The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. Here, we present the first developmental study on these organs and report that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality.     

A Drosophila salivary gland mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria

Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands. We are particularly interested in mucin immune functions and found that gp150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria. By RT-PCR and RNAi experiments, we identified gp150 as the previously described I71-7, an ecdysone-induced salivary glue protein. We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects. Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Kinetics of ingested host immunoglobulin G in hemolymph and whole body homogenates during nymphal development of Dermacentor variabilis and Ixodes scapularis ticks (Acari: Ixodidae)

Patterns in the utilization of host immunoglobulin G (IgG) during nymphal development differed between Dermacentor varibilis (Say) and Ixodes scapularis Say ticks. In unfed nymphs of D. variabilis, host IgG was readily detectable in both hemolymph and whole body homogenates. In unfed nymphs of I. scapularis, host IgG was absent in hemolymph and at very low concentrations in whole body homogenates. Host IgG in unfed nymphs was undoubtedly the remnants of IgG acquired during the larval bloodmeal that persisted through metamorphosis to the nymphal stage. In both tick species, host IgG crossed the midgut into the hemocoel during the latter phases of engorgement. Concentrations of host IgG in I. scapularis declined considerably after replete nymphs molted to the adult stage. In contrast, concentrations of host IgG in D. variabilis remained elevated throughout metamorphosis to the adult stage. When larval D. variabilis were fed on a rat, then 2 months later as nymphs on a rabbit, the rat IgG (“old IgG”) present in unfed nymphs was totally replaced by rabbit IgG (“new IgG”) within 2 d of nymphs attaching to the rabbit. Presumably, the old IgG acquired from a previous bloodmeal was secreted via saliva into the new host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and Properties of Ferritin from the Last Instar Larval Hemolymph of Protaetia brevitarsis (Coleoptera)

Ferritin, an iron storage protein, has been purified from the last larval hemolymph of Protaetia brevitarsis (coleoptera) by KBr density gradient ultracentrifugation and resource Q (anion exchange chromatography) using fast performance liquid chromatography (FPLC) system. The iron content of ferritin was determined by atomic emission spectroscopy and FereneS stain. Ferritin of P. brevitarsis is shown to have two different subunits presented on a SDSPAGE in normal (N) and ironinjected (I) hemolymph. SDS PAGE revealed that the ferritin consists of two major polypeptides of 27 and 28 kDa in normal hemolymph. Interestingly, however, 30 kDa subunit was substituted for 28 kDa when iron was injected into the hemolymph. Apporximate isoelectric points of 27 kDa, 28 kDa, and 30 kDa ferritin subunits were 6.7, 6.75, and 6.8, respectively. Ferritin of P. brevitarsis was detected by FereneS stain and confirmed by Western blotting using its polyclonal antibody. Other characteristics such as amino acid composition and Nterminal amino acid sequence were investigated. Amino acid composition of ferritin (N and I) was rich in alanine, glycine, glutamine or glutamic acid and serine, but poor in histidine, arginine, methionine and phenylalanine.     

Purification of Major Hemolymph Protein of Great Wax Moth, Galleria mellonella

Major hemolymph protein (MHP) was purified from larval hemolymph of Galleria mellonella by KBr density gradient ultracentrifugation, ion exchange chromatography (DEAE‐Trisacryl M), YM‐50 ultrafiltration and gel permeation chromatography (Sephadex G‐100). MHP is composed of two subunit (MHP‐1 and MHP‐2). The molecular weights of each subunit were determined (MHP‐1 = 86 kDa and MHP‐2 = 84 kDa). MHP is present in both hemolymph and fat body during developmental stages, indicating this protein is carrying out some functions different from other major protein such as storage protein and lipophorin.     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Nitrite and Nitrate Levels in Individual Molluscan Neurons: Single-Cell Capillary Electrophoresis Analysis

Abstract: Cell and tissue concentrations of NO₂^? and NO₃^? are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO₂^? and NO₃^? levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-µm separation capillary and the NO₂^? and NO₃^? were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO₂^? and NO₃^? were <200 fmol (<4 µM in the neurons under study). The NO₂^? and NO₃^? levels in individual neurons varied from 2 mM (NO₂^?) and 12 mM (NO₃^?) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO₂^? was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO₃^? averaged 1.8 ± 0.2 × 10^?3M. Hemolymph NO₃^? in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 ± 0.2 × 10^?5M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 × 10^?4M). Capillary electrophoresis methods provide utility and convenience for monitoring NO₂^?/NO₃^? levels in single cells and small amounts of tissue.     

异源病毒感染对银纹夜蛾血淋巴酯酶的影响

对染病昆虫酯酶同工酶（简称酯酶）的变化进行分析测定,已成为了解病毒进入虫体靶器官后的病理生化变化以及病毒复制与虫体新陈代谢之间关系的重要途径之一,这方面的报道“J侧重于同源病毒——寄主系统的研究,本研究则针对银纹夜蛾（AWrammaagnata）幼虫感染异源粉纹夜蛾核型多角体病毒（TnNPV）后血淋巴酯酶的变化进行了探讨。现将结果报告如下：材料和方法1材料11虫源实验室内用半人工饲料饲养三代的健康银纹夜蛾五龄村幼虫。l．2毒源由中山大学昆虫研究所生物工程室提供的已纯化的TnNPV病毒株。1．3幼虫血淋巴样品的制备挑选工龄…     

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996