Hemolymph Research Articles

A novel role for an insect apolipoprotein (apolipophorin III) in beta-1,3-glucan pattern recognition and cellular encapsulation reactions.

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and beta-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of beta-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

Proteomic Analysis of the Systemic Immune Response of Drosophila

Improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize proteins involved in a physiological process, such as the immune response of the insect model Drosophila melanogaster. Profiling of the proteins present in the hemolymph (insect blood) of noninfected flies versus flies infected with bacteria or fungi was performed by two-dimensional gel electrophoresis, silver or Coomassie staining, and image analysis. Through this differential analysis, more than 70 out of 160 spots were up- or down-regulated by at least 5-fold after microbial infection. Coomassie staining, in-gel digestion, and database searches yielded the identity of a series of proteins that are directly involved in the Drosophila immune system. This included proteases, protease inhibitors, and recognition molecules such as prophenoloxydase-activating enzymes, serpins, and Gram-negative binding protein-like. Proteins with a potential function in the immune response were also identified, such as an odorant binding protein, peptidylglycine alpha-hydroxylating monooxygenase, and transferrin, affording new candidates for further investigation of innate immune mechanisms. Moreover, several molecules resulting from the cleavage of proteins were detected after the fungal infection. Altogether, this first differential proteomic analysis of the immune response of Drosophila paves the way for the study of proteins affected during innate immunity.

The Effect of Unilateral and Bilateral Eyestalk Ablation on Haemolymph Osmotic and Ionic Regulation and Gill Na+, K+-atpase Activity In Marsupenaeus Japonicus (bate, 1888) (Decapoda, Natantia)

The effects of eyestalk ablation on osmotic and ionic regulation and Na + , K + -ATPase activity of Marsupenaeus japonicus were investigated in a period of 24 hrs. The osmolarity, potassium, and calcium concentration were high in the shrimps treated bilaterally, intermediate in the shrimps that were treated unilaterally, and low in the intact shrimps, while all those values of eyestalk ablated shrimps increased with increasing treatment time. However, the haemolymph sodium and chloride concentra- tion of eyestalk ablated shrimps were lowered, and also decreased further with increasing treatment time. The bilaterally and unilaterally ablated shrimps had higher total ATPase and Na + , K + -ATPase activities in the gills than the intact shrimp, and the ATPase activities of the ablated ones were raised with increasing treatment time up to 16 hrs. Therefore, eyestalk ablation of shrimps causes changes in the osmotic and ionic regulation, and also affects the Na + , K + -ATPase activity of the gills. Les effets de l'ablation du pedoncule oculaire sur la regulation osmotique et ionique et sur l'activite de la Na + , K + -ATPase de Marsupenaeus japonicus ont ete etudies sur une periode de 24 heures. L'osmolarite, la concentration en potassium et en calcium etaient elevees chez les crustaces traites bilateralement, intermediaire chez ceux traites unilateralement et faible chez les non traites, et chez les crevettes ayant subi une ablation complete, toutes ces valeurs augmentaient avec la duree du traitement. Cependant, le sodium de l'hemolymphe et la concentration en chlorures chez les specimens prives de pedoncule oculaire avaient diminue et ce encore plus lorsque la duree du traitement etait prolongee. Les crustaces traites bilateralement, et ceux traites unilateralement avaient des activites d'ATPase totale et de Na + , K + -ATPase plus elevees dans les branchies que les non traites, et les activites d'ATPase des specimens traites augmentaient avec la duree de traitement, jusqu'a 16 heures. Ainsi, l'ablation des pedoncules oculaires chez ces crevettes provoque des modifications de la regulation osmotique et ionique, et affecte egalement l'activite de la Na + , K + -ATPase des branchies.

Is the glucose concentration in the haemolymph a suitable indicator of circadian rhythmicity in Nephrops norvegicus (Decapoda, Nephropidae)?

