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     

Sperm morphology and arrangement along the male reproductive tract of the butterfly Euptoieta hegesia (Insecta: Lepidoptera)

Summary

The present study was undertaken to describe the morphological and organizational modifications that occur in apyrene and eupyrene spermatozoa along the male adult reproductive tract of the butterfly, Euptoieta hegesia. Testis, vas deferens, vesicula seminalis and ductus ejaculatorius were studied by transmission electron microscopy. In the testis, both sperm types are organized into cysts; apyrene sperm are devoid of extracellular structures while eupyrene ones have lacinate and reticular appendages. In the testis basal region, both sperm pass through an epithelial barrier and lose their cystic envelope. The eupyrene morphological and organizational modifications are more drastic than the apyrene ones. From the vas deferens to the ductus ejaculatorius, apyrene sperm are dispersed in the lumen and acquire several concentric layers that are formed by the folding of their abundant cell membrane. The apyrene distribution observed here suggests that their functions include eupyrene transportation. Eupyrene sperm, however, remain aggregated along the tract. In the vas deferens, they are covered by a filamentous material that develops into a homogeneous matrix surrounding the spermatozoa coat in the vesicula seminalis and the ductus ejaculatorius. Eupyrene sperm undergo complex morphological changes that include the loss of lacinate appendages and the formation of a dense and heterogeneous extracellular coat. The formation of the matrix and the coat in eupyrene extratesticular sperm is related to the loss of lacinate appendages. These changes are in general extracellular and are probably important for sperm maturation.     

Ultrastructure of the heart of the sturgeon Acipenser stellatus (Chondrostei)

Summary The ultrastructure of atrial and ventricular myocardial cells from Acipenser stellatus is described. The cells of the atrium are more loosely connected than those of the ventricle. Cell contact is by simple intercalated discs and by desmosomes. The cells are flattened, with peripheral myofibrils and a central region of mitochondria and the nucleus. The sarcoplasmic reticulum consists of subsarcolemmal tubules, that frequently extend towards the central mitochondria. Dyads are small and positioned at any sarcomeric level. No T-tubules are present. Specific granules are restricted to the atrial cell, and are sometimes present within the SR tubules.     

Modulation of the enkephalinergic phenotype of rat sympathetic neurons by hormonal and transynaptic mechanisms

Most sympathetic neurons contain one or more neuropeptides in addition to catecholamines. Although the regulation of catecholamines has been studied extensively, comparatively little is known about the regulation of neuropeptides. Since glucocorticoids and preganglionic innervation regulate catecholaminergic properties in chromaffin cells, we examined the effects of these factors on a co-localized neuropeptide, leucine enkephalin (L-Enk), in adult rat sympathetic neurons in vivo. Lowered serum glucocorticoid levels as a consequence of bilateral adrenalectomy resulted in a reduction of ganglionic L-Enk content that was reversed by exposure of adrenalectomized animals to the synthetic glucocorticoid, dexamethasone. Surgical denervation of the SCG eliminated L-Enk-IR preganglionic fibers and caused a dramatic increase in the number of L-Enk-IR neurons. Inhibition of the enkephalinergic component of the preganglionic innervation by chronic exposure to the opiate receptor antagonist naloxone increases the number of L-Enk-IR cell bodies and total ganglionic L-Enk content. None of the experimental manipulations noticeably altered the number or distribution of NPY-IR neurons, suggesting that the effects of glucocorticoids and the innervation on ganglionic L-Enk levels were specific and not simply an alteration of the biosynthetic state of the cells. These results demonstrate that circulating glucocorticoids and the preganglionic innervation regulate L-Enk levels in sympathetic neurons. Since both glucocorticoid levels and preganglionic activity are known to be altered by stressful stimuli, acute regulation of sympathetic L-Enk levels may constitute an important component of the autonomic response to stress. © 1993 John Wiley & Sons, Inc.     

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide – they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione. Accepted: 14 August 1999     

Ultrastructure and distribution of somatostatin-like immunoreactive neurons and nerve fibres in the coeliac ganglion of cats

Summary Somatostatin-like immunoreactivity was localized in nerve cell bodies and nerve terminals in the cat coeliac ganglion. Two types of somatostatin-immunoreactive cell bodies were revealed, the first being large (diameter 35 m), numerous and weakly labelled, where—as the second was considerably smaller (diameter 10.4 m), sparsely distributed and heavily stained. The immunoreactive nerve terminals were in synaptic contact with many immunonegative large neurons and dendrites. However, in a few cases, somatostatin-immunoreactive nerve terminals could also be observed on the surface of lightly stained neurons. Transection of vagal or mesenteric nerve failed to affect the distribution or density of somatostatin-like immunoreactive nerve terminals. These results demonstrate the existence of a synaptic input to the principal neurons of the coeliac ganglion of the cat by somatostatin-containing nerve terminals and suggest that this peptide may act as a neuromodulator or neurotransmitter. It is proposed that somatostatin-positive neurons provide intrinsic projections to other somatostatin-positive and to somatostatin-negative neurons throughout the coeliac ganglion, thereby creating a complex interneuronal system.     

