Tobacco Hawkmoth Manduca Sexta Research Articles

Somatic piRNAs and Transposons are Differentially Expressed Coincident with Skeletal Muscle Atrophy and Programmed Cell Death.

PIWI-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, ∼27 nt long, map antisense to transposons, are oxidation resistant, exhibit a 5’ uridine bias, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 19 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed coincident with the commitment of the muscles undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is correlated with the repression of several retrotransposons and the induction of specific DNA transposons. The developmentally-regulated changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death.

Learning lessons about muscle wasting from insect larvae

Muscle loss is an inevitable aspect of ageing, but disease and injury can also cause muscles to waste, leading to weakness and instability. ‘Unfortunately, low muscle mass limits normal daily activities – even simple tasks, such as eating or stair climbing – thus reducing quality of life’, says Erika Geisbrecht from Kansas State University, USA, who would like to understand why muscles waste with age in the hope of designing treatments to help people remain active for longer. However, investigating the molecular components of muscle deterioration is not ideal in a species that routinely lives for more than 70 years. Fortunately, Geisbrecht knew of an animal that experiences muscle wasting as a natural part of its 1–2 month life cycle, the tobacco hawk moth (Manduca sexta): ‘[muscle] atrophy occurs normally in a subset of muscles, called the intersegmental muscles, during the pupal stage’, says Geisbrecht, adding that the large pupae are ideal for identifying key components of the molecular machinery from individual animals.She and David Brooks set about investigating how the muscle proteins changed over the course of the tobacco hawk moth's metamorphosis. ‘The hardest part … was carefully controlling temperature and time points to make sure our developmental stages were precise’, says Geisbrecht, who had to collect muscles from the pupae first thing in the morning 15 days after the eggs were laid – just before the muscles began to waste – and then 3 days later, toward the end of the wasting process. Then, Brooks painstakingly removed the pupae's muscles and analysed the proteins that comprised them, identifying 244 that differed between the normal and wasted muscles. Not surprisingly, the insects had mobilised proteins that digest and dispose of proteins as the muscle broke down over 3 days, in addition to increasing the levels of the proteins that contribute to energy generation, to fuel protein digestion. They also discovered that the metamorphosing moth pupae were producing less of the proteins that comprise the contracting muscle filaments: actin and myosin. However, Geisbrecht was most surprised when she realised that the quantity of myosin in the muscle increased as the actin dwindled, suggesting that the wasting process is tightly coordinated.So, how does this help us to understand muscle loss in elderly and sick people, which look nothing like moth pupae? Fortunately, at a genetic level, humans and insects share ∼75% of the same genes, so, having identified some of the key proteins involved in muscle wasting in the tobacco hawk moth, Geisbrecht could switch to focus on muscle wasting in another insect, Drosophila, which comes complete with a sophisticated box of molecular tools that could be used to uncover the roles of specific genes in the process. But first she had to identify a gene in the versatile insect that she could switch off to prevent muscle wasting on demand. Kumar Vishal and Simranjot Bawa (both from Kansas State University) tested six Drosophila strains that had been adapted to allow specific genes to be switched off and struck gold when they found that deactivating an actin filament regulator, Ciboulot – which promotes the growth of actin filaments on one face of the actin molecule – prevented the muscle from wasting during metamorphosis.The team had found a gene in the insect that they can now use to control muscle wasting to begin identifying key components in the process. In addition, Giesbrecht is eager to find out whether Ciboulot is also a key player in muscle loss in elderly and ill people, in the hope of developing new treatments for people suffering from muscle loss.

Moths sense but do not learn flower odors with their proboscis during flower investigation.

ABSTRACTInsect pollinators, such as the tobacco hawkmoth Manduca sexta, are known for locating flowers and learning floral odors by using their antennae. A recent study revealed, however, that the tobacco hawkmoth additionally possesses olfactory sensilla at the tip of its proboscis. Here, we asked whether this second ‘nose’ of the hawkmoth is involved in odor learning, similar to the antennae. We first show that M. sexta foraging efficiency at Nicotiana attenuata flowers increases with experience. This raises the question whether olfactory learning with the proboscis plays a role during flower handling. By rewarding the moths at an artificial flower, we show that, although moths learn an odor easily when they perceive it with their antennae, experiencing the odor just with the proboscis is not sufficient for odor learning. Furthermore, experiencing the odor with the antennae during training does not affect the behavior of the moths when they later detect the learned odor with the proboscis only. Therefore, there seems to be no cross-talk between the antennae and proboscis, and information learnt by the antennae cannot be retrieved by the proboscis.

