarcinuslateralis) molt 6–8 wk after multiple leg autotomy (MLA).Autotomy of one or more of the 1° limb buds (LBs) that formafter MLA before a critical period interrupts proecdysis until2° LBs re-regenerate and grow to the approximate size ofthose lost. Based on these observations, Skinner proposed thatlimb buds produce two factors that control proecdysial events.Limb Autotomy Factor–Anecdysis (LAF_an), produced by 1°LBs when at least five legs are autotomized, stimulates anecdysialanimals to enter proecdysis. Limb Autotomy Factor–Proecdysis(LAF_pro), produced by 2° LBs in premolt animals when atleast one 1° LB is autotomized, inhibits proecdysial processes.Initial characterizations suggest that LAF_pro is a MIH-likepolypeptide that inhibits the synthesis and secretion of ecdysteroidby the Y-organs.     

Scorpion Sheds ‘Tail’ to Escape: Consequences and Implications of Autotomy in Scorpions (Buthidae: Ananteris)

Autotomy, the voluntary shedding or detachment of a body part at a determined cleavage plane, is a common anti-predation defense mechanism in several animal taxa, including arthropods. Among arachnids, autotomy has been observed in harvestmen, mites, and spiders, always involving the loss of legs. Autotomy of the opisthosoma (abdomen) was recently reported in a single species of the Neotropical buthid scorpion genus Ananteris Thorell, 1891, but few details were revealed. Based on observations in the field and laboratory, examination of material in museum collections, and scanning electron microscopy, we document autotomy of the metasoma (the hind part of the opisthosoma, or ‘tail’) in fourteen species of Ananteris. Autotomy is more common in males than females, and has not been observed in juveniles. When the scorpion is held by the metasoma, it is voluntarily severed at the joints between metasomal segments I and II, II and III, or III and IV, allowing the scorpion to escape. After detachment, the severed metasoma moves (twitches) automatically, much like the severed tail of a lizard or the severed leg of a spider, and reacts to contact, even attempting to sting. The severed surface heals rapidly, scar tissue forming in five days. The lost metasomal segments and telson cannot be regenerated. Autotomy of the metasoma and telson results in permanent loss of the posterior part of the scorpion’s digestive system (the anus is situated posteriorly on metasomal segment V) and the ability to inject venom by stinging. After autotomy, scorpions do not defecate and can only capture small prey items. However, males can survive and mate successfully for up to eight months in the laboratory. In spite of diminished predation ability after autotomy, survival allows males to reproduce. Autotomy in Ananteris therefore appears to be an effective, adaptive, anti-predation escape mechanism.     

Patterns of autotomy and regeneration in Hemigrapsus nudus

Leg autotomy and regeneration can have severe impacts on survival and reproduction, and these impacts may be even more pronounced in animals with multifarious legs, such as decapods. Thus, determining the patterns and frequency of autotomy and regeneration could reveal the effects of these processes on the individual and population level. We investigated whether some legs are lost more often than others and if all legs are equally likely to be regenerated. We sampled nearly 500 purple shore crabs (Hemigrapsus nudus) and showed that (1) most animals are found with at least one injured leg, (2) the patterns of autotomy differ between males and females, and (3) successful claw regeneration is unlikely in both males and females. Future work with H. nudus and other grapsid crabs will elucidate how patterns seen here relate to other developmental and ecological factors.     

Leg regeneration in insects. An experimental analysis in Drosophila and a new interpretation.

Fragments from prospective distal regions of Drosophila male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for 8 days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips, regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two categories of regulative fields observed in insect legs, the leg segment fields and the whole leg field, represent the units of regulation for two fundamentally different regulative pathways that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway, respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, contact one another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact.     

Characterization of limb autotomy factor-proecdysis (LAF(pro)), isolated from limb regenerates,that suspends molting in the land crab Gecarcinus lateralis

