Ultrastructural and histochemical study of collagen fibres types I and III in the venom gland of Bothrops jararaca during secretory cycle

Fragments of snake (Bothrops jararaca) venom gland were analysed by light and transmission electron microscopy in order to characterize the changes in collagen fibres types I and III in the intertubular gland septa during the secretory cycle. The snakes were sacrificed at 45 days (unmilked group), 6 h, 4 and 8 days after manual extraction of the venom. The fragments were fixed, processed according to standard histologic technique for embedding in paraffin, and stained with haematoxylin-eosin and Gomori's trichrome and submitted to Gomori's silver impregnation technique and picrosirius-polarization method. For transmission electron microscopy the fragments were fixed and processed for embedding in Spurr's medium. At the 45th day (the gland at rest), when the secretory activity was at a minimum, the septa were narrow and filled with densely packed collagen fibrils. At 6 h, the septa were enlarged and exhibited wide spaces filled with finely granular Alcian Blue-positive material. Until the 8th day, the septa were narrower and the histologic aspect resembled that of the gland at rest. The results demonstrated structural modifications in the glandular septa according to the different periods of the secretory cycle. These modifications can be associated with the transformation in the secretory epithelium during the venom synthesis cycle.     

Venom gland of the ichneumonid Diadromus collaris： morphology, ultrastructure and age-related changes

Females of the solitary parasitoid Diadromus collaris （Insecta： Hymenoptera： Ichneumonidae） lay eggs in the pupae of Plutella xylostella （Lepidoptera： Plutellidae）, and the venom is synchronously injected into hosts. The venom apparatus consists of two glandular tubules terminating in a common reservoir, A ductule connects the reservoir with the sting apparatus, by which the reservoir content enters the latter. Secretory units line the two glandular tubules. All secretory cells belong to dermal gland type Ⅲ. Dermal gland cells in glandular tubules are more abundant and developed than those in the reservoir. There are extensive rough endoplasmic reticulum and electrondense vesicles, and the microvilli are well developed. By the cuticle-lined central funnel secretion products of secretory units reach the reservoir. Moreover, the secretory apparatus undergoes age-related changes. The secretory units in the venom gland are better developed and more vigorous 7 days after eclosion than those 1 day after eclosion; autolytic processes occur 15 days after eclosion, and the tissue of the reservoir is more prostrate 15 day after eclosion than those 1 day after eclosion. The ovipostion peak of this parasitoid, about 3-7 days after eclosion, corresponds with the period when the venom gland is highly developed in the life span of the wasp.     

Morphology and ultrastructure of a secretory region enclosed by the venom reservoir in social wasps (Insecta,Hymenoptera)

The morphology and ultrastructure of the convoluted gland inside the venom reservoir of four species of social Vespidae are described. The cells of the venom gland (including the convoluted gland) can be divided into six groups: (1) epithelial cells, (2) glandular cells with the end apparatus secreting into the tubule inside the convoluted gland (internal or embedded tubule), (3) a continuous arrangement of glandular cells with the end apparatus secreting directly into the venom reservoir, (4) glandular cells that are loosely dispersed along the tubule lumen between the free tubules and the embedded tubule of the convoluted gland, (5) secretory cells of the free tubules and (6) duct cells. One kind of secretory cell, hitherto unknown and described in this paper (group 3), is characterized by the presence of a well-developed end apparatus, usually with enlarged extracellular spaces, but lacking the normally associated duct cells. The secretory cells contain several stacks of granular endoplasmic reticulum, but these are mainly concentrated in the middle of the cell. The basal half of the cells contains many lipid droplets. Although the function of the convoluted gland is not yet understood, an hypothesis is related to what is known of the function of reservoir secretory cells in solitary wasps. All wasp species studied showed the same organization of the convoluted gland, which clearly distinguishes their venom gland from that of Sphecidae.     

