Permeability of the peritrophic membranes of some Diptera to labelled dextrans

The permeability of the continuous, tube-like peritrophic membrane of some Diptera was investigated. Dyes, haemoglobin, cytochrome c, horseradish peroxidase, and especially dextran fractions labelled with the fluorescent dye fluorescein isothiocyanate were used as markers. The peritrophic membrane of larvae of Aedes aegypti was permeable only to dextran with a molecular weight of less than 2,400 Daltons; Evans Blue (960.8 Daltons) permeated only slowly through this peritrophic membrane. Labelled dextran with a mol. wt of 32,000 Daltons did not penetrate the peritrophic membrane of larvae of Anopheles stephensi. Dextrans larger than 32,000 Daltons did not permeate through the peritrophic membrane of Culex pipiens, Odagmia ornata, Anisopus (Phryne) cinctus, Sarcophaga barbata and Calliphora erythrocephala. Labelled dextran with mol. wt of 4,000–6,000 Daltons penetrated only slowly, and dextran of 6,200 Daltons did not penetrate the peritrophic membranes of adults of Sarcophaga barbata. The peritrophic membranes of the blowfly, Calliphora erythrocephala, were permeated slowly by dextran of 6,200 Daltons but not by dextran of 17,200 Daltons. Dextrans are readily soluble in water where the long chains form coils of round or oval shape. Charged protein molecules are more compact with smaller radii when compared to a dextran fraction of the same molecular weight. Therefore the results of investigations on permeability have to be compared in terms of the effective radii and not of the molecular weight.     

Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)

For the first time a sugar receptor (lectin) has been localized by electron microscopy in an invertebrate. The peritrophic membrane of the blowfly larva, Calliphora erythrocephala, is shown here to express lectins with high specificity for mannose. The lectin is restricted to the lumen side of the peritrophic membrane. The surface of the midgut epithelium is devoid of mannose-specific lectins. It is suggested that the midgut epithelium has lost these lectins during the course of evolution in favour of the peritrophic membrane which is secreted by specialized cells only at the beginning of the midgut.Peritrophic membranes and the midgut epithelium lack lectins specific for galactose. The lumen side of the peritrophic membrane of the larvae has mannose and/or glucose residues, and it is densely packed with two species of bacteria, Proteus vulgaris and P. morganii. These also have mannose-specific lectins as well as mannose residues on their pili. The existence of mannose-specific receptors and mannose residues on both, peritrophic membranes and bacteria, leads to the assumption of mutual adherence between the two surfaces.     

The fine structure of peritrophic membranes of the blowfly, Calliphora erythrocephala, grown in vitro under different conditions.

The formation of peritrophic membranes (PM) in vitro was studied in a flow chamber in order to avoid the accumulation of metabolic substances during prolonged incubation. During the first 8 to 10 hr of incubation the production of PM was nearly constant—3·5 ± 1·4 mm PM/hr. After about 10 hr it decreased and stopped after about 35 hr. Between 1 and 8 hr after the beginning of incubation the width of the periodic crossband pattern reached or nearly reached the values found in PM which had formed in vivo; afterwards it decreased more and more. During the first 20 min of incubation a ‘disturbed zone’ of PM without any regular crossband pattern is formed.In the cardia of adult Calliphora erythrocephala there are three formation zones forming three PM of different fine structures. The fine structure of PM 1 to 3 formed in vitro during the first 6 to 8 hr of incubation in Leloup's medium 1, with an osmolarity of 340 mOsmol, a pH of 6·8, and a temperature of 27°C, does not differ from the PM grown in vivo. PM 1–3 grown in vitro in Tyrode's solution with added glutamine or in Leloup's medium with added β-ecdysone show a considerable increase in thickness and a disturbed formation of the electron dense layer of PM 1.     

In vitro synthesis of peritrophic membranes of the blowfly, Calliphora erythrocephala.