Molting and limb regeneration are tightly coupled processes, both of which are regulated by ecdysteroid hormone synthesized and secreted by the Y-organs. Regeneration of lost appendages can affect the timing and duration of the proecdysial, or premolt, stage of the molt cycle. Autotomy of all eight walking legs induces precocious molts in various decapod crustacean species. In the land crab Gecarcinus lateralis, autotomy of a partially regenerated limb bud before a critical period during proecdysis (regeneration index <17) delays molting so that a secondary limb bud (2 degrees LB) forms and the animal molts with a complete set of walking legs. It is hypothesized that 2 degrees LBs secrete a factor, termed limb autotomy factor-proecdysis (LAF(pro)), that inhibits molting by suppressing the Y-organs from secreting ecdysone. Molting was induced by autotomy of eight walking legs; autotomy of primary (1 degrees ) LBs reduced the level of ecdysteroid hormone in the hemolymph 73% by one week after limb bud autotomy (LBA). Injection of extracts from 2 degrees LBs, but not 1 degrees LBs, inhibited 1 degrees LB growth in proecdysial animals, thus having the same effect on molting as LBA. The inhibitory activity in 2 degrees LB extracts was stable after boiling in water for 15 min, but was destroyed by boiling 15 min in 0.1 N acetic acid or incubation with proteinase K. These results support the hypothesis that LAF(pro) is a peptide that resembles a molt-inhibiting hormone.     

Leg Regrowth in Blaberus discoidalis (Discoid Cockroach) following Limb Autotomy versus Limb Severance and Relevance to Neurophysiology Experiments

Background

Many insects can regenerate limbs, but less is known about the regrowth process with regard to limb injury type. As part of our neurophysiology education experiments involving the removal of a cockroach leg, 1) the ability of Blaberus discoidalis cockroaches to regenerate a metathoracic leg was examined following autotomy at the femur/trochanter joint versus severance via a transverse coxa-cut, and 2) the neurophysiology of the detached legs with regard to leg removal type was studied by measuring spike firing rate and microstimulation movement thresholds.

Leg Regrowth Results

First appearance of leg regrowth was after 5 weeks in the autotomy group and 12 weeks in the coxa-cut group. Moreover, regenerated legs in the autotomy group were 72% of full size on first appearance, significantly larger (p<0.05) than coxa-cut legs (29% of full size at first appearance). Regenerated legs in both groups grew in size with each subsequent molt; the autotomy-removed legs grew to full size within 18 weeks, whereas coxa-cut legs took longer than 28 weeks to regrow. Removal of the metathoracic leg in both conditions did not have an effect on mortality compared to matched controls with unmolested legs.

Neurophysiology Results

Autotomy-removed legs had lower spontaneous firing rates, similar marked increased firing rates upon tactile manipulation of tibial barbs, and a 10% higher electrical microstimulation threshold for movement.

Summary

It is recommended that neurophysiology experiments on cockroach legs remove the limb at autotomy joints instead of coxa cuts, as the leg regenerates significantly faster when autotomized and does not detract from the neurophysiology educational content.     

Neurogenic pacemakers in the legs of Opiliones

ABSTRACT. After autotomy, the legs of all the species of Opiliones examined, and of a Kenyan Pholcid spider, twitched spontaneously at the femoro-patellar and tibio-basitarsal joints, for periods of up to an hour. These joints lack extensor muscles, extension being achieved at the femoro-patellar joint probably by haemolymph pressure, but at the tibio-basitarsal joint of Opiliones by a cuticular spring which can extend the joint fully. Comparable twitching activity could be evoked without autotomy if the central nervous system was burnt, or by asphyxiation. Electromyograms from the femur or tibia of an isolated twitching leg showed regular motor bursts which accompanied flexions, and sensory activity during extension. Forced movements of the joints did not perturb the rhythm of the motor bursts. An isolated proximal half of a femur could still generate the same bursting pattern whereas no other region showed this activity after its isolation. Bursts recorded in the tibia were shown to be dependent on the integrity of the femur. By stimulation of the femur with 1 -ms current pulses it was possible to reset the rhythm. Stimulation with 1-s pulses caused an acceleration or inhibition of the rhythm according to the direction of the current. Spontaneous bursts could be evoked in silent isolated legs, or in intact quiescent legs, by similar 1-s current pulses. It is postulated that the femur contains independent neurogenic pacemakers which are activated by injury current from the damaged leg nerve; they produce regular bursts of motor impulses without the interplay of proprioceptive loops, and are responsible for the movements observed.     

Foreleg Autotomy Reduces Mating Success of Male <Emphasis Type="Italic">Schizocosa ocreata</Emphasis> Wolf Spiders

Autotomy is a taxonomically widespread antipredator tactic that allows animals to escape life-threatening situations. Opposing the benefits of survival, animals that have autotomized appendages may later suffer reduced ability in important determinants of fitness. Male Schizocosa ocreata wolf spiders use their forelegs during courtship for visual displays, for tactile courtship, and to defend against attacks by females. In nature they are often found missing one, and sometimes both, forelegs. We found that autotomy of one foreleg has little effect on male ability to mate with virgin females, but that autotomy of both forelegs causes a significant reduction in mating success. Among males that mated, autotomy of one or both forelegs did not influence latency until mating, period spent mounted, probability that his mate would accept a subsequent suitor, or probability that his mate would kill a subsequent suitor.     