The effect of tobacco Hawkmoth caterpillar extract (Manduca sexta) on the growth and stress resistance of young animals

Article deals with the problem of reducing the level of stress and improving the growth and development of young animals during weaning from a nursing female. For this purpose, it is proposed to use an extract of larvae of tobacco hawkmoth (Manduca sexta), containing ecdysone and juvenile hormone. We studied the anxiety of animals in the experimental setup of the light-dark test. Authors conclude that the extract of tobacco hawk moth caterpillars reduces the anxiety of animals. This is manifested by an increase in the latency of entering the dark compartment of the light-dark dough, an increase in the time spent in the light compartment and a decrease in the number of boluses of the litter. It is possible to explain the large increase in body weight of experimental animals in comparison with the animals of the control group by reducing the level of stress and the anabolic effect of caterpillar extract.

High-resolution analysis of differential gene expression during skeletal muscle atrophy and programmed cell death.

Skeletal muscles can undergo atrophy and/or programmed cell death (PCD) during development or in response to a wide range of insults, including immobility, cachexia, and spinal cord injury. However, the protracted nature of atrophy and the presence of multiple cell types within the tissue complicate molecular analyses. One model that does not suffer from these limitations is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. Three days before the adult eclosion (emergence) at the end of metamorphosis, the ISMs initiate a nonpathological program of atrophy that results in a 40% loss of mass. The ISMs then generate the eclosion behavior and initiate a nonapoptotic PCD during the next 30 h. We have performed a comprehensive transcriptomics analysis of all mRNAs and microRNAs throughout ISM development to better understand the molecular mechanisms that mediate atrophy and death. Atrophy involves enhanced protein catabolism and reduced expression of the genes involved in respiration, adhesion, and the contractile apparatus. In contrast, PCD involves the induction of numerous proteases, DNA methylases, membrane transporters, ribosomes, and anaerobic metabolism. These changes in gene expression are largely repressed when insects are injected with the insect steroid hormone 20-hydroxyecdysone, which delays death. The expression of the death-associated proteins may be greatly enhanced by reductions in specific microRNAs that function to repress translation. This study not only provides fundamental new insights into basic developmental processes, it may also represent a powerful resource for identifying potential diagnostic markers and molecular targets for therapeutic intervention.

Pollination in the Anthropocene: a Moth Can Learn Ozone-Altered Floral Blends

Insect pollination is essential to many unmanaged and agricultural systems and as such is a key element in food production. However, floral scents that pollinating insects rely on to locate host plants may be altered by atmospheric oxidants, such as ozone, potentially making these cues less attractive or unrecognizable to foraging insects and decreasing pollinator efficacy. We demonstrate that levels of tropospheric ozone commonly found in many rural areas are sufficient to disrupt the innate attraction of the tobacco hawkmoth Manduca sexta to the odor of one of its preferred flowers, Nicotiana alata. However, we further find that visual navigation together with associative learning can offset this disruption. Foraging moths that initially find an ozone-altered floral scent unattractive can target an artificial flower using visual cues and associate the ozone-altered floral blend with a nectar reward. The ability to learn ozone-altered floral odors may enable pollinators to maintain communication with their co-evolutionary partners and reduce the negative impacts that anthropogenically elevated oxidants may have on plant-pollinator systems.

Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development.

The term programmed cell death (PCD) was coined in 1965 to describe the loss of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. While it was subsequently demonstrated that this hormonally controlled death requires de novo gene expression, the signal transduction pathway that couples hormone action to cell death is largely unknown. Using the ISMs from the tobacco hawkmoth Manduca sexta, we have found that Acheron/LARP6 mRNA is induced ∼1,000-fold on the day the muscles become committed to die. Acheron functions as a survival protein that protects cells until cell death is initiated at eclosion (emergence), at which point it becomes phosphorylated and degraded in response to the peptide Eclosion Hormone (EH). Acheron binds to a novel BH3-only protein that we have named BBH1 (BAD/BNIP3 homology 1). BBH1 accumulates on the day the ISMs become committed to die and is presumably liberated when Acheron is degraded. This is correlated with the release and rapid degradation of cytochrome c and the subsequent demise of the cell. RNAi experiments in the fruit fly Drosophila confirmed that loss of Acheron results in precocious ecdysial muscle death while targeting BBH1 prevents death altogether. Acheron is highly expressed in neurons and muscles in humans and drives metastatic processes in some cancers, suggesting that it may represent a novel survival protein that protects terminally differentiated cells and some cancers from death.