Molecular identification and immunohistochemical localization of atrial natriuretic peptide in the heart of the dromedary camel (Camelus dromedarius)

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.     

Interference of plant volatiles on pheromone receptor neurons of male Grapholita molesta (Lepidoptera: Tortricidae)

In moths, sex pheromone components are detected by pheromone-specific olfactory receptor neurons (ph-ORNs) housed in sensilla trichodea in the male antennae. In Grapholita molesta, ph-ORNs are highly sensitive and specific to the individual sex pheromone components, and thus help in the detection and discrimination of the unique conspecific pheromone blend. Plant odors interspersed with a sub-optimal pheromone dose are reported to increase male moth attraction. To determine if the behavioral synergism of pheromone and plant odors starts at the ph-ORN level, single sensillum recordings were performed on Z8-12:Ac and E8-12:Ac ph-ORNs (Z-ORNs and E-ORNs, respectively) stimulated with pheromone–plant volatile mixtures. First, biologically meaningful plant-volatile doses were determined by recording the response of plant-specific ORNs housed in sensilla auricillica and trichodea to several plant odorants. This exploration provided a first glance at plant ORNs in this species. Then, using these plant volatile doses, we found that the spontaneous activity of ph-ORNs was not affected by the stimulation with plant volatiles, but that a binary mixture of sex pheromone and plant odorants resulted in a small (about 15%), dose-independent, but statistically significant, reduction in the spike frequency of Z-ORNs with respect to stimulation with Z8-12:Ac alone. The response of E-ORNs to a combination of E8-12:Ac and plant volatiles was not different from E8-12:Ac alone. We argue that the small inhibition of Z-ORNs caused by physiologically realistic plant volatile doses is probably not fully responsible for the observed behavioral synergism of pheromone and plant odors.     

Identification of olfactory receptor neurons in Uraba lugens (Lepidoptera: Nolidae) and its implications for host range

Phytophagous insects detect volatile compounds produced by host and non-host plants, using species-specific sets of olfactory receptor neurons (ORNs). To investigate the relationship between the range of host plants and the profile of ORNs, single sensillum recordings were carried out to identify ORNs and corresponding active compounds in female Uraba lugens (Lepidoptera: Nolidae), an oligophagous eucalypt feeder. Based on the response profiles to 39 plant volatile compounds, 13 classes of sensilla containing 40 classes of ORNs were identified in female U. lugens. More than 95% (163 out of 171) of these sensilla contained 16 classes of ORNs with narrow response spectra, and 62.6% (107 out of 171) 18 classes of ORNs with broad response spectra. Among the specialized ORNs, seven classes of ORNs exhibited high specificity to 1,8-cineole, (±)-citronellal, myrcene, (±)-linalool and (E)-β-caryophyllene, major volatiles produced by eucalypts, while nine other classes of ORNs showed highly specialized responses to green leaf volatiles, germacrene D, (E)-β-farnesene and geranyl acetate that are not produced by most eucalypts. We hypothesize that female U. lugens can recognize their host plants by detecting key host volatile compounds, using a set of ORNs tuned to host volatiles, and discriminate them from non-host plants using another set of ORNs specialized for non-host volatiles. The ORNs with broad response spectra may enhance the discrimination between host and non-host plants by adding moderately selective sensitivity. Based on our finding, it is suggested that phytophagous insects use the combinational input from both host-specific and non-host specific ORNs for locating their host plants, and the electrophysiological characterization of ORN profiles would be useful in predicting the range of host plants in phytophagous insects.     