Tyrode solution containing added glutamine and Leloup's medium 1 has been used as a basic medium for the in vitro culture of the so-called proventriculus of adult Calliphora erythrocephala to elucidate some of the factors controlling the synthesis of peritrophic membranes (PM) in vitro. The formation rate was chosen as a quantitative criterion for the evaluation of the modifications of the incubation media.After systematic variation of osmolarity, pH, and temperature optimal formation rates were obtained in media with an osmolarity of 320 to 360 mOsmol, a pH of 6·8, and an incubation temperature of 27°C. Under these conditions the average rate of formation was in the modified Tyrode solution 3·0±1·1 mm PM/hr, and in Leloup's medium 3·6±0·8 mm PM/hr. In the modified Tyrode solution the formation of PM was complete after 5 to 7 hr, whereas in Leloup's medium it continued up to 24 hr. The addition of β-ecdysone caused an increase of the formation rate of PM to 4·5 to 5·5 mm PM/hr.The results obtained led to the hypothesis that an osmotically regulated enzyme system could be the limiting factor of the formation rate of peritrophic membranes, i.e. a system which could regulate the internal osmolarity of the formative cells by the interconversion of a bulk polymer and its monomer which are needed for the synthesis of PM.     

Experimentally induced in vivo increase in size of calcium citrate crystals in praying mantids

Regular, membrane-bound crystals of calcium citrate are secreted by a special gland in adult female Sphodromantis lineola, and reach a size of ca. 10 μm long by ca. 5 μm wide and ca. 4 μm high before they are extruded into the oötheca at each oviposition. Substantially larger, but still regular, crystals were obtained from the glands of insects which had been injected with laboratory prepared calcium or magnesium citrate, and from glands which had been artificially prevented from extruding their contents for extended periods (e.g. up to 70 days). Enlarged crystals retain the membranes which are probably responsible for maintaining the characteristic trapeziform shape.     

Traumatic Myiasis Caused by an Association of Sarcophaga tibialis (Diptera: Sarcophagidae) and Lucilia sericata (Diptera: Calliphoridae) in a Domestic Cat in Italy

We describe here a rare case of traumatic myiasis occurred in August 2014, caused by an association of 2 Diptera species, Sarcophaga tibialis Macquart (Diptera: Sarcophagidae) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), in a domestic cat in northern Italy. Species identification was based on adult male morphology. The present case is the first report of S. tibialis as an agent of myiasis in Italy, and also the first ever report of myiasis caused by an association of S. tibialis and L. sericata. The cat developed an extensive traumatic myiasis in a large wound on the rump, which was treated pharmacologically and surgically. The biology, ecology, and distribution of S. tibialis and L. sericata are also discussed. A literature review is provided on cases of myiasis caused by S. tibialis, and cases of myiasis by L. sericata involving cats worldwide and humans and animals in Italy.     

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

Summary The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microseopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5–10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthognal texture of bundles of microfibrils that are thought to contain chitin. The pesence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

Length variation in the non-transcribed spacer of Calliphora erythrocephala ribosomal DNA is due to a 350 base-pair repeat

The non-transcribed spacer regions in the ribosomal DNA cistrons of Calliphora erythrocephala vary in length. This length variation is shown to be due to variable numbers of small repeated units found in certain areas of the NTS² regions, as has been found in several other systems. In contrast to the other organisms, however, the length variation in C. erythrocephala leads to the formation of only two major size classes with a length difference of about 1 kb. We have investigated the nature of this length difference by means of electron microscope heteroduplex and restriction enzyme digestion analyses. We demonstrate that the length variation in C. erythrocephala is due to a variation in the number of 350 bp repeating units defined by the presence of sites for three restriction enzymes, HhaI, Sau3A and AluI. Furthermore, the existence of an XbaI site within one 350 bp unit and the lack of a HhaI restriction site within another 350 bp unit reveals that at least some sequence differences exist among the repeating units. These restriction site differences can be viewed as genetic markers and lead to the hypothesis that the longer NTS class originated from the shorter NTS class by an unequal crossover event that served to duplicate the region containing repetitive units. An interesting feature of our observation is that not all NTS length classes are equally represented, suggesting special rules for the unequal recombination events often hypothesized as the basis for such variation.     