Venom gland ontogeny in Formicinae, with special reference to the pulvinate convoluted gland (Hymenoptera, Formicidae)

 The primordia of the sclerites associated with the venom gland appear in third-stage larvae. The study aims to link the structure and function of this specialised venom structure in Formicinae, together with glandular ontogeny, and puts emphasis on the relevance of the distinguished glandular subunits contributing to the final secretion. The most conspicuous changes in glandular development occur in the pharate pupa. At this stage, all subunits of the venom gland (the tubule, the convoluted gland and reservoir) are visibly present. Formation of the glandular cuticle starts around day 4 of the pupal stage. Luminal cells in the convoluted gland are provided with abundant free ribosomes and apical microvilli that remain during adult life. Stacks of granular endoplasmic reticulum are also frequently found in these cells. The convoluted gland contains relatively few scattered secretory cells, belonging to type 3 according to Noirot and Quennedey (1974), which contain electron-dense material in their extracellular spaces during adult life. These cells strongly contrast with the apparently general non-glandular nature of the convoluted gland tubule. Histochemical investigation of the secretory cells in the pulvinate convoluted gland reveals that these cells contain lipoid material, most likely to correspond with lipoids demonstrated in earlier chemical analyses. This lipoidal material in minor quantities strongly contrasts with the bulk of acid constituting the secretion. The substances produced in the convoluted gland could act as insulators, thus protecting the insect against its corrosive venom. Accepted: 28 April 1998     

Asynchrony in the synthesis of secretory proteins in the venom gland of the snake Vipera palaestinae.

1. Venom of Vipera palastinae was subjected to isoelectrofocusing on polyacrylamide gel. The protein separation profiles were similar for different venom samples; more than 25 protein bands with a wide range of pI values could be demonstrated by this technique. 2. Labelled venom was obtained 8h after an intracardial injection of [3H]leucine. The relative radioactivities of four out of 12 main protein bands were significantly different in the venom synthesized during the 2nd day of the venom regeneration cycle as compared with the venom of the 4th day. The comparison was made in venom samples obtained from the two glands of the same snake at two different secretory stages. 3. It is concluded that the asynchronous synthesis of exportable proteins after the initiation of a new venom regeneration cycle is responsible for the non-parallel secretion of some venom proteins by the venom gland of Vipera palaestinae during the first few days after milking.     

Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects

Females of the parasitoid digger wasp species Liris niger hunt crickets as food for their future brood. The wasps paralyse the prey by injecting their venom directly into the CNS. The venom is produced in a gland consisting of two ramified glandular tubules terminating in a common reservoir. The reservoir contents enter the sting bulb via a ductus venatus. Secretory units of dermal gland type III line the two free gland tubules, the afferent ducts to the reservoir and the cap region within the reservoir. Secretion products of tubules reach the reservoir through the cuticle-lined central funnel. Secretory cells in the distal and middle parts of the tubules contain extensive rough endoplasmic reticulum and numerous electron-dense vesicles, whereas secretory cells of the afferent ducts and the cap region of the reservoir lack electron-dense vesicles and the endoplasmic reticulum is poorly developed. The secretory apparatus undergoes age-related changes. The secretory units in the venom gland tubules and inside the reservoir complete differentiation 1 day after imaginal ecdysis. After 30 days, massive autolytic processes occur in the secretory cells and in the epithelial cells of the reservoir. Analysis of the polypeptide composition demonstrates that the venom reservoir contains numerous proteins ranging from 3.4 to 200 kDa. A dominant component is a glycoprotein of about 90 kDa. In contrast the polypeptide composition of Dufour's gland is completely different and contains no glycoproteins. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets reveals that all of the major proteinaceous constituents become secreted. Thus the secreted venom contains exclusively proteins present in the soluble contents of the venom gland.     

Effect of topical application of a juvenile hormone mimic on the duration of the moulting cycle in fifth instar Rhodnius

Fifth instar Rhodnius prolixus given a single topical application of synthetic juvenile hormone (JH) on one of the first 12 days after feeding showed a sigmoid relationship between day of treatment and duration of the moulting cycle. A similar relationship was observed between day of treatment and mean ‘juvenile score’ in subsequently emerging insects. The linear relationship between mean ‘juvenile score’ and duration of the moulting cycle suggests that the increased time from feeding to ecdysis for late-treated insects is a result of the morphogenetic effects of the JH treatment. Some of the partially juvenilized insects obtained by treatment after day 5 produced an odour characteristically detected only from adult insects. The possible significance of this observation is discussed.     