The type of leg lost affects habitat use but not survival in a non‐regenerating arthropod

Finding shelter and surviving encounters with predators are pervasive challenges for animals. These challenges may be exacerbated after individuals experience bodily damage. Certain forms of damage arise voluntarily in animals; for instance, some taxa release appendages (tails, legs, or other body parts) as a defensive strategy (“autotomy”). This behavior, however, may pose long‐term negative consequences for habitat use and survival. Additionally, these putative consequences are expected to vary according to the function of the lost body part. We tested the effects of losing different functional leg types (locomotor or sensory) on future habitat use and survival in a Neotropical species of Prionostemma harvestmen (Arachnida: Opiliones) that undergo frequent autotomy but do not regrow limbs. Daytime surveys revealed that both eight‐legged harvestmen and harvestmen missing legs roosted in similar frequencies across habitats (tree bark, mossy tree, or fern), and perched at similar heights. Mark–recapture data showed that harvestmen that lost sensory legs roosted in tree bark less frequently, but on mossy trees more frequently. On the contrary, we did not observe changes in habitat use for eight‐legged animals or animals that lost locomotor legs. This change might be related to sensory exploration and navigation. Lastly, we found that recapture rates across substrates were not affected by the type of legs lost, suggesting that leg loss does not impact survival. This potential lack of effect might play a role in why a defensive strategy like autotomy is so prevalent in harvestmen despite the lack of regeneration.     

Autotomy of walking legs in the Pacific Shore crab Hemigrapsus oregonesis †

The ease of autotomy in Hemigrapsus oregonensis was tested by crushing the propodite of each walking leg. Individual curves of percent autotomy in groups of 18–21 animals were drawn for six different sequences of stimulation. Lumped data show peaks in ease of autotomy for the third anatomical leg of each side and for the second leg stimulated regardless of anatomical position. Later autotomies are more severely depressed when the stimulation sequence is from posterior to anterior. Segmental and lateral interaction and the characteristics of individual responses suggest that autotomy of legs in the crab, while depending upon specific structural features of the legs, is less a unisegmental reflex, than an accident occurring during escape.     

The effect of amputation and leg restraint on the free walking coordination of the stick insectCarausius morosus

Summary The stepping patterns of intact, amputated and leg restrained first instar stick insects were examined by analysing video tape records of their free walking behaviour. Amputation produced changes in the relative timing of protraction movements both along and across the body axis. Restraint of individual front or rear legs produced walking behaviour similar to that of the amputee animal but restraint of middle legs caused a breakdown in the coordination of front and rear legs. The changes in behaviour produced by leg autotomy and restraint were used to test certain assumptions of a model for generating the step pattern of these insects and to investigate how the tonic influence of proprioceptive input might be incorporated into the model.I would like to thank Professor P.N.R. Usherwood and Drs. M.D. Burns and W.J.P. Barnes for their comments and ideas on this work. A special acknowledgement goes to Dr. F. Delcomyn whose Fortran step analysis programs assisted greatly in the data reduction. I wish to thank S.R.C. for a returning scientist award and the support and equipment provided by grant B/SR/9774 to Professor Usherwood. A preliminary survey of some of the amputees was carried out at the Biology Department, Case Western Reserve University and I would like to acknowledge the support provided by a P.H.S. grant NB-06054 to Professor R.K. Josephson.     

