The salivary gland secretions of a neotropical bumblebee

Summary The larval salivary gland secretions ofBombus atratus were studied with cytochemical and cytophysical methods. In the young feeding larvae a proteic filamentous secretion depicting striations perpendicular to the long axis of its fibrillary threads and exhibiting a wellordered macromolecular array was found. It appears not to differ from the silk secretion described for the fully grown larvae of an europeanBombus species. However, in the fully grown larvae ofB. atratus changes which have not yet been reported for other bees occur involving the salivary gland secretion. Two secretion types are then distinguishable. One is composed of carboxylated and sulfated acid glycosaminoglycans and glycoprotein(s) (mucous secretion), and the other has the same composition as that of the filamentous secretion of the young larvae, although differing morphologically and in terms of macromolecular alignment (flocculent secretion). The filamentous secretion is assumed to be involved inB. atratus with the spinning of the silken partitions which at a relatively early stage separate the larvae reared within a common cell from one another. The mucous and flocculent secretions will participate in the cocoons which will cover the pupating larvae. The filamentous and flocculent secretions appear to contain an -helical fibroin, glycoprotein(s) and lipoprotein(s), but not collagen-type proteins.     

The salivary secretion of spinning larvae ofRhynchosciara americana

Summary The secretion present at the lumen of the salivary glands of spinning larvae ofRhynchosciara americana was studied cytochemically and with microspectrophotometry and fluorescence and quantitative polarization microscopy. It was found that structural proteins, including glycoproteins and lipoproteins, occur in this secretion. Findings involving spectral absorption profiles after xylidine ponceau staining, patterns of birefringence and dispersion of birefringence, and lack of dichroism after xylidine ponceau staining and of blue fluorescence after ANS staining are highly suggestive that the secretion ofR. americana differs from classical silks not only in terms of composition but also of macromolecular array. The silk secretion ofR. americana also appears to differ from that of another sciarid,Bradysia spatitergum. Part of the glycoproteins present at the glandular lumen is assumed to be extruded from cells of the posterior zone of the glands, whereas other glycoproteins (or their glycidic radicals) are probably removed from fat body cells via cells of the anterior zone of the glands. The salivary secretion of the spinning larvae ofR. americana contains calcium and is devoid of acid glycosaminoglycans.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

The duct connecting Malpighian tubules and gut: an ultrastructural and comparative analysis in various Ephemeroptera nymphs (Pterygota)

Malpighian tubules proper are connected to the gut by ducts called trunks, the organization of which is described at ultrastructural level in the nymphs of various mayfly species, namely Ecdyonurus venosus (Heptageniidae), Ephemerella ignita (Ephemerellidae), Choroterpes picteti (Leptophlebiidae), and Caenis luctuosa (Caenidae). Trunks are luminal tubes varying in arrangement, number, shape, and length. The main cell type of the trunk wall is represented by cells that are bordered by a thin cuticle along their luminal side (type-A cells). Whereas these cells are exclusive in the long trunks (such as those of C. picteti and C. luctuosa), in the shorter ones (such as those of E. venosus and Ephemerella ignita) cells with a microvillous luminal border (type-B cells) are also encountered. These cells are located close to the entrance of the collecting duct of the Malpighian tubules proper, and their long microvilli form a dense filamentous mesh filling up the lumen. Both cell types exhibit fine structural features that are characteristics of ion-transporting epithelia. Ultrastructural investigations show that trunks are not a simple conducting system but are involved in the regulation of the ionic composition of the primary urine for producing a fluid hypo-osmotic with respect to the hemolymph. Accepted: 30 May 2000     

Malpighian tubules of larval Aedes aegypti are hormonally stimulated by 5-hydroxytryptamine in response to increased salinity

Two environmental parameters, feeding status and salinity, are expected to affect water and ion balance of the aquatic larvae of Aedes aegypti. Evidence was obtained for regulation of Malpighian tubule fluid secretion rates in response to changes in each of these parameters. Exposure to increased salinity induces release into the hemolymph of material with diuretic effects on Malpighian tubules. Diuretic material is present in hemolymph of larvae raised in higher salinities, rapidly appears in the hemolymph of larvae following transfer from dilute water to higher salinity, and rapidly disappears from the hemolymph following transfer from higher salinity to dilute water. Feeding status affects diuretic properties of both hemolymph and Malpighian tubules. Feeding causes hemolymph to become diuretic relative to hemolymph from nonfeeding larvae. Malpighian tubules removed from feeding larvae have greater basal fluid secretion rates and also appear to have greater maximal fluid secretion capacity than do tubules removed from nonfeeding larvae. Larval hemolymph [5-HT] was found to increase fivefold in response to elevated salinity but was unaffected by feeding status. Methiothepin, a 5-HT receptor antagonist, inhibited stimulation of fluid secretion by 5-HT and blocked the diuretic effects of hemolymph from larvae exposed to higher salinity but was without effect on stimulation of fluid secretion by diuretic peptide. During the course of this investigation, a preliminary pharmacological characterization of the 5-HT receptor on Aedes Malpighian tubules, suggesting that this receptor may be pharmacologically distinct from other described insect 5-HT receptors, was obtained. Arch. Insect Biochem. Physiol. 34:123–141, 1997. © 1997 Wiley-Liss, Inc.     