扬子鳄胚胎背腺的发生及退化

本文在１４例扬子鳄Ａｌｌｉｇａｔｏｒｓｉｎｅｎｓｉｓ胚胎中观察了背腺的发生及退化过程。孵化第２８天,背中线左右两侧第二行鳞片处的表皮内陷形成实心的背腺腺芽;第３８天,背腺腺泡明显,腺上皮为复层上皮;从第４６天开始,腺上皮出现明显的退化,大量增殖的腺管上皮细胞逐渐堵塞腺管及腺孔。扫描电镜观察表明,孵化第３２—３６天的胚胎背部第二行鳞的各列鳞片表面均有背腺腺孔,以后逐渐出现少数不规则的退化,孵化第５８天以后,绝大多数背腺腺孔消失。对扬子鳄背腺的发生及退化现象作了讨论。     

High temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK

Pupal diapause in the flesh fly, Sarcophaga crassipalpis, can be terminated by exposure to high temperatures or, artificially, with a topical application of organic solvents. To analyze the molecular mechanisms involved in diapause termination we explored the possibility that the mitogen-activated protein kinases (MAPK) are involved in this response. Levels of phospho-ERK increased within 10 min after hexane application. Extracellular signal-regulated kinase (ERK) was also activated when pupae were transferred from 20 to 25 degrees C, thus suggesting that ERK activation is a likely component of the signal transduction pathway used to initiate development in response to diapause-terminating signals. 20-Hydroxyecdysone and cyclic GMP terminate diapause in this fly, and the juvenile hormone analog methoprene shortens the diapause, but none of these agents activated ERK. ERK was readily activated in isolated abdomens treated with hexane, thus we conclude that ERK is directly activated by the hexane treatment. ERK activation was evident in the brain, epidermis, midgut and fat body, but not in the ventral nerve mass or ring gland, thus suggesting that ERK does not act directly on the ring gland to promote ecdysteroid synthesis but exerts its effect through stimulation of the brain.     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

Morphology and ultrastructure of the convoluted gland in the ant Dinoponera australis (Hymenoptera: Formicidae)

The morphology and fine structure of the convoluted gland inside the venom reservoir of the ponerine ant Dinoponera australis (Hymenoptera: Formicidae) are described. The cells of the convoluted gland can be divided into 3 major groups: (1) epithelial cells, (2) glandular cells with end apparatus secreting into the tubule inside the convoluted gland, and (3) glandular cells with end apparatus secreting directly into the venom reservoir. A fourth group of cells belonging to the venom gland of this ant is also discussed as (4) secretory cells of the free tubule (not a part of the convoluted gland). The epithelial cells in the convoluted gland do not have many organelles. Most cells of group 3 are characterized by numerous mitochondria. In some of these cells, the mitochondria possess tubular cristae. Tubule cells of group 2 inside the convoluted gland, possess little rough endoplasmic reticulum when compared with cells of group 4, situated in the free tubule.     

Physiological and endocrine changes associated with polydnavirus/venom in the parasitoid-host system Chelonus inanitus-Spodoptera littoralis

As shown earlier, parasitization by the egg-larval parasitoid C. inanitus causes in its host the precocious onset of metamorphosis in the 5th instar followed by developmental arrest in the prepupal stage. Polydnavirus/venom were shown to be responsible for the developmental arrest. We investigated how polydnavirus/venom affect growth of the host larvae and found that head capsule widths were smaller from the 4th to 6th stadium and weights were lower in the 6th stadium in polydnavirus/venom-containing larvae than in non-parasitized larvae. In an attempt to identify endocrine parameters that are modified by polydnavirus/venom and might be responsible for the developmental arrest in the prepupa, we compared juvenile hormones, juvenile hormone esterase and ecdysteroids between non-parasitized and polydnavirus/venom-containing larvae from the 4th instar until pupation or developmental arrest, respectively. Obvious differences became manifest only in the 6th instar at the pupal cell formation stage, i.e. 12 days after entry of polydnavirus/venom into the host egg. Then, prothoracic glands of polydnavirus/venom-containing larvae released less ecdysteroids and ecdysteroid titres were lower than in non-parasitized larvae; this was followed by a delayed, reduced and desynchronized increase in prepupal juvenile hormones and juvenile hormone esterase and a slightly modified metabolism of ecdysone. This indicates that polydnavirus/venom affects the endocrine system of the host only after pupal commitment and that inhibition of prothoracic gland activity is the first detectable effect.     