Morphology of the male reproductive system and sperm ultrastructure of Leucoptera coffeella (Lepidoptera: Lyonetiidae)

The male reproductive tract of Leucoptera coffeella was processed for light and transmission electron microscopy. In the testis, the eupyrene cells are arranged in individual cysts, while the apyrene cysts form aggregates, never observed in other Lepidoptera. Both cysts contain 128 spermatozoa, which differ from the typical pattern. In the seminal vesicle, both types of spermatozoa are dispersed in the lumen, also different from other Lepidoptera. The apyrene spermatozoa are similar to those observed for other Lepidoptera. They present an anterior region covered by a dense cap and the flagellum is composed of a 9 + 9 + 2 axoneme and two mitochondrial derivatives. The eupyrene spermatozoa, however, differ from the typical pattern for Lepidoptera. Their anterior region contains a nucleus, an acrosome and a peculiar arc of eight accessory microtubules connected to the plasma membrane by dense bridges. In the nucleus–flagellum region, the ninth accessory microtubule is assembled between both mitochondrial derivatives, to participate in the axoneme. The flagellum comprises a 9 + 9 + 2 axoneme and two mitochondrial derivatives with paracrystalline cores. External to the plasma membrane and close to the accessory microtubules, there are tufts of an amorphous material, suggesting reduced lacinate appendages, while the reticular ones are absent. The reduction of lacinate appendages and the absence of sperm bundles in the seminal vesicle support the concept that the appendages of other Lepidoptera could be associated with the eupyrene aggregations. The characters ‘number of spermatozoa per cyst’ and ‘absence of bundles’ should be considered plesiomorphic, supporting the position of this taxon in the base of the Ditrysia.     

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

Summary The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine--hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 m) than those with both DBH-LI and NPY-LI (median diameter 20 m). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.     

Oviposition preferences of a threatened butterfly Leptidea morsei (Lepidoptera: Pieridae) at the western border of its range

The oviposition preferences of Leptidea morsei at the western border of its range (SE Slovenia) were studied with the aim of integrating this information into species habitat management recommendations. The characteristics of egg-laying habitat were examined at the landscape, patch, plant and leaf levels. Because sightings of adults in the field were infrequent, intensive searching for previously laid eggs on Lathyrus niger plants was chosen as the survey method. The main oviposition habitats of L. morsei in Slovenia were found to be the forest edges and light stands of thermophilous oak, oak-hornbeam and mesic beech forests (landscape level). The egg-laying sites were L. niger plants growing individually or in small stands, and oriented mostly towards W, SW, and S. The oviposition plants were between 33 and 120 cm high, with those growing in half to full shade preferred. Eggs were deposited singly, mainly on the underside of leaflets of terminal leaves, located mostly on the top quarter of plants irrespective of their height and growth form. The most important factors affecting egg-placement are sun exposure and the size of L. niger stands at ovipositing sites (patch level), leaf exposure on the plant in vertical and horizontal directions (plant level), leaf age and the leaf parts where the risk of leaf and egg desiccation is lower (leaf level). Management strategies for the main egg-laying habitats of the species are proposed.     

Larval growth and allometry in the cabbage butterfly Pieris brassicae (Lepidoptera: Pieridae)

By adopting a longitudinal study design and through geometric morphometrics methods, we investigated individual and ontogenetic variation in size, shape and timing during larval development of the cabbage butterfly Pieris brassicae under laboratory conditions. We found that ontogenetic size progression departs modestly, but significantly, from growth at a constant rate and that size at hatching contributes considerably to determine the size of the individual at all subsequent stages. As for the shape, ontogenetic allometry is much more conspicuous than static allometry, the latter in many cases being close to isometry. Analysis of developmental timing revealed a stage of apparently more effective developmental control at stage 3, supported by both the relatively small variance in cumulative developmental time up to stage 3 and by the pattern of correlation between duration of single stages. While presenting detailed quantitative aspects of growth in P. brassicae, in particular with respect to individual variation, this study and the associated dataset can provide a basis for further explorations of the post‐embryonic development in this insect and contribute to the ongoing investigations on growth regulation and control in insects.     

Ultrastructure of the larval corpus allatum of Hyphantria cunea drury (Insecta,Lepidoptera)

Summary The corpora allata of the three last larval instars were studied in newly molted animals, at the beginning, middle, and end of the feeding period, and during the molt period. They were found to consist of uniform gland cells, whose ultrastructure changes in the course of the instars.In gland cells considered to be resting, the outer and inner nuclear membranes run in parallel without forming a dilated perinuclear space. Mitochondria are small, polymorphic, with an electron-dense matrix. The smooth endoplasmic reticulum (SER) appears as stacks of parallel cisternae near the nuclear envelope and in the rest of the cytoplasm, and as accumulations of twisted profiles. Occasionally, the SER takes the form of paracrystalline bodies. There are few small smooth-surfaced vesicles in the cytoplasm.In cells considered as active, a dilated perinuclear space occurs. The peripheral ends of profiles forming the SER are swollen, and numerous vesicles and vacuoles bud off from them to fill the cytoplasm. Mitochondria are large, with a more transparent matrix. The plasma membrane of gland cells located just beneath the connective tissue sheath forms numerous small invaginations.The corpora allata consist of resting cells during the molt periods. At the beginning of each instar, few active gland cells appear. In the middle of the second to last and the third to last instars, the bulk of the gland cells is active. At the end of these instars, there are both active and inactive cells. In the middle of the last instar, the gland cells are inactive or subactive, and at its end, all gland cells are completely inactive.     