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of Drosophila melanogaster larvae

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance–associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically‐obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph. © 2010 Wiley Periodicals, Inc.     

Histochemistry,refractometry, and fine structure of excretory globules in larval Malpighian tubes ofMelipona quadrifasciata (Hym., Apoidea)

Summary The authors have studied excretory globules in the Malpighian tubes ofMelipona larvae. By means of histochemical methods for fats, micro-interferometry and electron microscopy, glycerides surrounded by a lipoprotein or phospholipid layer were tentatively recognized in the globules. They resemble in composition the brochosomes reported in Homoptera and are found in the cell borders and in the lumina of the tubes. Yellow-green autofluorescent granules are seen within the cells, in the honeycomb borders and in the lumina of the tubes. It was not determined whether there are two distinct kinds of globules or only one kind in different physiological stages.This research was partly supported by FundaÇÃo de Amparo à Pesquisa do Estado de SÃo Paulo.     

Studies on silk secretion in the trichoptera (F. Limnephilidae): I. Histology,histochemistry, and ultrastructure of the silk glands

As part of a study on trichopteran silk secretion, the histology, histochemistry, and ultrastructure of the silk glands of two species of limnephilid trichopteran larvae, Pycnopsyche guttifer (Walk.) and Neophylax concinnus McL., were investigated. The silk glands consist of three anatomically distinct regions: a long, posterior silk-secreting region; a shorter, anterior conducting tube; and a terminal press/common duct. In Pycnopsyche, there is also a modified bulbous region between the secreting and conducting areas. Each anatomical region has a distinct cell type. There are two structurally and histochemically different components of the secretion in the glandular lumen: a core and a peripheral layer. Both components are produced all along the gland and are principally proteinaceous. However, the peripheral layer is also PAS and alcian blue (pH. 2.5) positive and shows β-metachromasia with toluidine blue (pH 3.5), indicating the presence of both neutral and acidic polysaccharides.     

Fatty acid composition of fat body and malpighian tubules of the tenebrionid beetle, Zophobas atratus: Significance in eicosanoid-mediated physiology

Total and phospholipid fatty acid composition of fat body and Malpighian tubules from two larval stages, pupae and adults, of Zophobas atratus were analyzed. Saturated and unsaturated C16 and C18 fatty acids were major components and varied by life stage and tissue source. Eicosanoid-precursor fatty acids, including 20:3n-6, 20:4n-6 and 20:5n-3, were present in low quantities and varied by life stage and tissue source. 20:3n-6 was always present in the lowest proportions, indicating that eicosanoids derived from 20:4n-6 and 20:5n-3 (the 2- and 3-series) are likely to be of greater physiological significance in this insect. Fatty acid composition of Z. atratus fat body and Malpighian tubules was independent of diet, suggesting that this insect controls its fatty acid composition to meet the needs of individual tissues and ontogenetic constraints.     

Host population structure affects field sex ratios of the heteronomous hyperparasitoid, Coccophagus atratus

Abstract.

• 1 Second instar Filippia gemina de Lotto scale insects are the preferred hosts of female Coccophugus atrutus Compere larvae. These scale insects were found on their host plants, Chrysanthemoides monilifera Norlindh and Cliffortia strobilifera Mettenius, only at certain times during a 1 year sampling programme.
• 2 Late larval instars and prepupae of C.atratus, and a Metaphycus species, are the preferred hosts of male C.atratus larvae. These hosts, although they occurred on the same host plants as hosts for female C.atratus, were most numerous at different times during the sampling period.
• 3 The ratio of hosts suitable for C.atratus varied from a predominance of hosts suitable for females through to a predominance of hosts suitable for males. Sex ratios of adult C.atratus followed a similar trend but did not reflect, exactly, the ratio of available hosts. Differences in mortality between sexes and hyperparasitism may account for this anomaly.
• 4 Variable population sex ratios observed in C.atratus apparently result from the behaviour of individual females in which brood sex ratios are dependent on the relative availability of hosts for males and hosts for females. This behaviour, in turn, may result from variability in the host population structure but may also result from selection pressures operating at the time that heteronomous hyperparasitism evolved.