三种华枝断肢再生的研究

目(竹节虫目)的昆虫具有很强的断肢再生能力。该文通过对华枝属(Sinophasma spp)三种昆虫的实验,表明其再生能力与断肢发生的时间及数量有关。断肢1只或2只的1～4龄虫体发育至成虫期或至若虫末龄时,其再生足的长度与相应的正常足长度相近。若在5龄初时断肢1～2只,也具有再生能力,但至成虫期其再生足的长度则短于相对应的正常足。若在6龄及成虫时断肢,则无再生能力（若6龄时出现断肢再生,则若虫期多为7龄）。实验结果还表明,若断肢为3只或3只以上,则虫体不能存活,且多在断肢后2～3 d内死亡。观察中尚发现,再生足生长速度明显高于正常足。而且,断肢的龄期越高,再生足生长速度越快。再生足的伸长生长与正常足一样,均出现于虫体蜕皮时。     

Interactive effects of leg autotomy and incline on locomotor performance and kinematics of the cellar spider,Pholcus manueli

Leg autotomy can be a very effective strategy for escaping a predation attempt in many animals. In spiders, autotomy can be very common (5–40% of individuals can be missing legs) and has been shown to reduce locomotor speeds, which, in turn, can reduce the ability to find food, mates, and suitable habitat. Previous work on spiders has focused mostly on the influence of limb loss on horizontal movements. However, limb loss can have differential effects on locomotion on the nonhorizontal substrates often utilized by many species of spiders. We examined the effects of leg autotomy on maximal speed and kinematics while moving on horizontal, 45° inclines, and vertical (90°) inclines in the cellar spider Pholcus manueli, a widespread species that is a denizen of both natural and anthropogenic, three‐dimensional microhabitats, which frequently exhibits autotomy in nature. Maximal speeds and kinematic variables were measured in all spiders, which were run on all three experimental inclines twice. First, all spiders were run at all inclines prior to autotomization. Second, half of the spiders had one of the front legs removed, while the other half was left intact before all individuals were run a second time on all inclines. Speeds decreased with increasing incline and following autotomy at all inclines. Autotomized spiders exhibited a larger decrease in speed when moving horizontally compared to on inclines. Stride length decreased at 90° but not after autotomy. Stride cycle time and duty factor increased after autotomy, but not when moving uphill. Results show that both incline and leg autotomy reduce speed with differential effects on kinematics with increasing incline reducing stride length, but not stride cycle time or duty factor, and vice versa for leg autotomy. The lack of a significant influence on a kinematic variable could be evidence for partial compensation to mitigate speed reduction.     

The m. caudifemoralis longus and its relationship to caudal autotomy and locomotion in lizards (Reptilia: Sauna)

Although the phenomenon of tail autotomy has traditionally been viewed in a purely adaptive light, functional constraints imposed by the locomotor system appear to have influenced the presence and extent of autotomy in lizards. Them. caudifemoralis longus is an unsegmented hind limb retractor that originates from the caudal vertebrae. It does not participate in autotomy and thus limits the proximal position of autotomic septa. Variation in the extent of the m. caudifemoralis is correlated with locomotor type. The muscle is large and originates from a long series of caudal vertebrae in fast moving lizards with powerful limb retraction, as exemplified by taxa capable of bipedal running. In slower lizards with sprawling postures, such as geckos, the m. caudifemoralis is small and restricted to the first few postsacral vertebrae. Autotomy is typically restricted or absent in the former lizards, while in the latter only the most proximal vertebrae are incapable of autotomy. In the evolution of existing patterns of caudal autotomy, functional demands intrinsic to the tail may be subservient to locomotor constraints imposed on the tail base by the m. caudifemoralis longus .     

Intercalary regeneration in legs of crayfish: Proximal segments

An arthropod leg represents a protuberance of the body segmental integument which bears distinctive markers in both the mediolateral and the anteroposterior axes. To clarify the biaxial organization of the body segmental morphogenetic field, and to study the relation among the whole-limb, limb segmental, and body segmental fields previously recognized in arthropods, we have grafted a proximal leg segment into the ventral midline in crayfish. After this operation the majority of animals regenerated a mirror-symmetric pair of supernumerary legs at the host site. Some of these legs had the most proximal segment, the coxa, partially fused to the adjacent body surface. Minority patterns of regeneration included one midline leg with a gill, three midline legs with a gill, and two normal legs with a third double-half leg. These results are compatible with the principle that intercalary regeneration restores the continuity of positional information.     

