Ultrastructure of the muscles of the dorsal diaphragm in Locusta migratoria

Summary The alary muscles of Locusta migratoria adults make up the major tissue of the dorsal diaphragm which separates pericardial and perivisceral sinuses in the abdomen. The alary muscles are striated with a sarcomere at rest measuring about 9 m. The Z-line has a staggered-beaded arrangement with A-bands and I-bands readily discernable. Thick myofilaments are surrounded by 10 or more thin filaments. The sarcoplasm has few mitochondria near the area of the Z-line, dyads are present and sarcoplasmic reticulum is poorly developed. Axons which innervate the alary muscle are either contained within invaginated folds of the sarcolemma of the muscle cells or the muscle cells send finger-like projections to envelop the axons. The synaptic terminals contain synaptic vesicles between 40 and 45 nm in diameter and a few electron-dense granules near or less than 170 nm in diameter. Away from synaptic terminals the axon profiles show few or no granules. The axons are accompanied everywhere by well-developed glial cells. This then is not typical neurosecretomotor innervation, however, the presence of electron-dense granules suggests the possibility of peptidergic neurotransmission.     

Size and number of nerve fibres in the central nervous system connectives of the cockroach Blaberus craniifer

The size and number of axons in the ventral cord connectives of the cockroach Blaberus craniifer were determined from montages constructed of electron micrographs of the left connective of each of the connective pairs examined. The fibres were grouped into three main diameter categories: fine fibres from 0·2 to 1 μm, small fibres from 1 to 6 μm, and large fibres from 6 to 24 μm. In the five different left connectives examined, the fine fibres numbered from 2006 to 8535 and composed from 56·5 to 83 per cent of the total fibres. The small fibres numbered from 1269 to 2361 and composed from 16·5 to 41 per cent of the total fibre number. The large axons ranged between 29 and 220 in number which represented from 0·5 to 2·5 per cent of the fibre population.     

Bithorax Complex genes control alary muscle patterning along the cardiac tube of Drosophila

Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. We found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles.     

Control of the alary muscles of locust dorsal diaphragm

ABSTRACT. The heartbeat in whole, intact, adult Locusta migratoria R.F. was characterized by a regular rate but apparently irregular amplitude. Cutting segmental nerves often eliminated apparent amplitude fluctuations, and electrically shocking a segmental nerve in the whole animal evoked apparent amplitude changes corresponding to the shocks. Saline-perfused tissue preparations showed that the apparent amplitude fluctuations could be duplicated by segmental nerve stimulation, and that the fluctuations were due largely to contractions of the alary muscles of the dorsal diaphragm which shifted the position of the heart chamber without a change in volume. The alary muscles are each multi-terminally innervated by one motor axon. Neurally-evoked postsynaptic potentials facilitated and summated, and the diaphragm muscles began visibly contracting at stimulation rates as low as 2 Hz. Stimulation at higher frequencies caused greater depolarization of the muscle fibres with no indication of electrically-excited responses. The alary muscles were insensitive to perfusion with acetylcholine, l -glutamate, l -aspartate, dopamine, octopamine, noradrenaline, proctolin, 5-hydroxytryptamine, or gamma aminobutyric-acid in saline at concentrations up to 10^-3M. Larval or adult brain extracts of Locusta at 10 μg/μl and diluted 1:5 in saline caused uniform contractions of the alary muscle preparation, while perfusion of skeletal muscle extracts produced no response.     

On the mechanisms of transfilter induction of kidney tubules.

Induction of tubule formation in metanephrogenic mesenchyme by spinal cord, occurs across 15 μm thick nucleopore filters with pores greater than 0·1 μm in diameter, but not when the pores are less than 0·05 μm. Processes from the inducer cells penetrate the 0·1 μm pores but not those of 0·05 μm diameter. These results are usually interpreted in favor of contact-mediated induction, and against diffusion-mediated induction.In this communication, we establish the feasibility of a novel and third possibility, namely thát induction is mediated by short-range diffusion of material from the inducer, and that the necessary proximity (say 10 nm) between the interactants is brought about by penetration of the filter pores by cell-processes. When the pores are less than 0·1 μm in diameter, penetration does not occur and the inducer and target are kept 15 μm apart. It is argued that this distance is too great for diffusion mechanisms to achieve a high enough concentration of the inductive message at the target surface.     

