Larval Glands Research Articles

The influence of an analog of juvenile hormone on the silk gland of Africanized Apis mellifera larvae reared in vitro

As the larval silk glands of Africanized honey bees are well characterized in hive conditions, we compared the silk glands of larvae of Africanized honey bees from the hive (in vivo - literature information) with larvae reared under laboratory conditions according to a specific bioassay (in vitro rearing of Africanized honey bees). We also continuously exposed the larva to a juvenile hormone analog (JHA), the pyriproxyfen, in three different concentrations: 12.5 mg/L, 25 mg/L, and 50 mg/L. Concerning morphological and histochemical analyses, there were no characteristics in glands that indicated the acceleration of the cocoon spinning time and no features of cell death. The glands seemed to be in secretory activity. Pyriproxyfen-treated larvae had a slightly shorter silk synthesis period than control, although only in the 50 mg/L experimental group, the tactoids from silk were clearly visible in optical microscopy, probably due to a higher secretion activity as the response to JHA. We observed that the presence of lipids under artificial feeding was independent of the nurse bee presence. Thus, we found that pyriproxyfen alters the functioning of the silk gland, under laboratory conditions in morphological, histological, and/or morphometric aspects.

Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.

The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages.

Impact of seasonal variation on physiological traits and biochemical constituents in silk glands ofBombyx moriL.

The study was conducted during spring and summer seasons of 2010–2011 to examine the changes in biochemical constituents in silk gland contents and to identify the races with better growth of silk glands in various seasons. Larval weight and silk gland weight and size were significantly high in ‘SK-1’ and ‘SK-6’ and low in ‘CSR-2’ during both the seasons. The values were marginally high in spring than summer season. Biochemical contents in silk glands decreased more in summer than spring. There was no substantial variation in the expression of molecular weight of silk gland proteins in all the genotypes tested. Carbohydrate and protein contents were significantly high in race ‘SK-1’ during both the seasons but low values in race ‘CSR-2’. There was no substantial difference in the molecular weight of silk gland proteins in different breeds evaluated.

Ultrastructural evidence for completion of the entire miracidial maturation in intrauterine eggs of the digenean Brandesia turgida (Brandes, 1888) (Plagiorchiida: Pleurogenidae)

Results of this TEM study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs situated in the most posterior uterine regions of the pleurogenid trematode Brandesia turgida (Brandes, 1888). The ultrastructural characteristic of different larval organelles and cell types of these eggshell-enclosed, but fully formed, cilated miracidia is described. The body wall of the pyriform mature miracidium of B. turgida is composed of ciliated epidermis and underlying peripheral body musculature. Two miracidial flame cells of the protonephridial excretory system are localized in the central region of the ciliated larvae. Three types of miracidial glands were observed: a single apical gland, two lateral glands, and several small vesiculated glands; each gland type contains characteristic, but different types of secretory granules. The anterior end of each miracidium consists of an apical papilla on which are situated the exits of the three main larval glands: an exit of a single apical gland as well as the individual exits of two lateral glands. The exits of vesiculated glands, containing characteristic spherical membrane-bound and highly electron-dense granules, evidently different from the two other types of secretory granules of apical and lateral glands, were not identified. Germinative cells, grouped together in a sac-like germinative follicle, are situated in the medioposterior part of the larva, the germatophore. The germinative cells contain numerous electron-dense heterochromatin islands arranged in the form of a network or chain-like pattern and distributed mainly in the karyoplasm adjacent to the nuclear membrane. The thin layer of granular cytoplasm is rich in free ribosomes and contains a few small mitochondria. Both nuclear and cytoplasmic features if these cells indicate their great developmental potential for further growth and multiplication in postembryonic stages of the life cycle. In the mature eggs, the areas of focal cytoplasmic degradation were frequently observed and may be involved in the autolysis of some embryonic structures. Obtained results are compared with available literature data on the functional ultrastructure of the miracidia of other digeneans.