The haemolymph glucose concentration shows marked fluctuations over a 24-hour cycle in several decapods. In the present study, the influence of the diel light cycle as a modulator of glucose concentration was evaluated in the haemolymph of Nephrops norvegicus in the northwestern Mediterranean. Haemolymph samples were collected from animals caught by bottom trawls at two depths, 100 m and 400 m, during two different seasons, October and June. A time series analysis showed the absence of a rhythmic pattern in the glucose concentration at either depth and in either season. Knowing that fishing stress increases haemolymph glucose concentration, laboratory experiments were also planned under controlled conditions, under different photoperiod regimes (day-night: 12-12, 18-6, 6-18). Again, no rhythmic fluctuations in glucose concentration were detected. Field glucose concentrations were higher than those determined in laboratory experiments (t-test, p ≤ 0.05), indicating a reference value of the stress for animals caught by trawling nets. Therefore, the absence of rhythmicity in both field survey and laboratory experiments suggests that this physiological variable is not a suitable indicator of the circadian rhythmicity in animals dwelling at a depth greater than 100 m. We propose that rhythms in locomotor and emergence behaviour and their influence on the underlying physiology may explain the glucose arrhythmicity in this species. La concentracion de glucosa en hemolinfa muestra marcadas fluctuaciones diarias en varios decapodos. En este estudio se evalua la influencia del ciclo luminoso diario sobre la concentracion de glucosa en la hemolinfa de Nephrops norvegicus del Mediterraneo oeste. La hemolinfa fue extraida de ejemplares capturados por artes de arrastre en profundidades de 100 y 400 m, durante Octubre y Junio. El analisis de la serie temporal de datos no mostro ningun patron ritmico en la concentracion de glucosa, ni por profundidad ni por epoca del ano. Asumiendo que el efecto de estres de pesca incrementa la concentracion de glucosa, se realizaron tambien extracciones en condiciones controladas de laboratorio bajo distinto fotoperiodo (dia-noche: 12-12, 18-6, 6-18). Tampoco se detectaron variaciones ritmicas. Los resultados muestran que las concentraciones de glucosa de los animales procedentes del campo fue superior a la de los animales de laboratorio (t-test, p ≤ 0.05) indicando un valor de referencia de estres para animales procedentes de la pesca. La ausencia general de variaciones en la concentracion de glucosa en la sangre, tanto de los animales procedentes del campo como los estudiados en laboratorio sugieren que esta variable fisiologica no es un buen detector de la ritmicidad diaria en Nephrops. Se discute el papel que el comportamiento locomotor y de emergencia de las madrigueras en Nephrops pueda tener en la arritmicidad diaria de glucosa en esta especie.

Microbial infection causes the appearance of hemocytes with extreme spreading ability in monolayers of the tobacco hornworm Manduca sexta

The ability to adhere to and spread on a surface is a common property of insect blood cells. Spreading on a glass surface by insect hemocytes is often used as a measure of immune fitness that can be inhibited by some insect pathogens and parasites. Here, we report that upon infection of the tobacco hornworm Manduca sexta with either a fungus ( Beauveria bassiana) or a bacterium ( Photorhabdus luminescens), a new type of hemocyte, not previously observed in healthy insects, was found in hemocyte monolayers. These cells have a distinctive morphology, characterised by extreme spreading ability. They achieve a diameter of up to 120 μm after 1 h on glass coverslips and are therefore extremely thin. These hyper-spreading cells first appear in fungal-infected insects prior to hyphal growth. Their numbers later fall to zero as the pathogen begins to proliferate. The same hyper-spreading cells are induced after a 24 h delay following an injection of laminarin, a source of the fungal cell wall polymer β-1,3-glucans. Wounding, on the other hand, did not cause the appearance of hyper-spreading cells. Evidence is presented here that is consistent with these spreading cells having a role in the cellular immune response of nodule formation.

Insect blood cell engulfing bacteria.

Insect blood cell engulfing bacteria.

Cell of the month: An insect blood cell engulfing bacteria.

Cell of the month: An insect blood cell engulfing bacteria.

Cloning and analysis of Trypanosoma cruzi lanosterol 14alpha-demethylase.