Cardiac reflexes and their neural pathways in lepidopterous insects

Previously described salines for lepidoptera did not maintain a constant heart rate for a very long. We have been successful in maintaining a normal heartbeat for many hours in a newly designed saline. This saline was also suitable for maintaining normal neuromuscular junctional potentials. The cardiac reflexes studied in larvae of Bombyx and Agrius were five types of cardiac responses induced by mechanical stimuli to sensillar setae. The cardiac responses were caused by electrical stimulation of nerves in the reflex pathways. The antidromic heartbeat was triggered even in larvae before the 5th instar by stimulation of axons in the visceral nerve arising from the frontal ganglion and terminating at the aorta, while spontaneous heartbeat reversal started to occur in wandering larvae. Other axons in the visceral nerve terminate at the rear end of the heart. Electrical stimuli to the nerves caused cardiac inhibition of the orthodromic heartbeat. Nerves extending from the visceral nerve to the alary muscles of the 2nd abdominal segment contain axons to increase the tone of the muscles. Nerves extending from the 7th abdominal ganglion to the most posterior alary muscles also contain axons to increase the tone of the muscles, and were responsible for acceleration of the antidromic and orthodromic heartbeat, respectively.     

Fine structure and innervation of the honey stomach (crop) of the honeybee,Apis mellifera L. (Hymenoptera : Apidae)

The present paper describes the ultrastructure and innervation of the honey stomach (crop) of Apis mellifera (Hymenoptera : Apidae). The crop is innervated by 10–20 μm-thick nerves with numerous axons, running longitudinally, and small nerves (1–3 μm, often containing only one distinct axon), running latitudinally. Crop muscles are striated, often branched and have only few multidirectionally arranged myofilaments; they seem to be of the slow-contracting type. Longitudinal and circular muscles produce projections that connect synaptically to small nerves. At these neuromuscular junctions, a high density of synaptic vesicles is found in the axons. A small number of apparently multipolar neurones, stained with Procion Yellow, are irregularly distributed over the crop. The possibility that these neurones work as stretch receptor organs is discussed.     

Movement of a non-flagellate spermatozoon: A study of the male gamete of Nematospiroides dubius (Nematoda)

The spermatozoa of Nematospiroides dubius were studied using the scanning electron microscope and time-lapse cinematography. Spermatozoa undergo a profound change in morphology after insemination: they change from an elongate structure, 16–18 μm long, to a more rounded form about 5–10 μm in diameter. Spermatozoa from female worms stuck to, and migrated across a glass surface by the production of pseudopodia, but they adhered more readily to a glass surface coated with egg albumin. The average speed of a sample of six differentiated spermatozoa was 7·3 μm/min. Their locomotion is not considered to be amoeboid but resembles the movement of monopodial neutrophils. A hypothesis for the mechanism of movement is presented, and other possible functions of the pseudopodial region are discussed.     

Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus

The dorsal vessel (DV) in the abdomen of the blood-feeding insect Rhodnius prolixus was divided functionally into two regions, the heart, into which haemolymph entered the DV through four pairs of ostia located in abdominal segment VII, and the aorta, along which the haemolymph was propelled from abdominal segment VI to the thorax. Osmium-fixed whole mounts revealed the DV to consist of spirally arranged striated muscle fibers and to possess two rows of ventrally attached longitudinal fibers extending the length of the abdomen. Seven pairs of alary muscles were found attached to the DV in the posterior abdominal segments. Contractions of the alary muscles attached to the ventral surface of abdominal segments VII and VIII served to expand the heart. Electron microscopy revealed the DV to consist of a thin layer of contractile elements surrounded by an inner (intima) and outer (adventitia) connective tissue layer. Embedded in the intima along each lateral side of the DV were two large groups of endocardial cells extending the length of the DV. A small group of pericardial cells was embedded in the adventitia along the mid-ventral side of the DV, and clusters of pericardial cells were found attached to the alary muscles. Nerve terminals were found only on the heart: they contained agranular synaptic vesicles approximately 30 nm in diameter and densely stained granules approximately 100-120 nm in diameter. These structural components are discussed in relation to the role of the DV in circulation.     