Chemical skin defence in the Eastern fire-bellied toad Bombina orientalis: an ultrastructural approach to the mechanism of poison gland rehabilitation after discharge

Type I serous glands in the skin of the Eastern yellow-bellied toad Bombina orientalis released their product massively after 10 -3 M nor-adrenalin (NA) stimulation, mimicking orthosympathetic control on poison emission in chemical skin defence. Features of cutaneous glands involved in this bulk discharge were observed under light and electron microscopes. Furthermore, restoration of depleted glands was followed after 1, 2 and 3 weeks, and compared with serous biosynthesis during larval gland development. Bulk discharge was caused by contraction of myoepithelial cells (mecs) encircling the secretory units. Mec compression dramatically affected the secretory unit, but parts of this syncytial cytoplasm were saved from degeneration and cooperated in gland renewal with stem cells from the gland neck. These adenoblasts underwent proliferation and secretory cytodifferentiation, until merging with the syncytium. Cytoplasm that had resumed secretory activity showed the features typical of larval gland development: the endoplasmic reticulum (rer) cisterns were aligned in close parallel arrangement and Golgi stacks released minute type I granules. Secretory rehabilitation led to increasing amounts of granule content. In the meantime, rough cisterns decreased in number and assumed the less ordered pattern described in control specimens. Data collected in the present study revealed that chemical skin defence in anurans is a multi-factorial mechanism involving specific activities: mechanical from mecs, biosynthetic from secretory syncytium and proliferative from intercalated stem cells.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E—signaling through a nuclear receptor heterodimer consisting of EcR and USP—induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Comparative morphology of larval Camiarinae (Coleoptera: Leiodidae)Part I. Genus Paragyrtodes Szymczakowski 1966

The first, second and third larval instars of the Australian genus Paragyrtodes Szymczakowski, 1966 are described. Among three described species, larvae of two, P. candens Szymczakowski, 1966 and P. modestus Szymczakowski, 1966, are redescribed in detail, illustrated, photographed and measured. The chaetotaxy and porotaxy are given and their system is discussed. Morphological and chaetotaxic characters distinguishing mature larvae of two species of Paragyrtodes are provided; differences among mature larvae of the genera Agyrtodes Portevin, 1907 and Paragyrtodes are summarized. Pretergal glands, their glandular function, and tergal pores are documented in the family Leiodidae for the the first time. The problem of the identity of the larval glands in the family Leiodidae and superfamily Staphylinoidea is discussed. Data on presence, number and arrangement of the tergal and pretergal glands for all known larvae of Leiodidae and some representatives of Staphylinidae and Hydraenidae are gathered.

Development of serous cutaneous glands in Scinax nasica (Anura, Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families

We examined the development of serous (poison) cutaneous glands in larval and juvenile Scinax nasica (Hylidae) at the ultrastructural level. We describe the biosynthesis and maturation of the cutaneous poison in comparison with the corresponding processes in representatives of Discoglossidae, Leptodactylidae, Pelobatidae and Pipidae. Serous biosynthesis in S. nasica starts in discrete adenoblasts and continues in the syncytial secretory unit. Biosynthetic processes involve rough endoplasmic reticulum and the Golgi apparatus, that releases membrane-bounded material, varying from fine grained to flocculent. During the post-Golgian secretory phase, this material undergoes initial maturation, and two products are formed: dense granules and larger vesicles holding a thin substance that will later be structured into a three-dimensional, honeycomb-like net. Both the secretory granules and vesicles change into glomerular-like aggregates of bowed, rod-shaped subunits (modules). In adult frogs, formation of dense granules is bypassed. The modular granule substructure seems to be related to the merocrine release of small amounts of poison, involved in regulating skin homeostasis. Comparison with maturational changes in larval glands of species representing four anuran families discloses similar patterns in the Leptodactylidae, but production of opaque homogeneous granules occurs in the Discoglossidae, clear vesicles in the Pelobatidae and aggregates of dense bars in the Pipidae.

Fate mapping of the larval silk glands of Bombyx mori by UV laser irradiation of the egg at fertilisation.

