Flight duration and flight muscle ultrastructure of unfed hawk moths

Flight muscle breakdown has been reported for many orders of insects, but the basis of this breakdown in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes across their lifespans, yet how this change affects the ability of these insects to complete their life cycles is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (Manduca sexta). Flight duration was examined in young, middle-aged, and advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorsolongitudinal muscle) was collected and histologically prepared for transmission electron microscopy to compare several measurements of muscle ultrastructure among moths of different ages. Muscle function assays revealed significant positive correlations between muscle ultrastructure and flight distance that were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more complete understanding of the ecophysiology and resource allocation strategies of this species.     

Flight muscles polymorphism in a flightless bug, Pyrrhocoris apterus (L.): developmental pattern, biochemical profile and endocrine control

The flightless bug Pyrrhocoris apterus (L.) is polymorphic for both wing length and flight muscle development. The developed flight muscles of macropterous adults of both sexes first enlarge their volume during the first 5 days after adult emergence, but are then histolyzed in all males and females older than 10 and 14 days, respectively. The flight muscles of brachypterous adult males and females are underdeveloped due to their arrested growth. The total protein content of histolyzed dorsolongitudinal flight muscles from 21-day-old macropterous adults of both sexes is lower than that of developed dorsolongitudinal flight muscles in 5-10-days-old macropterous bugs, but substantially higher than the protein content of underdeveloped dorsolongitudinal flight muscles from adult brachypters. Histolyzed dorsolongitudinal flight muscles differ from the developed ones by decreased quantities of 18 electrophoretically separated proteins. Histolysis of developed dorsolongitudinal flight muscles is accompanied by significant decreases in citrate synthase, glyceraldehyde-3-phosphate dehydrogenase and β-hydroxyacyl-CoA dehydrogenase enzyme activities and an increase in alanine aminotransferase activity, and can be precociously induced by application of a juvenile hormone analogue. This is the first report of flight muscle polymorphism, histolysis of developed flight muscles and its endocrine control in insects displaying non-functional wing polymorphism.     

The developmental profile of paramyosin and other myofibrillar proteins of larval and imaginal thoracic muscles of Lepidoptera

The synthesis of paramyosin and other myofibrillar proteins of the thoracic muscles of the tobacco hornworm Manduca sexta was studied by immunological and electrophoretical methods during the histolysis of the larval thoracic muscles and the differentiation of the indirect flight muscles. Antigens of the myofibrillar proteins in the thoracic muscles of the last-larval stage cross reacted with those in the flight muscles of the adults against polyspecific antibodies from actomyosin and monospecific antibodies from paramyosin. After the breakdown of the larval thoracic muscles (2 days from larval-pupal ecdysis) these antigens can no longer be detected in the thorax. The results indicate an almost complete removal of the larval thoracic muscles. Paramyosin could be identified again in a homogenate of the thoracic muscles of animals on the 13th day from larval-pupal ecdysis. Paramyosin is the first protein found during the differentiation of the flight muscles. The other myofibrillar proteins could be identified in thoracic homogenates of pharate adults of Manduca sexta on the 14th and 15th day from larval-pupal ecdysis. On the 14th day from larval-pupal ecdysis the dorso-longitudinal muscle and the tergosternal muscles show cross-striation, and the appearance of most of the electrophoretical results are in accordance with immunological and morphological findings. The myofibrillar proteins of the indirect flight muscles of Manduca sexta are synthesized de novo during metamorphosis.     

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.     

A comparative study of motor patterns during pre-flight warm-up in hawkmoths

Summary Temporal patterns of activation of flight muscles were recorded by means of wires placed extracellularly in thoracic muscles. In the five species of hawkmoths studied, wingstrokes of small amplitude were produced during a preflight warm-up by synchronous contractions of certain groups of muscles which are antagonists in flight. The main depressor muscle, the dorsal longitudinal, was excited in synchrony with some or all of the indirect elevator muscles. Three direct muscles, the subalar, basalar and third axillary muscles, were usually excited out of phase with the dorsal longitudinal muscle. However, details of the motor pattern varied from species to species. During fixed flight phase changes comparable in magnitude to those which occur during the transition from warm-up to flight were observed in Manduca sexta and Smerinthus cerisyi. The results (summarized in Table 2) suggest that a variety of warm-up patterns evolved within the Sphingidae as modifications of a common mechanism generating flight motor patterns.I thank Dr. Harry Lange for assistance in the initial collecting of Manduca sexta and for identifying specimens of this species.     