Innervation of heart and alary muscles in Sphinx ligustri L. (Lepidoptera)

Summary The origin and orientation of the heart nerves in Sphinx ligustri and Ephestia kuehniella were investigated by scanning electron microscopy using a special technique which involved pinning the dissected specimens on a stabilizing metal pad. The heart and alary muscles in Sphinx particularly their caudal extremity were also examined by transmission electron microscopy. The alary muscles form an incomplete sheath around the heart with a mainly longitudinal fibre orientation, e.i. antagonistically to the fibres of the heart itself. The heart and alary muscles are multiterminally innervated by branches of the transverse segmental nerves. All branches contain a single electron lucent axon; the thickest branches also possess several neurosecretory axons. Swellings of the segmental nerves may indicate the position of nerve cell bodies. There are no lateral heart nerves. Only one type of neuromuscular junction is abundant in the alary muscles but less frequently found in the heart. The terminals originate from the central axon only. They are capped by glial cells, which interdigitate with the muscle cells. They penetrate into the T-system toward the Z-discs and form a complex intercellular space system. Exocytosis of dense-cored vesicles into this perisynaptic reticulum seems likely. Sites of neurohaemal release are distributed along the nerve branches and special nerve endings occur at the level of the ostia. The possible nervous influence upon heart activity is discussed.The transmission electron microscopic part of this investigation was supported by a research scholarship from the Deutsche Forschungsgemeinschaft     

Novel peripheral motor neurons in the posterior tentacles of the snail responsible for local tentacle movements

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15–35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5–7 µm diameter) and light neurons with 12–18 (T1 type) and 18–30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus–response pathway, consisting of olfactory stimulus—local motor neuron—motor response components, to generate local lateral movements of the tentacle tip (“quiver”). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus—distal segments of central motor axons—motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.     

Temporal variability in egg production rates of Acartia tonsa Dana in Long Island Sound

The relationship between week-by-week variations in the in situ egg production rates of Acartia tonsa Dana and changes in chlorophyll concentration in several size fractions was investigated by incubating adult females in natural sea water for 24-h periods. Our results indicate that the egg production of A. tonsa in Long Island Sound was better related to the 10 μm chlorophyll size fraction than to the total chlorophyll concentration. The < 10 μm size fraction comprised the greatest percentage of the chlorophyll during July and August when the water column was stratified. Egg production rates were lowest (8.7 eggs · female⁻¹ · day⁻¹) in early August when less than 0.5 μg chlorophyll 1 ⁻¹ was observed in the 10 μm chlorophyll a size fraction. Following destratification in late August, the “fall” diatom bloom occurred and egg production rates increased to the maximum observed rate of 56.6 eggs · female⁻¹ · day⁻¹. At this time, the concentration of the 10μm chlorophyll size fraction was 5.5 μg 1⁻¹. Maximum egg production rates were observed at chlorophyll concentrations as low as 0.8 μg 1⁻¹ in the 10 μm size fraction.     

THE ISOLATION AND LIPID COMPOSITION OF MYELIN-FREE AXONS FROM RAT CNS1

—Myelin-free axons were isolated from rat CNS using a modification of the method of De Vries et al. (1972). On a dry weight basis, the axons contained 15·2% lipid composed of 19·4% cholesterol, 56·9% phospholipid and 23·7% galactolipid with a weight ratio of cerebroside to sulfatide of 3·6-1. The phospholipid was composed of 11·0% ethanolamine phosphatides (44·4% in the plasmalogen form), 21·0% choline phosphatides (9·3% in the plasmalogen form), 4·5% sphingomyelin, 4·5% phosphatidyl serine, 4·3% phosphatidyl inositol, 3·0% diphosphatidyl glycerol and 8·5% unidentified phospholipid. The rat axons contained 0·18 μg ganglioside NeuNAc/mg dry wt. In addition to the 4 major brain gangliosides, the rat axons contained gangliosides G_D2 and G_D3. The axonal galactolipid could not be accounted for by myelin contamination as revealed by electron microscopy, absence of the characteristic ratio of myelin specific proteins in the axonal protein profile as shown by polyacrylamide gel electrophoresis, and the axonal level of the myelin marker enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The relationship between lipids of axons isolated from rat and bovine CNS, and rat whole brain and CNS myelin is discussed.     

Membrane potential of the corpus cardiacum neurosecretory cells of the blowfly, Calliphora erythrocephala

The corpus cardiacum neurosecretory cells (c.n.c.) of Calliphora are unipolar cells with slender projections (axonal length: 50 to 110 μ; diameter: 0·25 to 1·75 μ).The cell body, where production of neurosecretory material occurs, is electrically inexcitable (resting potential about 36 mV, inside negative), whereas the axon—responsible for controlled neurohormone release—is excitable. Spike potentials with a duration of 3 to 7 msec occur in volleys the number and duration of which are supposed to determine the amount of secretion released. Electrical activity may be stimulated via the brain. Resting and action potentials are compared with recordings from other cell types of the blowfly.     