Trypanosoma cruzi infection, transmitted by insect vectors or blood transfusions, is an important cause of morbidity and mortality in many Latin American countries. Treatments are toxic and frequently ineffective in curing patients with chronic manifestations of the infection (Chagas disease). Potentially exploitable chemotherapeutic targets of T. cruzi are enzymes of the sterol biosynthesis pathway. In particular, the P450 enzyme, lanosterol 14alpha-demethylase, has been implicated as the target of azole antifungal drugs that have potent anti-T. cruzi activity. In the work reported here, the T. cruzi lanosterol 14alpha-demethylase (Tc14DM) gene was cloned by degenerate PCR. The gene was found to be expressed in both insect and mammalian life-cycle stages of the parasite. Tc14DM was able to complement the function of the homologous gene in yeast (erg11) as demonstrated by restored ergosterol production in an erg11-deficient yeast strain. When the yeast strain was co-transfected with the P450 reductase gene from Trypanosoma brucei, the amount of ergosterol production was increased, indicating that the endogenous yeast P450 reductase was an inefficient partner with Tc14DM. Heterologous expression of Tc14DM in the baculovirus/Sf9 system resulted in a 52kDa product. The protein was observed to have the characteristic absorbance spectra of a P450 enzyme. A typical Type II binding spectrum was produced when the imidazole compound, ketoconazole, was mixed with the Tc14DM, demonstrating that ketoconazole binds the enzyme.

Invasion of insect blood tissue by Xenorhabdus bovienii

Xenorhabdus species are entomopathogenic Gram-negative bacteria that belong to the family Enterobacteriaceae. They exist in two main phenotypic variations referred to as phase I and phase II. Invasion studies showed that Xenorhabdus bovienii phase I, unlike phase II, adhered to and were phagocytosed by Drosophila melanogaster malignant blood neoplasm, mbn-2. The ingested bacteria within the phagosome multiplied and escaped into the mbn-2 cytoplasm. Further bacterial multiplication occurred within the cytoplasm with a longer postinvasion incubation period, resulting in mbn-2 lysis. X. bovienii multiplied more rapidly in artificial media than in mbn-2. The generation time of phase I X. bovienii in the artificial media was 2.5- to 3-fold that in mbn-2. Cell free extract of X. bovienii phase I fluoresced under UV light, unlike that of phase II.

Effects of climate on variability in Lyme disease incidence in the northeastern United States.

Numbers of reported Lyme disease cases have increased dramatically over the past decade in the northeastern United States, but the year-to-year variability is sizable (average standard deviation approximately 30% of the mean). An improved understanding of the causes of such variability would aid in prevention and control of the disease, which is transmitted by a spirochete carried in the "black-legged" tick, Ixodes scapularis. In this study, the variability in reported Lyme disease incidence between 1993 and 2001 in seven northeastern US states was analyzed as an outcome of weather variability. For all seven states analyzed, significant (p < 0.05) positive relations were found for the correlation of early summer disease incidence with the June moisture index (Palmer Hydrological Drought Index) in the region 2 years previously. The correlations may reflect enhanced nymph tick survival in wetter conditions. Few significant relations were found with same-year moisture index, which suggests that moisture has a greater effect on nymph tick survival following the insect's blood meal than before. In some states, significant correlations were observed related to warmer winter weather a year and a half prior to disease incidence, which may have been due to higher survival and activity levels of the white-footed mouse, the main host for Lyme disease-infected ticks.

Roles of Aromatic Residues in the Structure and Biological Activity of the Small Cytokine, Growth-blocking Peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects.

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta.

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.

Trialysin, a Novel Pore-forming Protein from Saliva of Hematophagous Insects Activated by Limited Proteolysis

We have characterized a pore-forming lytic protein from the saliva of the hematophagous insect Triatoma infestans, a vector of Chagas disease. This protein, named trialysin, has 22 kDa and is present in the saliva at about 200 microg/ml. Purified trialysin forms voltage-dependent channels in planar lipid bilayers with conductance of 880 +/- 40 pS. It lyses protozoan parasites and bacteria indicating that it has a role in the control of microorganism growth in the salivary glands. At higher concentrations, but below those found in saliva, trialysin can also permeabilize and lyse mammalian cells, suggesting that it might also facilitate insect blood feeding by interfering with the cell response of the host. The translated cDNA sequence of trialysin shows a basic, lysine-rich protein in which the N-terminal region is predicted to form an amphipathic alpha-helical structure with positive charges on one side and hydrophobic amino acids on the opposite side. A synthetic peptide corresponding to this cationic amphipathic alpha-helix induces protozoan lysis and mammalian cell permeabilization, showing that this region is involved in lytic activity. However, the lytic peptide G6V32 is 10-fold less efficient than trialysin in lysing parasites and 100-fold less efficient in permeabilizing mammalian cells. Trialysin activity is about 10-fold reduced in salivary gland homogenates prepared in the presence of an irreversible serine-protease inhibitor. Since trialysin precursor contains an anionic pro-sequence of 33 amino acids contiguous to the cationic amphipathic putative alpha-helix, we propose that removal of the acidic pro-sequence by limited proteolysis activates trialysin by exposing this lytic basic amphipathic motif.