Antagonistic effects of floral scent in an insect–plant interaction

In southwestern USA, the jimsonweed Datura wrightii and the nocturnal moth Manduca sexta form a pollinator–plant and herbivore–plant association. Because the floral scent is probably important in mediating this interaction, we investigated the floral volatiles that might attract M. sexta for feeding and oviposition. We found that flower volatiles increase oviposition and include small amounts of both enantiomers of linalool, a common component of the scent of hawkmoth-pollinated flowers. Because (+)-linalool is processed in a female-specific glomerulus in the primary olfactory centre of M. sexta, we hypothesized that the enantiomers of linalool differentially modulate feeding and oviposition. Using a synthetic mixture that mimics the D. wrightii floral scent, we found that the presence of linalool was not necessary to evoke feeding and that mixtures containing (+)- and/or (−)-linalool were equally effective in mediating this behaviour. By contrast, females oviposited more on plants emitting (+)-linalool (alone or in mixtures) over control plants, while plants emitting (−)-linalool (alone or in mixtures) were less preferred than control plants. Together with our previous investigations, these results show that linalool has differential effects in feeding and oviposition through two neural pathways: one that is sexually isomorphic and non-enantioselective, and another that is female-specific and enantioselective.     

Myofilament proteins in the synchronous flight muscles of Manduca sexta show both similarities and differences to Drosophila melanogaster

Insect flight muscles have been classified as either synchronous or asynchronous based on the coupling between excitation and contraction. In the moth Manduca sexta, the flight muscles are synchronous and do not display stretch activation, which is a property of asynchronous muscles. We annotated the M. sexta genes encoding the major myofibrillar proteins and analyzed their isoform pattern and expression. Comparison with the homologous genes in Drosophila melanogaster indicates both difference and similarities. For proteins such as myosin heavy chain, tropomyosin, and troponin I the availability and number of potential variants generated by alternative spicing is mostly conserved between the two insects. The exon usage associated with flight muscles indicates that some exon sets are similarly used in the two insects, whereas others diverge. For actin the number of individual genes is different and there is no evidence for a flight muscle specific isoform. In contrast for troponin C, the number of genes is similar, as well as the isoform composition in flight muscles despite the different calcium regulation. Both troponin I and tropomyosin can include COOH-terminal hydrophobic extensions similar to tropomyosinH and troponinH found in D. melanogaster and the honeybee respectively.     

Abdicating power for control: a precision timing strategy to modulate function of flight power muscles

Muscles driving rhythmic locomotion typically show strong dependence of power on the timing or phase of activation. This is particularly true in insects' main flight muscles, canonical examples of muscles thought to have a dedicated power function. However, in the moth (Manduca sexta), these muscles normally activate at a phase where the instantaneous slope of the power-phase curve is steep and well below maximum power. We provide four lines of evidence demonstrating that, contrary to the current paradigm, the moth's nervous system establishes significant control authority in these muscles through precise timing modulation: (i) left-right pairs of flight muscles normally fire precisely, within 0.5-0.6 ms of each other; (ii) during a yawing optomotor response, left-right muscle timing differences shift throughout a wider 8 ms timing window, enabling at least a 50 per cent left-right power differential; (iii) timing differences correlate with turning torque; and (iv) the downstroke power muscles alone causally account for 47 per cent of turning torque. To establish (iv), we altered muscle activation during intact behaviour by stimulating individual muscle potentials to impose left-right timing differences. Because many organisms also have muscles operating with high power-phase gains (Δ(power)/Δ(phase)), this motor control strategy may be ubiquitous in locomotor systems.     

Thermal biology of flight in a butterfly: genotype,flight metabolism,and environmental conditions