Localised UV laser irradiation of Bombyx eggs at the fertilisation stage causes localised cuticle defects in the integument of the resultant larvae. Based on the correlation between the site of irradiation and the site of defects, I previously established a fate map of the larval integument of Bombyx mori. Although most of the internal organs of the irradiated specimens were eliminated in the acidic fixative used for cuticle preparation, silk proteins which had accumulated in the gland lumen were resistant to the acid treatment and silk gland morphology was thus preserved. Taking advantage of this phenomenon, I continued fate mapping, now of the larval silk glands of Bombyx mori, utilising the same specimens that were prepared and analysed in the previous study. Of 710 irradiated specimens analysed, 452 showed two normal glands, 27 showed one normal and one deformed gland, 21 showed one normal gland only, and 210 showed no silk gland. The sites of irradiation that caused the deletion of a silk gland were located within a limited area that entirely overlapped the ventral half of the area where irradiation caused defects in the labium. These results are consistent with previous histological descriptions stating that the silk glands develop from the paired invaginations in the labial segment. Based on the data, the presumptive region for the larval silk glands was located on the previously established Bombyx fate map.

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca 2+- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca 2+ influx, Ca 2+-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca 2+-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.

Thyroid hormone-induced apoptosis of larval cells and differentiation of pepsinogen-producing cells in the stomach of Xenopus laevis in vitro

It is generally known that the anuran stomach begins to express pepsinogens (Pg) during metamorphosis. To clarify the mechanisms of differentiation of Pg-producing cells, we examined immunohistochemically the epithelial transformation from larval to adult form in Xenopus laevis stomach at the cellular level. At the beginning of metamorphic climax, concomitantly with the modification of the basement membrane, apoptotic cells labelled by TUNEL suddenly increased in number in the entire epithelium except for the primordia of adult epithelial cells in the basal region of larval glands. Subsequently, with the development of connective tissue, the adult epithelial cells actively proliferated and replaced the larval cells from the basal to the luminal region. Following the start of morphogenesis of adult glands, Pg-producing cells became differentiated in newly formed adult glands, but not in the adult surface epithelium. We then developed an organ culture system and examined effects of thyroid hormone (TH) on the differentiation of Pg-producing cells in X. laevis stomach in vitro. In the presence of TH, just as in spontaneous metamorphosis, Pg-producing cells differentiated from the adult epithelial primordia after the apoptosis of larval epithelial cells. In contrast, in the absence of TH, neither apoptotic larval cells no Pg-producing cells were detected. Therefore, we conclude that TH triggers organ-autonomously the entire process leading to the differentiation of Pg-producing cells in X. laevis stomach. In addition, the strict localization of Pg-producing cells in the adult glands both in vivo and in vitro suggests the correlation between the differentiation of Pg-producing cells and morphogenesis of the glands surrounded by the developed connective tissue.

A review of the Palaearctic blackfly subgenus Simulium (Rubzovia) with the emphasis on S. (R.) paraloutetense, a species confined to Gran Canaria Island (Diptera: Simuliidae)

AbstractThe larval and pupal stages of Simulium (Rubzovia) paraloutetense Crosskey are described following their first discovery in a spring trickle in Gran Canaria Island where the species is endemic; data from the polytene chromosomes of the larval silk glands are included. Subgeneric placement of the species is revised in light of all the findings. A diagnosis for the subgenus Rubzovia, a discussion of its geographical distribution and habitat, and a key to the four known species are included.

Early development and morphogenesis of the intracapsular veliger of Haminaea navicula (Gastropoda: Opisthobranchia: Bullomorpha)

Summary The development of Haminaea navicula was studied by anatomical and histological methods. This species shows typical spiral cleavage, which leads to a stereoblastula. Gastrulation is reached by epiboly. A fully developed veliger larva is reached within the egg capsule that is not only capable of swimming when artificially hatched but is also provided with the same larval organs (larval shell, velum, operculum, larval cephalic sensory organ, anal cells, anal gland, nephrocyst-protonephridial complex, epipodial and pallial gland cells) that are present in pelagic larvae of related species. Aspects of the development of H. navicula are compared with observations of the pelagic larvae of H. exigua and H. vesicula. Additional data from the literature are discussed.

