Glycosphingolipids in insects. Chemical structures of ceramide monosaccharide, disaccharide, and trisaccharide from pupae of Calliphora vicina (Insecta: Diptera)

The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(beta 1-1)Cer, Man(beta 1-4)-Glc(beta 1-1)Cer [with minor component Gal(beta 1-4)Glc(beta 1-1)Cer] and GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)-Cer. The ceramide composition of the parent insect glycosphingolipids is dominated by the 20:0 fatty acid, arachidic acid, and the sphingoid tetradecasphing-4-enine.     

Glycosphingolipids in insects. The amphoteric moiety, N-acetylglucosamine-linked phosphoethanolamine, distinguishes a group of ceramide oligosaccharides from the pupae of Calliphora vicina (Insecta: Diptera)

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as: (PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1- 4)Glc beta Cer; Gal(beta 1-3)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1- 3)Man(beta 1-4)Glc beta Cer; GlcNAc(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn- 6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer.     

Glycosphingolipids in insects. Chemical structures of two variants of a glucuronic-acid-containing ceramide hexasaccharide from a pupae of Calliphora vicina (Insecta: Diptera), distinguished by a N-acetylglucosamine-bound phosphoethanolamine sidechain

The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high-performance liquid chromatography. The acidic moiety was identified as glucuronic acid by beta-glucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometric and 1H-nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i.e. N-acetylglucosamine. The composition of the ceramide moiety was dominated by a C20:0 fatty acid (arachidic acid) and a C14:1 sphingoid base (tetradecasphing-4-enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be: GlcA(beta 1-3)Gal-(beta 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man (beta 1-4)Glc(beta 1-1)Cer; GlcA(beta 1-3)Gal(beta 1-3)GalNAc(beta 1-4)[2AeP-6]-GlcNAc(beta 1-3) Man(beta 1-4)Glc(beta 1-1)Cer. Such glucuronic-acid-containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.     

Campaniform sensilla of Calliphora vicina (Insecta,Diptera)

Summary A classification scheme of campaniform sensilla using morphological criteria was developed. All variations of the two most important outer structural elements, the cuticular cap and the cuticular collar, were taken into consideration: (a) the external shape of the cuticular cap; (b) the position of the cuticular cap in relation to the remaining cuticle; (c) the position of the cuticular collar in relation to the cuticular cap. This resulted in a classification of campaniform sensilla into 24 types. This typology was applied to the campaniform sensilla of Calliphora, which show considerable variations in their outer structures. According to SEM (scanning electron microscope) pictures and TEM (transmission electron microscope) sections we found only 9 out of 24 different types of campaniform sensilla in the fly.     

Development of labellar taste hairs in the blowfly,Calliphora vicina (Insecta,Diptera)

Summary The early development of taste sensilla has been studied with special emphasis on cilia, dendrite, and pore formation.In the 39-h stage (the first stage investigated) differentiation of sensilla is already under way. The mechanisms of differentiation of dendrites (39–48 h) deviate from the mechanisms described for differentiation of true cilia. In taste hairs the centrioles meet in the tip of the narrowed apical region of the sensory neuron. Together they sink deeper into this region and line up coaxially, thus forming the basal body complex. Thereafter, lateral contacts between this complex and the plasma membrane of the neuron are established. Formation of open connections between the trichogen lumen of the hair and the environment, or the dendrite lumen, was not observed.Electrophysiological data indicate that the sensilla become functional from 3 days before emergence onwards.List of Abbreviations B Basal body complex - bc Basal compartment - bs Basal sheath - bl Basement lamina - D Dendrite(s) - EPC Non-sensillar epithelial cells - ES Ecdysial sheath - GERL Granular endoplasmatic reticulum-lysosomes - HS Hemolymph space - P Protrusions - PRN Prospective receptor neuron - RER Rough endoplasmatic reticulum - RN Receptor neuron - n Neck - THC Thecogen cell - TOC Tormogen cell - TRC Trichogen cell - TRS Trichogen sprout     

Chemical distribution of glycosphingolipids in third-instar larval organs of the blowfly, Calliphora vicina (Insecta: Diptera).

As a first approach to testing the working hypothesis that glycosphingolipids are functionally involved in the ontogeny of insects, their chemical distribution in larval organs was determined and any stadium-correlated differences documented. Selected organs, i.e., the fatbody, striated muscle, intestinal tract, salivary glands, imaginal discs, and central nervous system, were dissected from seven-day-old larvae of the blowfly, Calliphora vicina, and their glycolipids isolated. Two-dimensional, high-performance thin-layer chromatography was used to separate the neutral and acidic glycolipids of each organ. Significantly different total glycolipid component-patterns were obtained for the individual organs, whereby, except for a number of additional uncharacterized components in the intestinal tract, the neutral glycolipids of all organs were found to be qualitatively similar. However, major quantitative differences between the selected organs were found in their total glycolipid-carbohydrate contents, as well as the respective quantitative neutral glycosphingolipid-component distributions. The acidic glycolipids showed pronounced qualitative as well as quantitative organ-dependent variations. Whereas the highest proportion of uncharged glycolipids was characteristic of the fatbody, a high proportion of zwitterionic glycolipid-components was observed to be typical of the central nervous system and imaginal discs, i.e., of organs persisting during larval life and throughout metamorphosis. Imaginal disc glycolipids were distinguished by their high content of acidic glycolipids, a putative reflection of the functional role of these glycoconjugates in regulated cell reorganization during metamorphosis.     