Knowledge of the effects of thermal conditions on animal movement and dispersal is necessary for a mechanistic understanding of the consequences of climate change and habitat fragmentation. In particular, the flight of ectothermic insects such as small butterflies is greatly influenced by ambient temperature. Here, variation in body temperature during flight is investigated in an ecological model species, the Glanville fritillary butterfly (Melitaea cinxia). Attention is paid on the effects of flight metabolism, genotypes at candidate loci, and environmental conditions. Measurements were made under a natural range of conditions using infrared thermal imaging. Heating of flight muscles by flight metabolism has been presumed to be negligible in small butterflies. However, the results demonstrate that Glanville fritillary males with high flight metabolic rate maintain elevated body temperature better during flight than males with a low rate of flight metabolism. This effect is likely to have a significant influence on the dispersal performance and fitness of butterflies and demonstrates the possible importance of intraspecific physiological variation on dispersal in other similar ectothermic insects. The results also suggest that individuals having an advantage in low ambient temperatures can be susceptible to overheating at high temperatures. Further, tolerance of high temperatures may be important for flight performance, as indicated by an association of heat‐shock protein (Hsp70) genotype with flight metabolic rate and body temperature at takeoff. The dynamics of body temperature at flight and factors affecting it also differed significantly between female and male butterflies, indicating that thermal dynamics are governed by different mechanisms in the two sexes. This study contributes to knowledge about factors affecting intraspecific variation in dispersal‐related thermal performance in butterflies and other insects. Such information is needed for predictive models of the evolution of dispersal in the face of habitat fragmentation and climate change.     

Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix ??-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix ??-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene.We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.     

Drosophila neural pathways

Summary TheDrosophila giant axon pathways cervical connective — thoracic indirect flight muscles were studied by a combined electrophysiological and genetic analysis. A functional coupling of the left and right giant axon pathways was revealed by intracellular recordings of electrical responses of the thoracic indirect flight muscles, when evoked by electrical stimulation of cervical connective (Fig. 2). This functional coupling was demonstrated in wild-type flies and in flies of the single gene, temperature-sensitive paralytic mutation,para ^ts . The functional coupling was evident also in selected bilateral gynandromorph flies, mosaics for thepara ^ts mutation (Fig. 1), even at restricted elevated ambient temperature (Tables 1–3). Analysis of neurally evoked electrogenic muscle responses of wild-type flies, following injection of picrotoxin, verifies the notion that both the dorsoventral and the dorsolongitudinal flight muscles share a common activating pathway (Fig. 3). Picrotoxin application to gynandromorph flies demonstrated the existence of neuronal elements additional to the giant axon pathways, that evoke the indirect flight muscles in response to cervical stimulation (Figs. 4, 5). An unexpected finding was the poor correlation between the mosaic external phenotype of the gynandromorph flies ofpara ^ts mutation and the genotype of neural pathways activating their thoracic flight muscles, as evidenced by the intracellular recordings.Abbreviations GA giant axon - DVM dorsoventral muscle - DLM dorsolongitudinal muscle - PSI peripherally synapsing interneuron - ts temperature sensitive     

THE FINE STRUCTURE OF THE VENTRAL INTERSEGMENTAL ABDOMINAL MUSCLES OF THE INSECT RHODNIUS PROLIXUS DURING THE MOLTING CYCLE : I. Muscle Structure at Molting

Rhodnius prolixus, a South American insect, molts five times in its development to an adult after emerging from the egg. Each molting cycle is triggered with a blood-meal. The ventral intersegmental abdominal muscles of Rhodnius develop during each molting cycle and are functional at molting. The fine structure of these fully developed muscles from fourth stage larval insects is studied. They have the characteristic structure of slow muscles. They have multiple motor nerve endings, and the myofibrils are poorly defined in cross-section. Longitudinal sections show long sarcomeres (8–10 µ), irregular Z-lines, and no apparent H zones. No M line is seen. Transverse sections through the A-band region show that each hexagonally arranged thick filament is surrounded by 12 thin filaments. Two thin filaments are shared by two neighboring thick filaments. The ratio of thin to thick filaments is 6:1. This structure is related to that found in vertebrate skeletal muscle and insect flight muscle.     

Dual Dimensionality Reduction Reveals Independent Encoding of Motor Features in a Muscle Synergy for Insect Flight Control