Cyclic AMP is a requisite messenger in the action of big PTTH in the prothoracic glands of pupal Manduca sexta

Prothoracicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. Results of the present study indicate that PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS, an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of [ 32P]8-N 3cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development.

Metamorphic changes in the stomach of the frog Rana temporaria tadpoles

The histological transformation of amphibian stomach during metamorphosis was studied in the frog Rana temporaria. The earliest metamorphic changes occur shortly before regression of the cloacal piece of tail and appearance of forelegs. Autolysis of primary, larval epithelial cells and activity of phagocytes lead to regression of the apical mucosa, which is shed into the gastric lumen. Histogenesis takes place from the very beginning of metamorphosis in the basal region of the mucosa; undifferentiated, regenerative cells, arranged in small compact cords and surrounded by a thick basement membrane, give rise to secondary lining epithelium and glands. Lining epithelial cells differentiate into a typical mucosecretory epithelium. Oxyntic cells present in larval glands are substituted by both ion- and protein-secreting oxyntic-peptic cells. During metamorphosis, connective and muscular tissues markedly increase, a submucosa, not present in larval tadpoles, gradually develops. A muscularis mucosae is also formed and the muscular propria becomes thicker. At late metamorphosis, folded structures involving both mucosa and submucosa develop, increasing the luminal surface as in adults. Removal of the larval gastric mucosa and its replacement by a new, adult-type definitive one, together with development of peripheral connective and muscular tissues, account for metamorphosis of tadpole stomach.

Structure-activity relationships in corpora allata of the cockroach Diploptera punctata: roles of mating and the ovary.

Morphometric studies were made on corpora allata of the cockroach Diploptera punctata from animals in which increasing gland size is not coupled to hormone synthesis (ovariectomized mated females; last-instar larvae) and in which gland size is coupled to hormone synthesis (normal mated and virgin females; penultimate-instar larvae). Cell number, gland volume, and juvenile hormone synthesis were measured. From electron micrographs, nuclear, cytoplasmic, and extracellular volumes; and cell membrane area were calculated; and fine structure described. Low-activity glands of ovariectomized mated females resembled high-activity glands from mated females in high cell number, large overall and cytoplasmic volume, and low nuclear-cytoplasmic ratio; they differed in having organelles typical of low-activity glands, mitochondria with dense matrices and large whorls of smooth endoplasmic reticulum. Inactive last-instar larval glands resembled mated ovariectomized female glands in increased cell number and organelles characteristic of inactive glands; however, their nuclear-cytoplasmic volume ratio was much higher. Penultimate larval glands with high activity per cell resembled active glands of normal mated females. Ovariectomy did not change morphometric parameters of virgin female glands; thus mating results in increase in size of adult female glands whereas the growing ovary is needed for changes in mitochondria and endoplasmic reticulum associated with high juvenile hormone synthesis.

Competence of blastomeres for the expression of molecular tissue markers is acquired by diverse mechanisms in the embryo of Platynereis (Annelida).

This paper is devoted to the role of cell divisions for the establishment of histospecificity in the embryo of the spiralian, Platynereis dumerilii (Annelida). We have incubated successive cleavage stages in cytochalasin B (CCB) and observed whether the cells thereafter were able to acquire the competence for expressing histospecific antigens of larval gland cells (labelled by the monoclonal antibody OI64) and of neural components of the ventral nerve cord (labelled by mAb OI7 or by testing acety1cholinesterase activity), respectively. Incubation in CCB results in permanent cleavage arrest, but does not necessarily interfere with biochemical differentiation of such markers. Synthesis of the differentiation marker specific for larval gland cells does not require any cleavages but this capacity becomes restricted to the 1a and 1b cell lines if cleavages are allowed to occur. In contrast, the progenitors of neural cells need at least 6.5 h of normal development before they acquire the competence to synthesize two neural differentiation markers, which can be demonstrated after at least two more days of development. Thus, prespecifying and diversifying cleavages are a prerequisite for neurogenesis and production of the investigated neural markers. Competence for the expression of histospecific antigens may also depend on cell-cell interactions. If 20-24 h old embryos are treated with puromycin, pioneering fibres fail to grow out from a pair of posterior nerve cell progenitors as they would have done normally 24-48 h after fertilization. Concomitantly, a number of potential nerve cells which now do not come into contact with pioneering fibres do not express the neural antigen. This suggests that a local inductive stimulus from the pioneering fibres normally imprints cell fate onto ventral plate cells and turns them into neuroblasts.