Microbiota Associated with the Gastrointestinal Tract of the Common House Cricket, Acheta domestica

The location and morphology of the bacteria associated with the gastrointestinal tract of Acheta domestica were studied, and these bacteria were partially characterized. Bacteria were associated with the peritrophic membrane in the midgut and with the gut wall and cuticular structures of the hindgut. No bacteria were associated with the fat bodies. Colony-forming unit determinations indicated that there were three times more cultivatable bacteria in the hindgut than in the midgut. Of these bacteria, 40 to 85% cleared uric acid anaerobically, and 90 to 100% cleared uric acid aerobically. Of the 25 isolates obtained, 21 belonged to the genera Citrobacter, Klebsiella, Yersinia, Bacteroides, and Fusobacterium.     

Cometoides pechumani sp. n. (Protozoa: Eugregarinida), a gregarine parasite of salt marsh deerflies (Diptera: Tabanidae)

Cometoides pechumani sp. n. is described from larvae and adults of Chrysops fuliginosus and from a pupa of C. atlanticus. Stages of the gregarine were found in the fore-, mid-, and hindguts. The globular to spherical epimerite possessed at least 14 long filaments. The cephalic sporadins were elongate, cylindrical, and tapered. Mean length was 998 μm. Mean diameters of the gametocysts were 519.8 × 558 μm. Oocysts (spores) are hexagonal in outline with polar spines and two bands of equatorial spines. Their mean length and width were 7.35 × 4.32 μm. Incidence of infection of field-collected C. fuliginosus larvae was greatest in summer when rates were as high as 89%. Infection during winter ranged from 30 to 58%. Incidence of infection of adult C. fuliginosus never exceeded 7%.     

Rearing bacteria and maggots concurrently: a protocol using Lucilia sericata (Diptera: Calliphoridae) as a model species

Maggot debridement therapy using live Lucilia sericata (Meigen) larvae is an efficient and cost-effective way to treat chronic wounds. The recent increase in studies to assess the antibacterial properties of L. sericata has created a need for a simple, low-cost, and comprehensible rearing and investigative method for researchers with little or no entomological experience. This paper describes and evaluates a reproducible protocol for sterilising and rearing blowfly larvae utilising two sterile artificial diets (blood–yeast agar and pre-prepared blood agar plates) that is suitable for directly investigating the effect of larvae on microbial growth. Using Lucilia sericata as a model, the results show that larval growth on the pre-prepared blood agar diet is detrimental to larval growth and survival, whereas larval growth and survival on the blood–yeast agar diet are comparable to those of larvae raised on porcine liver. This diet is proposed as a standard for blowfly and bacteria interaction studies investigating clinical microbial strains. Developmental data are provided for L. sericata larvae raised on both sterile and nonsterile diets so that researchers can determine the effect of treatment based on the length of time for larvae to reach the required life stage at 25 ± 2 °C. Information on larval ageing (instars at an average of 1, 2, 3 and 4 days), oviposition times (4–5 days after adult emergence) and adult longevity on the diets (102–116 days) is also given.     

Novel Myxobolus and Ellipsomyxa species (Cnidaria: Myxozoa) parasiting Brachyplatystoma rousseauxii (Siluriformes: Pimelodidae) in the Amazon basin,Brazil