     

Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins

Summary Monoclonal antibody PCBC3, raised against stylar extracts fromNicotians, alata flowers, was deduced from enzyme-linked immunosorbent assays and inhibition of immuno-gold labelling on tissue sections to bind specifically to carbohydrate epitopes on arabinogalactan proteins (AGPs) but not to other arabinose-containing cell wall polysaccharides. When pollen grains ofN. tabacum were hydrated in fixative, PCBC3 bound to vesicles in the vicinity of the endoplasmic reticulum but, when grains were hydrated for 20 min in culture medium before fixation, binding was restricted to the plasma membrane. The generative-cell plasma membrane was also labelled in grains ofLycopersicon peruvianum. In pollen tubes ofN. tabacum grown in liquid culture, the AGPs detected by PCBC3 were located in several regions, including the plasma membrane, tubular-vesicular structures (plasmalemmasomes) at and under the plasma membrane, and multilamellar bodies within vacuoles, features generally associated with endocytosis. Labelling was not evident in secretory vesicles or the plasma membrane at the pollen-tube tip. The AGPs detected with PCBC3 were also present in pollen-tube walls, near the interface between the inner, callosic layer and the outer, fibrillar, pectic layer. Pollen tubes ofN. tabacum grown in medium lacking added CuSO₄ produce a wall with an abnormally thickened fibrillar layer, and this layer was uniformly labelled with PCBC3. The disposition of wall AGPs thus changes in pollen tubes of different morphologies.Abbreviations AGP arabinogalactan protein - -L-Araf -L-arabinofuranose - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - PBS phosphate-buffered saline     

大麦虫及其肠道微生物对聚乙烯的生物降解

【背景】废旧塑料聚乙烯因具有较高的化学惰性,不易被自然降解而形成长期污染。【目的】探究聚乙烯泡沫塑料对大麦虫生长发育的影响,为大麦虫作为降解聚乙烯泡沫塑料的昆虫推广提供理论依据。【方法】以大麦虫幼虫为研究对象,选用常见的泡沫塑料(聚乙烯),采用4种不同的饲喂方式T1 (麦麸)、T2 (泡沫塑料)、T3 (泡沫塑料+麦麸)、T4 (不饲喂)进行驯化,处理30 d后对大麦虫进行解剖,取肠道内容物于LB培养基中进行富集培养,将富集培养后的菌液加入以聚乙烯(polyethylene,PE)为唯一碳源的LCFBM培养基进行选择性培养,从中筛选分离得到对PE塑料有降解能力的菌株。【结果】取食泡沫塑料30d后,与单一饲喂PE相比,麦麸和PE混合饲喂后大麦虫幼虫的存活率为76%。采用傅里叶变换红外光谱检测发现虫粪组分中主要官能团中峰值明显变化,表明PE长链有断裂现象,并从肠道中分离得到3株可以对PE薄膜边缘造成明显侵蚀的菌株。【结论】大麦虫可取食并消化PE塑料,其肠道内的微生物对PE塑料的降解起到关键作用,研究结果为塑料污染的生物降解提供了科学证据。     

Stellate cells in the Malpighian tubules of Drosophila hydei and D. melanogaster larvae (Insecta, Diptera)

 The Malpighian tubules of Drosophila hydei and D. melanogaster larvae are composed of two types of cell, principal cells and stellate cells. In the anterior larval Malpighian tubules approximately 26% (D. hydei) and 18% (D. melanogaster), respectively, of all cells are stellate cells. In the larvae of D. melanogaster, the stellate cells are fenestrated and the hemolymph space and tubule lumen are separated only by the basal lamina. Injection of dyes into the hemolymph did not indicate any facilitated transfer of substances through the fenestrated cells. The principal cells of the distal segment are carbonic anhydrase positive indicating transport activity, whereas the stellate cells lack this enzyme. In the stellate cells of the transitional segment, the sodium content is strikingly high in comparison to the neighbouring principal cells and lumen where no sodium was detected. This finding indicates that stellate cells reabsorb sodium as supposed earlier in 1969 by Berridge and Oschman (Tissue Cell 1:247–272). Accepted: 12 February 1999     

Some observations on variegation inDrosophila

Attempts to modify experimentally the variegated phenotypes of the mutantsz inDrosophila melanogaster andw ^m2 inD. hydei, were largely unsuccessful. Transplanted eyes ofz were found to be more intensely pigmented, but this effect was not influenced by treatment of the imaginal disks or by the host's genotype. Inw ^m2, increased pigmentation was observed after treatment with 5-bromo-uracil.Thew ^m2 phenotype is strongly modified by supernumerary Y chromosomes. One Y shifts the eye colour from yellow to brown, two Y's, to dark red. In the Malpighian tubules, two Y's have a similarly strong effect, but one Y a slight effect only. Malpighian tubules ofw ^m2 larvae were used for a quantitative analysis of mottling. An average clonal cluster size of 1.1 was found. This indicates that the state of pigmentation is determined around the last cell division in the embryo.     