The myonuclear domain is not maintained in skeletal muscle during either atrophy or programmed cell death.

Skeletal muscle mass can increase during hypertrophy or decline dramatically in response to normal or pathological signals that trigger atrophy. Many reports have documented that the number of nuclei within these cells is also plastic. It has been proposed that a yet-to-be-defined regulatory mechanism functions to maintain a relatively stable relationship between the cytoplasmic volume and nuclear number within the cell, a phenomenon known as the "myonuclear domain" hypothesis. While it is accepted that hypertrophy is typically associated with the addition of new nuclei to the muscle fiber from stem cells such as satellite cells, the loss of myonuclei during atrophy has been controversial. The intersegmental muscles from the tobacco hawkmoth Manduca sexta are composed of giant syncytial cells that undergo sequential developmental programs of atrophy and programmed cell death at the end of metamorphosis. Since the intersegmental muscles lack satellite cells or regenerative capacity, the tissue is not "contaminated" by these nonmuscle nuclei. Consequently, we monitored muscle mass, cross-sectional area, nuclear number, and cellular DNA content during atrophy and the early phases of cell death. Despite a ∼75-80% decline in muscle mass and cross-sectional area during the period under investigation, there were no reductions in nuclear number or DNA content, and the myonuclear domain was reduced by ∼85%. These data suggest that the myonuclear domain is not an intrinsic property of skeletal muscle and that nuclei persist through atrophy and programmed cell death.

Orientation-Dependent Changes in Single Motor Neuron Activity during Adaptive Soft-Bodied Locomotion

Recent major advances in understanding the organizational principles underlying motor control have focused on a small number of animal species with stiff articulated skeletons. These model systems have the advantage of easily quantifiable mechanics, but the neural codes underlying different movements are difficult to characterize because they typically involve a large population of neurons controlling each muscle. As a result, studying how neural codes drive adaptive changes in behavior is extremely challenging. This problem is highly simplified in the tobacco hawkmoth Manduca sexta, which, in its larval stage (caterpillar), is predominantly soft-bodied. Since each M. sexta muscle is innervated by one, occasionally two, excitatory motor neurons, the electrical activity generated by each muscle can be mapped to individual motor neurons. In the present study, muscle activation patterns were converted into motor neuron frequency patterns by identifying single excitatory junction potentials within recorded electromyographic traces. This conversion was carried out with single motor neuron resolution thanks to the high signal selectivity of newly developed flexible microelectrode arrays, which were specifically designed to record from M. sexta muscles. It was discovered that the timing of motor neuron activity and gait kinematics depend on the orientation of the plane of motion during locomotion. We report that, during climbing, the motor neurons monitored in the present study shift their activity to correlate with movements in the animal's more anterior segments. This orientation-dependent shift in motor activity is in agreement with the expected shift in the propulsive forces required for climbing. Our results suggest that, contrary to what has been previously hypothesized, M.sexta uses central command timing for adaptive load compensation.

Scaling of midgut perimeter and amino acid transporter expression in Manduca sexta larvae

Metabolic rate of tobacco hawkmoth (Manduca sexta) larvae increases with body mass to the approximately 0.90 power, greater than the 0.67 and 0.75 scaling exponents predicted by isometric increase of surface area and fractal geometry of exchange networks. We evaluated whether increased midgut folding and/or nutrient transporter expression in larger larvae contribute to this discrepancy. In 3rd through 5th instar larvae, midgut wet mass scaled with an estimated exponent of 0.85. Exponents for anterior, middle, and posterior midgut were 0.96, 0.70 and 0.85, respectively. Composite micrographs of transverse sections revealed progressively increased folding of the midgut perimeter in 1st through 5th instar larvae. These results suggest that relative midgut surface area declines less than predicted by theoretical models as larvae grow. Also, expression of mRNA encoding a potassium amino acid transporter (KAAT1) increased 2 to 3‐fold in 5th instar compared to 4th instar larvae. To assess the consequences of altering the expression of KAAT1, we used RNA interference to decrease midgut KAAT1 expression. Delivery of a 453bp dsRNA encoding KAAT1 through direct injection into the hemolymph resulted in an approximately 6‐fold reduction in KAAT1 mRNA expression, while oral delivery in a sucrose droplet did not reduce KAAT1 expression. Knockdown of KAAT1 mRNA expression did not affect larval growth. (NSF‐DMS0827208)