We describe two novel myxosporean parasites from Brachyplatystoma rousseauxii, an economically important freshwater catfish from the Amazon basin, Brazil. Myxobolus tapajosi n. sp., was found in the gill filaments of 23.5% of 17 fish, with myxospores round to oval in frontal view and biconvex in lateral view: length 15 (13.5–17) μm and width 10.7 (9.6–11.4) μm; polar capsules equal, length 5.8 (4.6–7.1) μm and width 3 (2.3–3.8) μm containing polar tubules with 6–7 turns. Ellipsomyxa amazonensis n. sp. myxospores were found floating freely or inside plasmodia in the gall bladder of 23.5% of fish. The myxospores were ellipsoidal with rounded extremities: length 12.8 (12.3–13.6) μm and width 7.6 (6.7–8.7) μm; with two equal, slightly pyriform polar capsules, length 3.8 (3.8–4.0) μm and width 3.1 (2.5–3.4) μm, containing polar tubules with 2–3 turns. We combined spore morphometry, small-subunit ribosomal DNA data, specific host, and phylogenetic analyses, to identify both of these parasites as new myxozoan species. Maximum likelihood and Bayesian analyses showed that Myxobolus tapajosi n. sp. clustered in a basal branch in a subclade of parasites from exclusively South American pimelodid fishes. Ellipsomyxa amazonensis n. sp. clustered within the marine Ellipsomyxa lineage, but we suspect that although the parasite was collected in freshwater, its hosts perform a large migration throughout the Amazon basin and may have become infected from a brackish/marine polychaete host during the estuary phase of its life.     

A size analysis of planktic foraminifera from the Arabian Sea

Planktic foraminiferal faunas from different environments in the Arabian Sea were size fractionated using 14 sieves with meshes between 100 and 710 μm, to assess the effect of the sieve mesh size cut off level on the faunal composition and to determine the size frequency distribution of individual species. Nine samples from a plankton pump and a towed net, a sediment trap, a box-core and a piston core were selected, to cover living and settling flux faunas as well as fossil faunas from the sediment. In living faunas, most species show an exponential size frequency distribution, with highest numbers in the finest interval of the size spectrum. In sediment trap and core samples, individual species size frequency distributions may consist of: (1) an exponential distribution of relatively small pre-adult specimens; (2) a Gaussian-shaped distribution of larger specimens, which may be classified as adult or terminal; or (3) a combination of both. The distributions are separated using a best fit technique. The composition of the total planktic foraminiferal fauna strongly changes along the size spectrum. Dominant taxa in >355 μm fractions are Orbulina universa, Globorotalia menardii, Globorotalia tumida, Globigerinella siphonifera and Globigerinoides sacculifer, in 125–355 μm fractions Globigerina bulloides, Globigerinoides ruber, Neogloboquadrina dutertrei and Globigerinita glutinata, and in <125 μm fractions Dentigloborotalia anfracta, Tenuitella compressa, Tenuitella iota, Turborotalita quinqueloba and the immature specimens of larger species. Consequently, the choice of the sieve mesh size strongly determines the percent composition of the assemblage and in turn the paleoceanographic interpretations based on these counts. Species richness and the Shannon diversity increase with decreasing sieve mesh size, while equitability generally decreases with decreasing size. In the water column approximately 60% of the fauna (>100 μm) is present in the 100–125 μm fraction and 1–6% is larger than 250 μm. In samples representing a settling flux (sediment trap and sediment samples) 29–57% of the fauna is present in the 100–125 μm fraction, while 6–23% is larger than 250 μm. Size frequency distributions of the dextral Neogloboquadrina complex (= Neogloboquadrina dutertrei and Neogloboquadrina pachyderma + P–D intergrades) show a bimodal pattern; a smaller peak reflecting dextral Neogloboquadrina pachyderma, and a larger peak of adult Neogloboquadrina dutertrei. By applying a best fit technique to the data, the two species may be separated from each other. In size fractions larger than 150 μm most species have reached the adult stage of ontogeny and we recommend this mesh size for standard faunal analysis. In addition, sieve mesh sizes of 125 and 250 μm have to be used to obtain a reliable estimate of the abundance of small and large species, respectively.     