Effect of DEHP exposure in Calliphora vicina (Diptera: Calliphoridae)

Analysis of a possible delay between a person’s death and insect colonization plays a crucial role in forensic entomology and could be affected by numerous factors. Di- (2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor with the ability to target various organs, intervene with their normal function and cause harmful impact on organisms. The current paper points out the effects of exposure to DEHP and/or its metabolites on the larval stages of Calliphora vicina. We observed significantly lower larval length of the second instars (p = 0.04) and width of the third instars (p = 0.04) exposed directly by parent DEHP. Length of the first instars on the board of significance (p = 0.057) was also recorded. We found discrepancy in development, but development rate and larval weight did not significantly differ from the control group. In the experiment focusing on the effect of DEHP metabolites throughout mice cadavers exposed to this diester during life, we detected a statistically significantly increased larval weight of the post-feeding third instars in the experimental group (p = 0.04). Hypothetically, our preliminary results indicate a disruptive effect of DEHP and/or its metabolites on the metric parameters of larvae of the forensically important Calliphora vicina species, but further studies are needed.     

Mechanisms of wing rotating regulation in Calliphora erythrocephala (Insecta,Diptera)

Summary The blowfly Calliphora erythrocephala rotates its wings, i.e. changes the geometrical angle of attack, generating forces and moments for flight steering. There are two possibile ways to regulate this angle. The mechanisms for these movements are described. (1) The leading edge and the anterior part of the wing — between the costal vein and radial vein 4 — are pronated automatically due to the interaction of the moving parts during the downstroke. They are supinated during the upstroke. This is basic automatic regulation. (2) The posterior part of the wing — behind the anterior cross vein — is pronated and supinated independently of wing-drive. This is wing-drive independent additional regulation.Abbreviations a.c anterior cross vein - a.n anal veins - a.t.l anterior tergal lever - a.w anterior part of the wing - b.z bending zone - co costal vein - cr crossing of the tendons of the posterior notal wing process - c.s cross section - cu cubital vein - f fit or turning point of ventral radial vein 1 - h.a horizontal axis of pterale III - h.c humeral cross vein - h.co head of costal vein - h.r head of radial vein - k Klöppel - l.a longitudinal axis - me median vein - mp middle plate - ms mesoscutum - p anterior process of the anal veins - p.c posterior cross vein - pl pleurum - p.n.w.p posterior notal wing process - p.n.w.p 1–4 muscles 1–4 of the posterior notal wing process - pt I–III pterale I–III - p.t.l posterior tergal lever - p.w. posterior part of the wing - p.w.j pleural wing joint - r 1–4 radial veins 1–4 - r.s. ring stiffenings - sc subcostal vein - s.p semicircular part of the middle plate - s.t subalar tendon - t.c tip cross vein - te tegula - t.st thin strips - t.v.r tooth of ventral radial vein - v.a. vertical axis of pterale III - w wing - III 1–4 muscles 1–4 of pterale III     

Hybrid carrot seed crop pollination by the fly Calliphora vicina (Diptera: Calliphoridae)

Many insect species can contribute to crop pollination; however, most growers remain highly dependent on the managed honey bee (Apis mellifera L.) for this service. The European Blue Blow Fly Calliphora vicina Robineau‐Desvoidy, 1830 is one species with potential use as a pollinator. It occurs worldwide and is easy to rear. Caged trials conducted within a hybrid carrot (Daucus carota L.) seed crop found C. vicina to be an effective pollinator. Seed yield (number and weight) from field‐grown carrot plants caged with C. vicina, but excluding all other large flower visitors (body width > 3 mm), was similar to seed yield from uncaged plants in the presence of honey bees (Apis mellifera L.) and other insects. In contrast, caged plants without C. vicina produced 10‐fold less seed. Under open field conditions, C. vicina spent an average of 71.0 s per umbel compared to 54.4 s for honey bees; however, under caged conditions, C. vicina spent more time on average per umbel (128.9 s). Counts of C. vicina and honey bees on umbels outside of cages and C. vicina inside cages found that honey bees were most abundant on days with maximum temperature > 25°C, while C. vicina was more abundant on cooler days around 20°C. C. vicina may therefore be a useful pollinator of crops grown in isolation cages for plant breeding purposes as well as in open fields when climatic conditions are less favourable for optimal honey bee activity.     