What are the features of movement encoded by changing motor commands? Do motor commands encode movement independently or can they be represented in a reduced set of signals (i.e. synergies)? Motor encoding poses a computational and practical challenge because many muscles typically drive movement, and simultaneous electrophysiology recordings of all motor commands are typically not available. Moreover, during a single locomotor period (a stride or wingstroke) the variation in movement may have high dimensionality, even if only a few discrete signals activate the muscles. Here, we apply the method of partial least squares (PLS) to extract the encoded features of movement based on the cross-covariance of motor signals and movement. PLS simultaneously decomposes both datasets and identifies only the variation in movement that relates to the specific muscles of interest. We use this approach to explore how the main downstroke flight muscles of an insect, the hawkmoth Manduca sexta, encode torque during yaw turns. We simultaneously record muscle activity and turning torque in tethered flying moths experiencing wide-field visual stimuli. We ask whether this pair of muscles acts as a muscle synergy (a single linear combination of activity) consistent with their hypothesized function of producing a left-right power differential. Alternatively, each muscle might individually encode variation in movement. We show that PLS feature analysis produces an efficient reduction of dimensionality in torque variation within a wingstroke. At first, the two muscles appear to behave as a synergy when we consider only their wingstroke-averaged torque. However, when we consider the PLS features, the muscles reveal independent encoding of torque. Using these features we can predictably reconstruct the variation in torque corresponding to changes in muscle activation. PLS-based feature analysis provides a general two-sided dimensionality reduction that reveals encoding in high dimensional sensory or motor transformations.     

Influence of interganglionic interactions on steroid-mediated dendritic reorganization and death of proleg motor neurons during metamorphosis in Manduca sexta

In Manduca sexta, the larval abdominal prolegs and their muscles degenerate at pupation. The proleg motor neurons undergo a period of dendritic regression, after which a specific subset of them dies. The surviving motor neurons undergo dendritic outgrowth during pupal-adult development, and most die after adult emergence. All of these events are regulated hormonally by ecdysteroids and juvenile hormone, but interactions of the motor neurons with other cells may potentially contribute as well. To investigate the possible influence of interganglionic neural interactions, we chronically isolated individual abdominal ganglia by severing the adjacent rostral and caudal connectives in the larval stage. Subsequent metamorphic changes in proleg motor neurons were examined in the isolated ganglia and ganglia adjacent to the isolated ganglia. Two abnormalities were observed: (1) some imprecision in the timing of motor neuron death, both at pupation and after adult emergence, and (2) the growth of ectopic neurites outside the neuropil boundaries during pupal-adult development (in ganglia with or without neuromas caused by connective transections). Other aspects of proleg motor neuron metamorphosis, including the segment-specific death of motor neurons at pupation, were the same as that in intact and sham-operated insects. Thus, interganglionic interactions appear to play a relatively minor role in the steroid-mediated metamorphic transformation of proleg motor neurons. © 1994 John Wiley & Sons, Inc.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

The activity of pleurodorsal muscles during flight and at rest in the moth Manduca sexta (L.)

In the moth Manduca sexta, the paired mesothoracic flight steering muscle II PD2m takes part in the generation of the flight rhythm and is spontaneously active in the non-flying animal. This spontaneous activity is modulated by optomotor stimuli and directionally selective. The directional response characteristics are analyzed. Another spontaneously active steering muscle pair, the III PD2c, is situated in the metathorax. The activities of this pair and of a third muscle pair, the III PD3 are also influenced by visual stimulation.The responses of all 6 muscles to optomotor stimuli which simulate the flight situations yaw, roll, thrust and lift are analyzed. Each situation elicits a unique pattern of activation/deactivation within this set of muscles. The activity pattern in non-flying animals allows the prediction of flight steering mechanisms such as changes of wing area in flight turns and provides a useful basis for the analysis of visuo-motor pathways.     

Flight muscle myofibrillogenesis in the pupal stage of Drosophila as examined by X-ray microdiffraction and conventional diffraction

In the asynchronous flight muscles of higher insects, the lattice planes of contractile filaments are strictly preserved along the length of each myofibril, making the myofibril a millimetre-long giant single multiprotein crystal. To examine how such highly ordered structures are formed, we recorded X-ray diffraction patterns of the developing flight muscles of Drosophila pupae at various developmental stages. To evaluate the extent of long-range myofilament lattice order, end-on myofibrillar microdiffraction patterns were recorded from isolated quick-frozen dorsal longitudinal flight muscle fibres. In addition, conventional whole-thorax diffraction patterns were recorded from live pupae to assess the extent of development of flight musculature. Weak hexagonal fluctuations of scattering intensity were observed in the end-on patterns as early as approximately 15 h after myoblast fusion, and in the following 30 h, clear hexagonally arranged reflection spots became a common feature. The result suggests that the framework of the giant single-crystal structure is established in an early phase of myofibrillogenesis. Combined with published electron microscopy results, a myofibril in fused asynchronous flight muscle fibres is likely to start as a framework with fixed lattice plane orientations and fixed sarcomere numbers, to which constituent proteins are added afterwards without altering this basic configuration.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.