Ortholinea scatophagi (Myxosporea: Ortholineidae), a novel myxosporean infecting the spotted scat,Scatophagus argus (Linnaeus 1766) from southwest coast of India

A new species of myxosporean, Ortholinea scatophagi n. sp. infecting the urinary bladder of the spotted scat, Scatophagus argus (Linnaeus 1766) is described. O. scatophagi n. sp. is characterized by spherical myxospores with a slightly flattened anterior end and equal spore valves with extra sutural ridges on its surface; measured 7.34 ± 0.67 μm in length, 6.90 ± 0.71 μm in width and 6.48 ± 0.37 μm in thickness. Two polar capsules, equal, spherical to oval in shape, arranged diametrically opposite and measured 2.59 ± 0.42 μm in length and 2.24 ± 0.35 μm in width. Polar filaments, 21.84 ± 2.86 μm long, with four to five coils. Scanning electron microscopy revealed the presence of extra sutural ridges on spore surface. Pansporoblasts spherical to irregular in shape, measured 31.08 ± 2.67 μm in length and 13.88 ± 5.40 μm in width; Monosporic, disporic and polysporic plasmodial stages were observed; plasmodia spherical or irregular in shape with granular cytoplasm containing refractile granules. The species was compared with 23 existing nominal species of Ortholinea, based on morphology and morphometry. Molecular analysis resulted in a 1773 bp long SSU rDNA sequence (GenBank accession number MN 310514). In phylogenetic analyses the present parasite clustered with other members of Ortholinea, under the freshwater urinary clade. Considering the morphologic, morphometric and molecular differences with previously described species of Ortholinea, and differences in host and geographic locations, the present species is treated as new and the name Ortholinea scatophagi n. sp.is proposed.     

Cellular organization and peritrophic membrane formation in the cardia (Proventriculus) of Drosophila melanogaster

The peritrophic membrane of Drosophila melanogaster consists of four layers, each associated with a specific region of the folded epithelial lining of the cardia. The epithelium is adapted to produce this multilaminar peritrophic membrane by bringing together several regions of foregut and midgut, each characterized by a distinctively differentiated cell type. The very thin, electron-dense inner layer of the peritrophic membrane originates adjacent to the cuticular surface of the stomadeal valve and so appears to require some contribution by the underlying foregut cells. These foregut cells are characterized by dense concentrations of glycogen, extensive arrays of smooth endoplasmic reticulum, and pleated apical plasma membranes. The second and thickest layer of the peritrophic membrane coalesces from amorphous, periodic acid-Schiff-positive material between the microvilli of midgut cells in the neck of the valve. The third layer of the peritrophic membrane is composed of fine electron-dense granules associated with the tall midgut cells of the outer cardia wall. These columnar cells are characterized by cytoplasm filled with extensive rough endoplasmic reticulum and numerous Golgi bodies and by an apical projection filled with secretory vesicles and covered by microvilli. The fourth, outer layer of the peritrophic membrane originates over the brush border of the cuboidal midgut cells, which connect the cardia with the ventriculus.     

5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

Visual observations on the process of digestion and the production of faecal pellets in the chaetognath Sagitta hispida Conant

The passage of food through the gut of Sagitta hispida Conant is described as a batch process and the formation of membranes which surround the faecal material is demonstrated photographically. The utility of such a peritrophic membrane system is considered in the light of the marine ecosystem as a whole.     

Protein components of two different regions of an intestinal epithelial cell membrane: Regional singularities

The two different regions of the plasma membrane, i.e. apical and basolateral membranes, of intestinal epithelial cells were analyzed as to their proten components. They showed very contrasting profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apical membranes possessed several major components with apparent molecular weights larger than 108 000, most of which were also periodic acid-Schiff reagent positive. In contrast, there were no protein components with corresponding molecular weights in the basolateral membrane. The electrophoretic profile of the latter was strinkingly simple. The dominant band was assigned a molecular weight of 101 000 and was periodic acid-Schiff reagent negative. No major components were shared by the two membranes.     

The interaction of calcium and procaine with hepatocyte and hepatoma tissue culture cell plasma membranes studied by fluorescence spectroscopy

The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca²⁺ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K_D = 120,μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca²⁺ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K_D (40–50 μm). Procaine was also shown to completely displace Ca²⁺ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K_D = 74 μM) from the hepatocyte system. The addition of Ca⁺² resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K_D values of 15 μm and 50 μm. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca²⁺. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.