Steroid hormone regulation of the voltage‐gated, calcium‐activated potassium channel expression in developing muscular and neural systems

A precise organization of gene expression is required for developing neural and muscular systems. Steroid hormones can control the expression of genes that are critical for development. In this study we test the hypothesis that the steroid hormone ecdysone regulates gene expression of the voltage-gated calcium-activated potassium ion channel, Slowpoke or KCNMA1. Late in adult development of the tobacco hawkmoth Manduca sexta, slowpoke (msslo) levels increased contributing to the maturation of the dorsal longitudinal flight muscles (DLMs) and CNS. We show that critical components of ecdysteroid gene regulation were present during upreglation of msslo in late adult DLM and CNS development. Ecdysteroid receptor complex heterodimeric partner proteins, the ecdysteroid receptor (EcR) and ultraspiracle (USP), and the ecdysone-induced early gene, msE75B, were expressed at key developmental time points, suggesting that ecdysteroids direct aspects of gene expression in the DLMs during these late developmental stages. We provide evidence that ecdysteroids suppress msslo transcription in the DLMs; when titers decline msslo transcript levels increase. These results are consistent with msslo being a downstream gene in an ecdysteroid-mediated gene cascade during DLM development. We also show that the ecdysteroids regulate msslo transcript levels in the developing CNS. These results will contribute to our understanding of how the spatiotemporal regulation of slowpoke transcription contributes to tailoring cell excitability to the differing physiological and behavioral demands during development.

Regulation of muscle differentiation and survival by Acheron

Acheron (Achn), a phylogenetically-conserved member of the Lupus antigen family of RNA binding proteins, was initially identified as a novel cell death-associated gene from the intersegmental muscles of the tobacco hawkmoth Manduca sexta. C 2C 12 cells are a standard model for the study of myogenesis. When deprived of growth factors, these cells can be induced to: form multinucleated myotubes, arrest as quiescent satellite-like reserve cells, or undergo apoptosis. Achn expression is induced in myoblasts that form myotubes and acts upstream of the muscle specific transcription factor MyoD. Forced expression of ectopic Achn resulted in the formation of larger myotubes and massive reserve cell death relative to controls. Conversely, dominant-negative or antisense Achn blocked myotube formation following loss of growth factors, suggesting that Achn plays an essential, permissive role in myogenesis. Studies in zebrafish embryos support this hypothesis. Reduction of Achn with antisense morpholinos led to muscle fiber loss and an increase in the number of surviving cells in the somites, while ectopic Achn enhanced muscle fiber formation and reduced cell numbers. These results display a crucial evolutionarily conserved role for Achn in myogenesis and suggest that it plays key roles in the processes of differentiation and self-renewal.

Differential control of cell death and gene expression during two distinct phases of hormonally-regulated muscle death in the tobacco hawkmoth Manduca sexta

In larvae of the tobacco hawkmoth Manduca sexta, the intersegmental muscles (ISMs) span eight abdominal segments and represent the major muscle group. Following pupation, the ISMs in the first two and last two segments undergo programmed cell death (PCD), while the remaining four segments persist until the time of adult eclosion, when they too undergo PCD. ISM death at adult eclosion is initiated by a decline in the circulating ecdysteroid titer and requires de novo gene expression. In this study we have investigated the hormonal regulation and the patterns of gene expression that accompany both early and late ISM death. We find that distinct endocrine cues regulate these two periods of muscle death. Even though the middle segments of ISMs are exposed to the same endocrine environment as the adjacent cells that die following pupation, they do not express death-associated transcripts until they are specifically signaled to die following adult eclosion. These data indicate that subsets of homologous muscles appear to make segment-specific decisions to couple their endogenous cell death programs to distinctly different developmental cues. Nevertheless, once cell death is initiated, they utilize many of the same molecular components.

Application of digital particle image velocimetry to insect aerodynamics: measurement of the leading-edge vortex and near wake of a Hawkmoth

Some insects use leading-edge vortices to generate high lift forces, as has been inferred from qualitative smoke visualisations of the flow around their wings. Here we present the first Digital Particle Image Velocimetry (DPIV) data and quantitative analysis of an insect’s leading-edge vortex and near wake at two flight speeds. This allows us to describe objectively 2D slices through the flow field of a tethered Tobacco Hawkmoth (Manduca sexta). The near-field vortex wake appears to braodly resemble elliptical vortex loops. The presence of a leading-edge vortex towards the end of the downstroke is found to coincide with peak upward force production measured by a six-component force–moment balance. The topology of Manduca’s leading-edge vortex differs from that previously described because late in the downstroke, the structure extends continuously from wingtip across the thorax to the other wingtip.