Prothoracicotropic hormone regulates the phosphorylation of a specific protein in the prothoracic glands of the tobacco hornworm, Manduca sexta

Particulate (membrane) fractions were obtained from homogenates of prothoracic glands from staged larvae and pupae of the tobacco hornworm, Manduca sexta. The addition of cAMP to these fractions stimulated the in vitro phosphorylation of at least three phosphoproteins. This cAMP-mediated phosphorylation was blocked by the addition of the specific inhibitor of the cAMP-dependent protein kinase. When intact prothoracic glands were preincubated with [ 32P]O 4 to label endogenous ATP, one of these proteins (34kDa) was phosphorylated in response to dibutyryl cAMP, forskolin, as well as the brain neurohormone, big prothoracicotropic hormone (PTTH). Neither cyclic GMP nor small PTTH stimulated 34 kDa protein phosphorylation, although the latter elicited increased ecdysteroid synthesis by larval glands. Further, injection of big PTTH into intact larvae previously loaded with [ 32P]O 4 also resulted in enhanced phosphorylation of the same prothoracic gland protein. In prothoracic glands from animals at all developmental stages examined except diapausing pupae the protein was phosphorylated in vitro, although glands activated at the time of dissection showed a reduced phosphorylation response in vitro. The intensity of phosphorylation in intact glands was big PTTH dose-dependent, and the dose-response curve of phosphorylation closely followed the curve for PTTH-stimulated ecdysteroid secretion. The time course of phosphorylation is consistent with a role for this phosphoprotein as an intermediate between PTTH stimulation of adenylate cyclase and ecdysteroid biosynthesis.

Cytoskeletal F-actin patterns in whole-mounted larval and adult salivary glands of the fleshfly, Sarcophaga bullata

The patterns of filamentous actin were analysed in different larval, pupal and adult stages in the salivary glands of the fleshfly Sarcophaga bullata. Using the rhodamine labelled phalloidin staining method in combination with detergent extraction specific actin filament distribution was detected. The salivary glands which are histolysed during the process of metamorphosis show distinct cellular morphology and actin filament patterns in larvae and adults. The large third instar larval salivary gland cells contain a well developed apicolateral microvillar zone. In third instar larvae this microvillar zone invaginates and expands in the basal part of the lateral membranes. Larval salivary gland cells also contain numerous parallel basal actin bundles. The larval glands are histolysed during metamorphosis and adult glands are formed out of the imaginal cell group. At the onset of metamorphosis these basal actin bundles form a network of crossing bundles. The filamentous actin patterns of the proximal part of adult gland cells is confined to the apicolateral microvillar membranes. The cells in the distal, tubular part of the adult salivary glands show intense staining of their folded lateral membranes.

A new aspect of the role of larvae in the pharaoh's ant society (Monomorium pharaonis (L.)-Formicidae, Myrmicinae): Producer of fecundity-increasing substances to the queen

Observations of undisturbed brood chambers of the pharaoh's ant showed that mated, mature queens regularly selected large larvae lying on the bottom of the chambers. The queens obviously obtained liquid secretions by licking various parts of these larvae. It was mainly stomodeal secretion, most likely originating from the larval labial glands, but also anal secretion and gut liquid from larvae becoming prepupae on shedding the meconium.