Fungal Decomposition of Oat Straw during Liquid and Solid-State Fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei6/16) were grown on oat straw–based liquid and solid media, as well as in a bench-scale reactor, either individually or as cocultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mold Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely,Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the coculture of Coriolus hirsutus andCerrena maxima,caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw–based liquid medium, the basidiomycetes consumed up to 40% of the polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% of the lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid-state straw fermentation, white rot fungi consumed up to 75% of cellulose and 55% of lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for cocultures ofMycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide–monitored solid-state fermentation of oat straw by the coculture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Water vapour uptake from subsaturated atmospheres by engorged immature ixodid ticks

Contrary to current opinion, fully engorged and detached larvae and nymphs of some ixodid ticks consistently take up substantial amounts of atmospheric water vapour and thereby display their regulative capacity for maintaining water balance in subsaturated air. Net uptake of vapour generally begins some days after detachment and the capability persists until shortly after initiation of apolysis, a period which in diapausing specimens may extend up to several months. This was shown forIxodes ricinus, Haemaphysalis punctata, and the North AmericanI. dammini. Apparently, some other engorged ixodid immatures fail to exhibit net vapour uptake, as was shown for both larvae and nymphs ofDermacentor marginatus and nymphs ofHyalomma anatolicum excavatum. But there is some evidence for engorged nymphs ofD. marginatus that active uptake of vapour does occur, masked by spiracular transpiration. Net uptake of vapour is apparently not possible during the pharate phases. InI. ricinus both teneral nymphs and adults are capable of achieving net water gains by active vapour uptake on the first day following ecdysis. There is new evidence from fully engorgedI. ricinus immatures for the decisive role of agranular alveoli in the production of the salivary secretion involved in vapour uptake.     

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties

Summary EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H₁B clone form branching cords devoid of a central lumen, K₃A₃ cells from cords enclosing small multifocal lumina, and J₃B₁ cells form large cavitary structures containing a wide lumen. I₃G₂ cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Be1a cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell-lined cavitary structures, except H₁B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesis.     

Ultrastructural and histochemical observations on spermathecal gland of Dytiscus marginalis L. (Coleoptera)

Summary Histochemical studies have been carried out on the composition of feltwork and tubule in the spermathecal gland cells of Dytiscus marginalis L.These extracellular structures represent the secretion-collecting apparatus of the cells of the spermathecal gland, through which the secretion passes externally into the lumen of the spermatheca.The presence of a particular protein, resilin, also called arthropod elastin, at the level of the tubule and the feltwork, and of neutral polysaccharides present only in the feltwork have been demonstrated.Special consideration is given to the silver-methenamine reaction, the results of which have been interpreted in terms of the amino-acid composition of resilin itself.The behavior of this reaction, as a function of the type of fixation solution employed, is also discussed.     

New physiological activities of myosuppressin,sulfakinin and NVP-like peptide in <Emphasis Type="Italic">Zophobas atratus</Emphasis> beetle

Three neuropeptides Zopat-MS-2 (pEDVDHVFLRFa), Zopat-SK-1 (pETSDDYGHLRFa) and Zopat-NVPL-4trunc. (GRWGGFA), recently isolated from the neuroendocrine system of the Zophobas atratus beetle, were tested for their myotropic and hyperglycaemic activities in this species. These peptides exerted differentiated dose-dependent and tissue specific physiological effects. Zopat-MS-2 inhibited contractions of the isolated heart, ejaculatory duct, oviduct and hindgut of adult beetles and induced bimodal effects in the heart contractile activity of pupae in vivo. It also increased the haemolymph free sugar level in larvae of this species, apart from myotropic activity. Zopat-SK-1 showed myostimulatory action on the isolated hindgut of the adult beetles, but it decreased contractions of the heart, ejaculatory duct and oviduct. Injections of this peptide at a dose of 2 μg also caused delayed cardioinhibitory effects on the heartbeat of the pupae. Together with the ability to increase free sugar level in the haemolymph of larvae these were new physiological activities of sulfakinins in insects. Zopat-NVPL-4trunc. inhibited the muscle contractions of the two organs: hindgut and ejaculatory duct but it was inactive on the oviduct and the heart of the adult beetles. This peptide also increased free sugar level concentration in the haemolymph of Z. atratus larvae. These physiological actions are the first biological activities discovered for this group of the insect peptides. The present work showed pleiotropic activity of three neuropeptides and indicates that the visceral muscle contractions and the haemolymph sugar homeostasis in Z. atratus are regulated by complex mechanisms.