Contact behaviour of tenent setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae)

To enable strong attachment forces between pad and substrata, a high proximity between contacting surfaces is required. One of the mechanisms, which can provide an intimate contact of solids, is a high flexibility of both materials. It has been previously presumed that setae of hairy attachment pads of insects are composed of flexible cuticle, and are able to replicate the surface profile. The aim of this work was to visualise the contact behaviour of the setae by freezing-substitution technique to understand setal mechanics while adhering to a smooth surface. This approach revealed considerable differences in the area of the setal tips between contacting and non-contacting pulvilli. Based on the assumption that setae behave like a spring pushed by the tip, a spring constant of 1.31 N m(-1) was calculated from direct measurements of single setae by atomic force microscopy. In order to explain the relationship between the behaviour of the attachment setae at a microscale and leg movements, high-speed video recordings were made of walking flies. This data show that some proximal movement of the leg is present during contact formation with the substrate.     

A villin-like protein in the intestinal brush border of Calliphora vicina R.D. larvae (Diptera : Calliphoridae)

The brush border of the larval midgut and hindgut in Calliphora vicina (Diptera : Calliphoridae) shows a positive staining with a rabbit antiserum directed against pig villin. This protein has been extensively studied in chicken and in several mammals; it is known to bind together the actin filaments of the microvillar cytoskeleton. The presence of a villin-like protein in insect microvilli has never been reported. The immunodetection of villin in both hind- and midgut points out that villin exists in ectodermal tissues as well as in endodermal or mesodermal ones. Moreover, the “insect villin” seems to present the polypeptidic sequence of 13 amino acids, which has been found in both mammalian and avian villin. That may indicate that villin is a conserved molecule in several taxa.     

Morphology of the first instar of Calliphora vicina, Phormia regina and Lucilia illustris (Diptera, Calliphoridae)

Scanning electron microscopy documentation of first instar Calliphora vicina Robineau-Desvoidy, Phormia regina (Meigen) and Lucilia illustris (Meigen) (Diptera: Calliphoridae) is presented for the first time, and the following morphological structures are documented: pseudocephalon; antenna; maxillary palpus; facial mask; labial lobe; thoracic and abdominal spinulation; spiracular field; posterior spiracles, and anal pad. Light microscopy documentation and illustrations are provided for the cephaloskeleton in lateral and ventral views. New diagnostic features are revealed in the configuration of the facial mask, cephaloskeleton and posterior spiracles. The first instar morphology of C. vicina, Ph. regina and L. illustris is discussed in the light of existing knowledge about early instars of blowflies.     

Growth modulating and cytotoxic effects of the peptide from hemolymph of blow fly Calliphora vicina (Diptera, Calliphoridae) in vitro

Biological activity of alloferon (AF), peptide, consisting of 13 a. a. isolated from hemolymph of experimentally infected blow fly Calliphora vicina was studied. AF in concentrations form 1 x 10(-5) to 250 microg/ml was added into the culture medium of the target cell lines K562, J-96, P388DI, Hep22a and 3T3B-SV40. First two days the peptide in concentrations 1 x 10(-5) and 1 x 10(-3) microg/ml in most cases stimulated the cell proliferative activity and suppressed the cell growth when applied in concentrations 10 and 100 microg/ml. Trend in growth modulating effect was dependent on duration of AF treatment. The peptide did not expressed cytotoxic effect with the exception of destruction of P388D1 cells that was registered after 96 h incubation in the medium initially contained 100 microg/ml AF. Simultaneously, cytotoxic and growth modulating effects of doxorubicin and cytosinarabinoside, as well as hybrid molecules, AF--cytosinarabinoside (cytal) and AF--doxorubicin (doxal) have been studied. Doxorubicin and cytosinarabinoside expressed greater growth inhibition effect compared to the hybrid molecules and AF itself. The results obtained with mass cell cultures were supported by experiments where P388D1 cells clonogenic capacity was tested. Besides, it has been demonstrated that AF rapidly penetrates into cytoplasm of J-96 cells and concentrates mainly into and around the cell nuclei.     

Induction of larval diapause in the blowfly, Calliphora vicina R.-D. (Diptera, Calliphoridae) under field and laboratory conditions

Field experiments conducted in the environs of St. Petersburg (Russia) with a local population of Calliphora vicina showed that induction of larval diapause under natural conditions was significantly dependent on day lengths and temperature. The maternal photoperiodic response had a distinct threshold: the first diapausing larvae hatched from the eggs laid in the middle of August when the day length was 16 h; at shorter photoperiods, the fraction of diapausing larvae depended only on temperature. At the mean temperature of 16°C, larval diapause was rarely recorded; at 12–13°C, about 50% of the larvae entered diapause; at 7–9°C, nearly all the larvae entered diapause. These results of the field experiments agree well with the parameters of photoperiodic and thermal responses studied in the laboratory at constant temperatures and photoperiods.     