Recent developments in the chemotherapy of Chagas disease.

Chagas disease remains an important health problem in the Americas. Advances are being made in parts of South America in blocking transmission from insect vectors or blood transfusion, but more effective chemotherapy is needed for the millions who are already infected. This is especially important since recent results have indicated that treatment is beneficial for the elimination of the chronic course of the disease. The rational development of new drugs depends on the identification of differences between human metabolism and that of the causative parasite, Trypanosoma cruzi. Recent developments in the study of the basic biochemistry of the parasite have allowed the identification of novel targets for chemotherapy, such as sterol metabolism, protein prenylation, proteases, and phospholipid metabolism, and these are the subject of this review.

Adipokinetic hormones of insect: Release, signal transduction, and responses

Flight activity of insects provides an attractive yet relatively simple model system for regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a well-accepted model insect. Peptide adipokinetic hormones (AKHs) are synthesized and stored by neurosecretory cells of the corpus cardiacum, a neuroendocrine gland connected with the insect brain. The actions of these hormones on their fat body target cells trigger a number of coordinated signal transduction processes which culminate in the mobilization of both carbohydrate (trehalose) and lipid (diacylglycerol). These substrates fulfill differential roles in energy metabolism of the contracting flight muscles. The molecular mechanism of diacylglycerol transport in insect blood involving a reversible conversion of lipoproteins (lipophorins) has revealed a novel concept for lipid transport in the circulatory system. In an integrative approach, recent advances are reviewed on the consecutive topics of biosynthesis, storage, and release of insect AKHs, AKH signal transduction mechanisms and metabolic responses in fat body cells, and the dynamics of reversible lipophorin conversions in the insect blood.

A Novel Protein That Binds Juvenile Hormone Esterase in Fat Body Tissue and Pericardial Cells of the Tobacco HornwormManduca sexta L.

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm moth Manduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and native M. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.

Isolation from an Ant Myrmecia gulosa of Two Inducible O-Glycosylated Proline-rich Antibacterial Peptides

Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.

The Amino Acid Composition ofAnopheles stephensi(Diptera: Culicidae) Infected withNosema algerae(Microsporida: Nosematidae)

The amino acid composition of larvae, pupae, and adult mosquitoes ofAnopheles stephensiinfected withNosema algeraeand noninfected insects was analyzed using an amino acid analyzer. The increase and decrease in the concentration of single amino acids during the development of the microsporidium were compared with the spread of meronts, sporonts, and spores within the host. The appearance of meronts and sporonts coincided mainly with an increase in the concentration of alanine and histidine. The amount of alanine, proline, and tyrosine decreased during spore formation. Tyrosine appeared in the hemolymph of infectedA. stephensi,whereas this amino acid could not be found in the blood of uninfected insects. The spores themselves contained predominantly the glycogenic amino acid alanine, glutamic acid, aspartic acid, serine, and glycine. Proline, methionine, or tyrosine could not be detected within the spores.

Caterpillars have evolved lungs for hemocyte gas exchange

Since insect blood usually lacks oxygen-carrying pigments it has always been assumed that respiratory needs are met by diffusion in the gas-filled lumen of their tracheal systems. Outside air enters the tracheal system through segmentally arranged spiracles, diffuses along tubes of cuticle secreted by tracheal epithelia and then to tissues through tracheoles, thin walled cuticle tubes that penetrate between cells. The only recognized exceptions have been blood cells (hemocytes), which are not tracheated because they float in the hemolymph. In caterpillars, anoxia has an effect on the structure of the hemocytes and causes them to be released from tissues and to accumulate on thin walled tracheal tufts near the 8th (last) pair of abdominal spiracles. Residence in the tufts restores normal structure. Hemocytes also adhere to thin-walled tracheae in the tokus compartment at the tip of the abdomen. The specialized tracheal system of the 8th segment and tokus may therefore be a lung for hemocytes, a novel concept in insect physiology. Thus, although as a rule insect tracheae go to tissues, this work shows that hemocytes go to tracheae.