The aerodynamics ofManduca sexta: digital particle image velocimetry analysis of the leading-edge vortex

Here we present the first digital particle image velocimetry (DPIV) analysis of the flow field around the wings of an insect (the tobacco hawkmoth Manduca sexta, tethered to a 6-component force-moment balance in a wind tunnel). A leading-edge vortex (LEV) is present above the wings towards the end of the downstroke, as the net upward force peaks. Our DPIV analyses and smoke visualisations match the results of previous flow visualisation experiments at midwing, and we extend the experiments to provide the first analysis of the flow field above the thorax. Detailed DPIV measurements show that towards the end of the downstroke, the LEV structure is consistent with that recently reported in free-flying butterflies and dragonflies: the LEV is continuous across the thorax and runs along each wing to the wingtip, where it inflects to form the wingtip trailing vortices. The LEV core is 2-3 mm in diameter (approximately 10% of local wing chord) both at the midwing position and over the centreline at 1.2 m s(-1) and at 3.5 m s(-1) flight speeds. At 1.2 m s(-1) the measured LEV circulation is 0.012+/-0.001 m(2) s(-1) (mean +/-S.D.) at the centreline and 0.011+/-0.001 m(2) s(-1) halfway along the wing. At 3.5 m s(-1) LEV circulation is 0.011+/-0.001 m(2) s(-1) at the centreline and 0.020+/-0.004 m(2) s(-1) at midwing. The DPIV measurements suggest that if there is any spanwise flow in the LEV towards the end of the downstroke its velocity is less than 1 m s(-1). Estimates of force production show that the LEV contributes significantly to supporting body weight during bouts of flight at both speeds (more than 10% of body weight at 1.2 m s(-1) and 35-65% of body weight at 3.5 m s(-1)).

Remodeling of an identified motoneuron during metamorphosis: Hormonal influences on the growth of dendrites and axon terminals

During metamorphosis of the tobacco hawkmoth Manduca sexta, the femoral depressor motoneuron (FeDe MN) undergoes remodeling of its dendrites and motor terminals. Previous studies have established that remodeling of MNs during metamorphosis is mediated by the same hormones that control metamorphosis: the ecdysteroids and juvenile hormone (JH). During the pupal stage, the ecdysteroids promote adult-specific growth of MNs in the absence of JH, but JH or its synthetic mimics can interfere with ecdysteroid-mediated growth if applied during early sensitive periods. Hence, the application of a JH mimic (JHM) either systemically or locally to a target muscle has been used to distinguish those aspects of motor-terminal remodeling that are controlled by ecdysteroid action on the CNS from those that are influenced by ecdysteroid action on the peripheral targets. Here, we have extended the analysis of central and peripheral hormonal influences on MN remodeling by injecting JHM locally into the CNS thus altering the hormonal environment of the FeDe MN soma without altering the hormonal environment of its target muscle. Our results demonstrate that adult dendritic growth and motor-terminal growth can be experimentally uncoupled, suggesting that each is regulated independently. JHM application to the CNS perturbed dendritic growth, but had no measurable impact on motor-terminal growth. Peripheral actions of ecdysteroids, therefore, appear sufficient to promote the development of adult-specific motor terminals but not the development of an adult-specific dendritic arbor.

Biosynthesis of sex pheromones in moths: stereochemistry of fatty alcohol oxidation in Manduca sexta

Six chiral deuterium labelled metabolic probes, ( R) and ( S)-enantiomers of [1,10,10- 2H 3]-hexadecan-1-ol, (11 E)-[1,10,10- 2H 3]-hexadec-11-en-1-ol and (11 Z)-[1,10,10- 2H 3]-hexadec-11-en-1-ol, were synthesized to examine the stereospecificity of the fatty alcohol oxidase from the female pheromone gland of the tobacco hawk moth ( Manduca sexta, Sphingidae). Both in vitro and in vivo oxidations were found to proceed by selective removal of the C1–H R hydrogen or deuterium atom ( Re-specificity) to yield the corresponding aldehydes. ( R) and ( S)-enantiomers of deuterium labelled salicyl alcohol and 2-thienyl-methanol, compounds entirely chemically diverse from the natural pheromone precursors, were also oxidised Re-specifically to salicylaldehyde and 2-thiophenecarb-aldehyde, respectively.

Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta.

The actions of various peptides and other compounds on fluid secretion by Malpighian tubules in the tobacco hawkmoth Manduca sexta sexta are investigated in this study. Using a newly developed pharate adult Malpighian tubule bioassay, we show that three tachykinin-related peptides (TRPs), leucokinin I, serotonin (5-HT), octopamine, the cardioacceleratory peptides 1a, 1b and 2c, cGMP and cAMP each cause an increase in the rate of fluid secretion in pharate adult tubules. Whereas the possible hormonal sources of biogenic amines and some of the peptides are known, the distribution of TRPs has not been investigated previously in M. sexta. Thus we performed immunocytochemistry using an anti-TRP antiserum. We show the presence of TRP-like material in a small subset of cells in the M. sexta central nervous system (CNS). The larval brain contains approximately 60 TRP-immunopositive cells and there are approximately 100 such cells in the adult brain including the optic lobes. Every ganglion of the ventral nerve cord also contains TRP-like immunoreactive cells. No TRP-containing neurosecretory cells were seen in the CNS, but endocrine cells of the midgut reacted with the antiserum. We propose the hypothesis that the control in insects of physiological systems by hormones may not always involve tissue-specific hormones that force stereotypical responses in their target systems. Instead, there may exist in the extracellular fluid a continuous broadcast of information in the form of a chemical language to which some or all parts of the body continuously respond on a moment-to-moment basis, and which ensures a more effective and efficient coordination of function than could be achieved otherwise.

Changes in contractile properties of skeletal muscle during developmentally programmed atrophy and death.

Skeletal muscle atrophy and death are protracted processes that accompany aging and pathological insults in mammals. The intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta are composed of giant fibers that undergo distinct hormonally-regulated programs of atrophy and death at the end of metamorphosis. Atrophy occurs during the 3 days preceding adult emergence and results in a 40% reduction of mass, whereas death takes place during the subsequent 30 h and results in the complete loss of the fibers. There are no significant changes in tetanic force or calcium sensitivity in skinned fiber preparations during atrophy. However, the size of caffeine-induced contractions fell by about 50%. With the onset of the death phase, dramatic reductions occur in ISM: tetanic force, twitch amplitude, resting potential, caffeine-induced contractions, calcium sensitivity, and Hill coefficients. Several lines of evidence suggest that ISM atrophy is caused by an increase in protein turnover without significant modification of fiber organization. In contrast, ISM death is accompanied by disorganization of the contractile apparatus and concomitant loss of contractile function.

A Member of the Phylogenetically Conserved CAD Family of Transcriptional Regulators Is Dramatically Up-Regulated during the Programmed Cell Death of Skeletal Muscle in the Tobacco HawkmothManduca sexta

The intersegmental muscles (ISMs) of the tobacco hawkmothManduca sextaparticipate in the emergence behavior of the adult moth at the end of metamorphosis and then die during the subsequent 30-hr period. The trigger for this death is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone (20-HE). Previous work has demonstrated that the ability of the ISMs to die is dependent on new gene expression. Using a differential hybridization cloning strategy, a cDNA library made from the ISMs committed to die was screened, and four up-regulated clones were isolated. One clone, 18-56, was selected for this study. Northern and Western analysis demonstrated that while clone 18-56 was expressed in all tissues examined and during every stage of ISM development, there was a dramatic increase in expression at both mRNA and protein levels when the ISMs became committed to die. If ISM death was delayed by an injection of 20-HE on the day proceeding adult emergence, 18-56 expression remained at basal levels. Immunocytochemistry demonstrated that 18-56 protein was located predominantly in nuclei prior to the commitment of the ISMs to die and then accumulated to high levels in cytoplasm at the time of cell death. DNA sequence analysis revealed that 18-56 protein shares 74% identity with yeast SUG1 and 92% with human Trip1, both of which are members of the conserved CAD (Conserved ATPase-containing Domain) family of putative transcriptional regulators. To verify that these genes shared functional as well as sequence homology,Manducaclone 18-56 was transformed into a yeast mutant for SUG1 function.Manduca18-56 was able to both complement the lethal SUG1 phenotype and to suppress the transcriptional activity of a SUG1 mutation in yeast. Taken together, these data support the hypothesis that members of the phylogenetically conserved CAD family participate in important basal and developmental processes.