Pattern formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae

Embryos and first instar larvae of Drosophila melanogaster were X-irradiated in order to study pattern formation in discs damaged at early stages. After treatment, appendages were found in which some pattern elements were duplicated and others were absent. In some strains legs were preferentially duplicated, and in others, antennae were preferentially duplicated. Duplicated appendages were mirror images and resulted most frequently when animals were irradiated during late embryonic or early larval stages. Appendages varied from those which showed complete duplication of only the distal parts (claws or aristae), to those which showed duplication of almost the entire appendage. Examination of the cuticular patterns in duplicated mesothoracic legs showed that in legs with complete duplication solely in distal regions, only extreme lateral leg parts were duplicated, and medial leg parts were absent. In legs with duplication extending into proximal regions, much of the lateral side was duplicated, and only extreme medial parts were missing. The situation for partially duplicated antennae was similar. Prothoracic legs were found fused in some X-rayed flies. The cuticular patterns were almost perfect mirror images, although the amount of fusion varied widely between different individuals. Apparently the pattern forming processes in the right and left first leg discs are coordinated in X-rayed animals. The results were consistent with a model embracing a gradient of developmental capacity in the early disc similar to that postulated to exist in the late third instar leg disc. This model is also consistent with results of various surgical experiments reported in the literature. Several predictions of the model are explained including the possible mode of action of a specific class of mutants which may affect pattern formation by altering a morphogenetic gradient.     

Regeneration of Walking Legs in the Fiddler Crab Uca pugilator

SYNOPSIS. Regeneration of walking legs in the fiddler crab Ucapugilator is most efficient when it follows autotomy (the reflexiveloss of a limb). Closure of the wound and would healing occurimmediately following autotomy and visible regeneration beginswithin a few days. Regeneration of the walking leg occurs intwo distinct stages: The first stage, called Basal Growth, involvesmitosis and differentiation. The second stage involves primarilyprotein synthesis and water uptake and is called ProecdysialGrowth. Proecdysial Growth is, in part, under direct hormonalstimulation.     

Dependence of transdetermination frequency on the developmental stage of cultured imaginal discs of Drosophila melanogaster

Male foreleg tissue from prepupal stages of Drosophila melanogaster was tested for its capacity to grow when cultured in the adult fly hemocoel and for its capacity, after culture, to produce adult cuticular structures when differentiated in a metamorphosing larva. Evaginated, segmented leg tissue from 8-hr-old prepupae (at 25°C), still retained the capacity to grow well in culture. Growth was, however, restricted to cells of the proximal half of the leg. Tissue from 11- and 24-hr stages (pupal ecdysis at 11 hr) was not successfully cultured. Cultured proximal halves of 8 hr prepupal legs frequently differentiated not only proximal structures, but also distal structures, such as sex combs and claws, indicating regeneration of missing leg structures during the culture period. Transdetermination to wing tissue occurred only rarely (once in 90 implants) whereas third-instar leg tissue in culture transdetermined frequently (50% of the implants) to wing, even though growth of tissue of the two stages was equivalent. Identical results were obtained with third-instar foreleg discs evaginated in vitro with β-ecdysone. This is the first in vitro treatment reported to reduce transdetermination frequency, without affecting growth proportionately. These results indicate that cell proliferation in culture, while probably a necessary condition for transdetermination, is not a sufficient condition. The developmental stage of the cultured tissue strongly affects the frequency of transdetermination.     

Change in take‐off elevation angle after limb autotomy mitigates the reduction in jumping distance in rice grasshoppers Oxya yezoensis

Autotomy is the ability to spontaneously self‐amputate a limb or other appendage, often as a reflexive action. This limb amputation typically occurs as a specialized defensive response to an attack from a predator and thereby enables the prey to escape from predation. Despite the benefits of escape, autotomized organisms lose the body part and its associated function. Here, we investigated the jumping behavior and performance of one‐leg‐autotomized and intact rice grasshoppers, Oxya yezoensis, to examine changes in jumping behavior after autotomy. The take‐off elevation of autotomized grasshoppers was 7.8° lower than in intact grasshoppers, resulting in nearly a 45° angle of take‐off, which maximized the jumping distance. Kinematic analyses of the jumping manner revealed that the angle of the femur during jumping differed between intact and autotomized grasshoppers, suggesting that the grasshoppers behaviorally change the take‐off elevation after autotomy. According to analyses of jumping performance, the degree of decline in performance differed between horizontal distance and vertical height. Even though they jumped on only one hind leg, one‐leg‐autotomized grasshoppers realized 69% performance along a horizontal distance relative to intact grasshoppers. In contrast, autotomized grasshoppers realized only a 44% performance in vertical height compared to intact grasshoppers. The difference in take‐off elevation between autotomized and intact grasshoppers is likely related to the observed difference in the magnitude of the decline in performance between horizontal distance and vertical height. These results suggest that rice grasshoppers may alter their take‐off elevation after limb autotomy to minimize the reduction in jumping distance.