Distribution of F-actin in the compound eye of the blowfly,Calliphora erythrocephala (Diptera,Insecta)

Summary Sexual stimulation of males has been reported to affect hypothalamic oxytocinergic systems. In the present study we used radioimmunoassays of micro-dissected forebrain regions and immunocytochemical analysis of Vibratome sections to study the oxytocin systems of naive males, males killed after one mating, and males mated daily with different receptive females for 3 weeks. In males that had mated once, less oxytocin-immunoreactive neurons were observed in the paraventricular (PVN), supraoptic (SON) and periventricular (NPE) nuclei than in naive males. However, after repeated matings, the number of immunoreactive neurons and their staining intensity was increased in these regions. Furthermore, additional oxytocinergic neurons could be found in the lateral subcommissural nucleus, the zona incerta and the ansa lenticularis of repeatedly mated males. Oxytocin-immunoreactive neurons were only occasionally seen in these areas in unmated males or in animals that had been killed after initial mating. Radio-immunoassays of microdissected PVN, SON, NPE and the lateral hypothalamus confirmed the reduction in oxytocin-immunoreactive levels after a first mating by a male and the increase after repeated matings. It is likely that oxytocin secretion into peripheral and portal circulation is stimulated by the endocrine conditions associated with initial mating. These immediate effects may be followed by the activation of synthesis in oxytocin neurons in several sites of the basal forebrain.     

Neurogenic and septic inductions of synthesis of peptide antibiotics in larvae of Calliphora vicina R.-D. (Diptera: Calliphoridae)

Synthesis of antimicrobial peptides in diapausing larvae Calliphora vicina can be induced by two different pathways. One pathway is well known in insects and includes recognition of microbial particles by the pattern-recognizing receptors. The other pathway includes perception and transduction of stress signal to immunocompetent cells by neuroendocrine system. This phenomenon consists in stimulation of synthesis of defensins, cecropins, and diptericins under effect of chromic stimulation of mechanoreceptors with ligature applied on the larva head end. Formation of immune response in brain is established to need less than 30 s, after which isolation of the neuroendocrine complex does not eliminate activation of immune response As judging from rate of the neurogenic induction, transduction of the stimulating signal from brain to the immune system cells can be connected with release into hemolymph of biogenic amines or other neurohormones stored preliminarily in the neurohemal organ. The nature of this inductor at present remains unknown, as analysis of role of octopamine, dopamine, and adipokynetic hormone did not reveal stimulating effect on synthesis of bactericidal peptides. Physiological mechanism of this phenomenon is not finally understood, its key links seem to be CNS, hormonal factor of cardial bodies, and system of antimicrobial peptides. Synthesis of antimicrobial peptides is directly regulated by the neuroendocrine system that can produce both stimulating and stress action by reminding in this aspect the known immunoneuroendocrine interrelations in vertebrates. The existence of similar integrating mechanisms in such polar animal kingdom groups which are insects and vertebrates indicate that they are more ancient than this was considered earlier.     

Glycosphingolipids in cestodes. Chemical structures of ceramide monosaccharide, disaccharide, trisaccharide and tetrasaccharide from metacestodes of the fox tapeworm, Taenia crassiceps (Cestoda: Cyclophyllidea).

The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal-phase TLC pattern of the neutral-fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Gal beta 1Cer, Gal beta 6Gal beta 1Cer and Gal beta 6Gal beta 6Gal beta 1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Gal beta 6Gal beta 6Gal beta 6Gal beta 1Cer and the component Gal alpha 4Gal beta 6Gal beta 6Gal beta 1Cer (both occurring in approximately equimolar proportions). The long-chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse-phase TCL, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.     

The rate of preimaginal development of the blowfly, Calliphora vicina R.-D. (Diptera, Calliphoridae) under field and laboratory conditions

Open field experiments with the blowfly, Calliphora vicina originating from the environs of St. Petersburg showed that the correlation between the rate of its preimaginal development under the natural conditions and the mean daily temperature could be very closely approximated by linear regression. The sum of effective temperatures required for development from the egg to the puparium constituted ca 140 degree-days and from the egg to the adult, ca 320 degree-days with the lower thresholds of 5.8 and 4.8°C, respectively. The minimum duration of development (6–8 days from the egg to the puparium and 16–20 days from the egg to the adult) was recorded during the period of the highest mean temperatures (22–23°C) from July 8 to August 15 of 2010 and 2011. The rate of development under natural thermorhythms was not siggnificantly different from that under the laboratory conditions at constant temperatures from 12 to 23°C.