Kinetics of microtubule formation after cold disaggregation of the mitotic apparatus

The rate of spindle-fiber reformation following cold treatment of the giant amoeba, Chaos carolinensis, has been determined and used to test a single growth point, subunit incorporation model of microtubule assembly. Mitotic apparatuses isolated at one-minute intervals after rewarming contain progressively longer spindle fibers; re-assembly begins at the kinetochore region, proceeds at a rate of 1·5 μm per minute, then slows as the normal length of 5 μm is approached. From information on microtubule ultrastructure, the total number of 40-Å subunits in mitotic apparatuses per amoeba, and hence the concentration released during disassembly, was calculated to be 1·0 × 10¹⁵ molecules per cm³. Calculation of diffusion and assembly kinetics indicates that this concentration of microtubule subunits is equal to the concentration required to produce a growth rate of 1·5 μm per minute by diffusion of single subunits to one assembly point per microtubule.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Tip growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis> upon resource shortage: Disturbances of the coordination of elongation,branching, and septation

The characteristics of elongation, branching, septation, and nuclear morphology in hyphal tips (of ~400 μm in length) of the mycelial fungus Neurospora crassa isolated from the mycelium and cultivated for several hours have been investigated using intracellular fluorescent markers. The newly formed branches had the following characteristic features: (1) the predefined orientation was conserved, whereas the diameter decreased (from 10–20 to 6.5 ± 0.4 μm), as did the elongation rate (from 24 ± 1 to 6.7 ± 0.5 μm/min); (2) a disturbed branching pattern with abnormally large internodal distances (up to 1471 μm) and developmental arrest of part of the buds of lateral branches; and (3) a conserved septation pattern and a relatively constant length of hyphal segments (68 ± 2 μm). The size of the nucleus-free zone at the tip (5–33 μm) and the distance between the first septum and the growth point (210 ± 15 μm) in the daughter branches of the isolated fragments were almost the same as in hyphae connected to the mycelium, whereas the average distance between the growth point and the first lateral branch (492 ± 127 μm) and the variability of this parameter were higher in the isolated fragments. The morphology of the nuclei and the size of the nucleus-free zone near the growth point did not differ from those reported for normal vegetative hyphae of N. crassa. The experimental model developed may be used for the elucidation of details of molecular genetic mechanisms that underlie the regulation of interactions between the intracellular structures that provide tip growth of the hyphae in N. crassa.     

Neuroendocrine plexus in the pericardial septum of the stable fly,stomoxys calcitrans (L.) (diptera : muscidae)

An ultrastructural study of the pericardial septum (dorsal diaphragm) of the stable fly revealed that it consists of a central band of longitudinal muscle fibers upon which the paired alary muscles attach. A new neurohemal site was discovered that extends the length of the pericardial sinus. The neurosecretory axons terminating in this site originate from the segmental nerves. These nerves are also the source of axons that form 4 morphological types of neurosecretomotor junctions with the longitudinal muscle of the pericardial septum. Since the heart of the stable fly is myogenic, the neurohormones released into this septum may modulate the activity of the heart and/or be available for circulation to other sites of activity in this insect.     

Contractile properties of the isolated rat soleus muscle and its single skinned soleus fibers at the early stage of gravitational unloading: Facts and hypotheses

The contractile properties of the postural soleus muscle were studied in rats at the early stage of gravitational unloading (three-day hindlimb suspension) with regard to different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle and calcium-induced contraction of isolated skinned fibers). A significant (p < 0.01) enhancement of the peak twitch tension of the muscles of suspended rats without changes in time-dependent characteristics was observed, although the half-relaxation time tended to decrease. The fiber diameter did not change (42.37 ± 0.76 vs. 43.43 ± 1.15 μm in controls). The calcium-induced peak isometric tensions in control and unloaded soleus muscles were 37.6 ± 1.52 and 32.1 ± 1.05 mg, respectively (decrease significant at p < 0.05). No changes in threshold calcium concentration were recorded, but the pCa₅₀ value in unloaded muscles decreased from 6.05 ± 0.02 in controls to 5.97 ± 0.02 (p ≤ 0.05), indicating loss of myofibrillar calcium sensitivity. The cooperativity coefficient ηn in control animals was 3.46 ± 0.16, and in suspended ones it decreased to 3.08 ± 0.11 (p < 0.05). Analysis with the Fluo-4AM calcium probe demonstrated that the intracellular Ca²⁺ concentration increased significantly after hindlimb suspension, whereas the relative contents of titin or nebulin did not change.