Adaptive radiation and convergence in subdivisions of the butterfly genus Heliconius (Lepidoptera: Nymphalidae)

The process of adaptive radiation and convergence, usually regarded as a feature of macro-evolution, can be seen in the mimetic colour patterns of the butterflies within the confines of the South American genus Heliconius. This can be shown by dividing the genus into subgroups on the basis of adult, pupal and larval morphology: the theory that the mimicry between species results solely from close systematic relationships is thereby refuted, as members of the same morphological group can display widely divergent mimetic patterns, and conversely mutual mimics may belong to several different morphological groups. Various forms of parallel and convergent evolution are thought to account for the present pattern of mimicry, the process is known to start even before full speciation has taken place. A new subgenus (Neruda) is created to contain three atypical members of the genus.     

World travellers: phylogeny and biogeography of the butterfly genus Leptotes (Lepidoptera: Lycaenidae)

Leptotina butterflies (Lycaenidae, Polyommatiinae) are found mostly in tropical and subtropical areas around the globe, marginally penetrating into temperate regions. Here, we investigated phylogenetic and biogeographical relationships of most representatives of the subtribe, using both likelihood and Bayesian approaches. We also estimated the timing of their diversification. And lastly, we studied phylogeographic patterns of the most widespread species, Leptotes pirithous. DNA sequences from two mitochondrial (COI, COII) and two nuclear genes (wingless, Ef1α) were analysed for 13 species of the genus Leptotes Scudder and one species of the genus Cyclyrius Butler. Both genera together form a monophyletic clade, and Cyclyrius is rooted deep inside Leptotes. Therefore, we designate Cyclyrius to be a junior synonym of Leptotes. According to our study, the genus Leptotes originated between the late Eocene and early Oligocene (35–31 Ma). During the Miocene it dispersed to the rest of the southern hemisphere, with further speciation events within the Indo‐Australian region, and separate radiations in the Americas and the Afrotropics. Leptotes webbianus from the Canary Islands turned out to be sister to the American clade from which it split c. 12 Ma. Leptotes pirithous originated in Madagascar c. 4 Ma and invaded the whole of Africa and southern Europe, including numerous surrounding islands. Populations of L. pirithous from Mauritius and Madagascar turned out to represent a distinct species (Leptotes durrelli sp.n. ) and the same applies to the Australasian populations of Leptotes plinius (Leptotes lybas stat. rev. ). This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:20308930‐988B‐4327‐A35F‐CC983D46263B .     

Ultrastructural study of the postembryonic development of the neural lamella of Galleria mellonella L. (Lepidoptera)

Summary The neural lamella encapsulating the brain of the wax moth Galleria mellonella develops from a very thin (80–120 nm) layer in the first larval instar, resembling the basal lamina, to a thick (1–4 m) sheath composed of two zones in the seventh (last) instar. After its breakdown at the time of larval-pupal ecdysis the neural lamella is reconstructed in the pupa, 2–3 days before pupaladult ecdysis.The cells of the perineurium seem to be responsible for the formation of the neural lamella, both in the larva and pupa, even though its ultrastructure differs at these stages.This paper is dedicated to the memory of doc. dr hab. Andrzej B. Dutkowski who proposed this studyPreliminary results of these studies were presented at the XII-th Congress of the Polish Zoological Society, Pozna (Osiska, 1979)The authors previous surname was Dybowska. She wishes to express her gratitude to Prof. Aleksandra Przeecka for valuable assistance in the preparation of this paper. She is also greatly indebted to Joanna Koodziejczyk, M.Sc. for technical aid     

Physiological differences between two sugar-sensitive neurons in the galea and the maxillary palp of the spruce budworm larva Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

The L1 contact-chemoreceptor sensillum on the maxillary palp of the spruce budworm larva Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae) was examined electrophysiologically for its responses to stimulation by various pyranose and furanose sugars. The results were compared to those from previous work on the sugar-sensitive neuron of the LST sensillum on the galea. We show that the L1 contact-chemoreceptor sensillum contains one sugar-sensitive neuron with furanose but no pyranose sites. It has response characteristics that differ from those of the sugar-sensitive neuron in the LST. Behavioural 2-choice feeding experiments show that, even with both known sugar-sensitive neurons disabled, larvae can still discriminate between disks treated with either distilled water or alpha-D-glucose. We conclude that the epipharyngeal sensilla must thus also contain a sugar-sensitive neuron.