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta,Hymenoptera, Sphecidae): Structural and biochemical aspects

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 μm (∅ 125 μm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.     

Immunocytochemical localization and secretion process of the toxin CSTX-1 in the venom gland of the wandering spider Cupiennius salei (Araneae: Ctenidae)

Fluorescein and horseradish peroxidase-labeled monoclonal antibodies were used to localize the predominant toxic peptide CSTX-1 in the venom gland of the spider Cupiennius salei. There was no polarity of CSTX-1 expression in repleted glands, whereas the glands of previously milked spiders showed a decreasing immunofluorescent response from the distal to the proximal portion. Detailed investigation revealed a new structure in the venom-secreting epithelium, which is postulated to be an evolutionary adaptation to increasing gland volume. CSTX-1 was found to be synthesized and stored as a fully active toxin within complex units, composed of long interdigitating cells running perpendicular to the muscular sheath and extending into the central lumen of the gland. These venom-producing units were found in all sectors of the gland, including the transitional region between the main gland and the venom duct. The venom is liberated from the venom-producing units into the glandular lumen following the contraction of the surrounding muscle layer. Free nuclei or other cellular fragments, which would have provided evidence for a holocrine secretion process, were not found in the glandular lumen or in the crude venom obtained by electrical stimulation. The fine regulation of the spider's venom injection process is postulated to be the function of the bulbous ampulla, situated in the anterior third of the venom duct.     

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.     

Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo.     

Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly, Sarcophaga crassipalpis, and the tobacco hornworm, Manduca sexta

ABSTRACT. Alkanes, diethyl ether, and various other organic solvents proved to be potent stimulants of development in diapausing pupae of Sarcophaga crassipalpis Macquart and of Manduca sexta (Johansson). Topical application of 2μl or vapour exposure for 1–2h was sufficient stimulation for the flies, but the solvent had to be injected to elicit the response in the hornworms. In flies, oxygen consumption increased nearly 100-fold within 15 min of hexane application, and thereafter persisted at non-diapause levels. Sensitivity of fly pupae to hexane remained high throughout diapause but acetone sensitivity dropped sharply after the second day in diapause. Acetone applied the day before the onset of diapause averted diapause in flies, and topical application to hornworm larvae 3 days before pupation likewise prevented pupal diapause. Debrained pupae failed to respond to solvent treatment, and we conclude that it is the brain, rather than the prothoracic gland, that responds directly to stimulation.     

Development of secretory activity in the long hyaline gland of the male migratory grasshopper,Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins.Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.     

Exocrine glands of the ant Myrmoteras iriodum

This paper describes the morphological characteristics of nine major exocrine glands in workers of the formicine ant Myrmoteras iriodum. The elongate mandibles reveal along their entire length a conspicuous intramandibular gland, which contains both class‐1 and class‐3 secretory cells. The secretory cells of the mandibular glands show a peculiar appearance, with a branched end apparatus, which is unusual for ants. The other major glands (pro‐ and postpharyngeal gland, infrabuccal cavity gland, labial gland, metapleural gland, venom gland and Dufour gland) show common features for formicine ants. The precise function of the glands could not yet be experimentally demonstrated, and to clarify this will depend on the availability of live material of these enigmatic ants in future.     

Effect of 1-(4-phenoxyphenoxypropyl)imidazole (KS-175) on larval growth in the silkworm Bombyx mori

1-(4-Phenoxyphenoxypropyl)imidazole (KS-175), which has two types of characteristic moieties of insect growth regulators (IGRs), the phenoxyphenoxyalkyl group of juvenile hormone analogs (JHAs) and imidazole of 1,5-disubstituted imidazole such as KK-42, was tested for its biological activity on the silkworm, Bombyx mori. Penultimate (4th) instar larvae topically treated with KS-175 did not molt for more than 20 days. This activity was different from that reported for any IGRs. After the treatment, ecdysteroid levels in the hemolymph did not increase and the cells of the prothoracic gland had shrunk. When the treated penultimate larvae were fed an artificial diet supplemented with 20 ppm of 20-hydroxyecdysone, the larvae molted to the ultimate (5th) instar with a timing similar to that of control larvae fed a diet with or without 20-hydroxyecdysone. These results suggest that topical application of KS-175 irreversibly damages ecdysone biosynthesis in the prothoracic glands.