Hemolymph Research Articles

Subchronic effects of environment-like cadmium levels on the bivalve Anodonta anatina (Linnaeus 1758): III. Effects on carbonic anhydrase activity in relation to calcium metabolism

The effects of subchronic cadmium (Cd exposure at environment-like levels (water- and algae-borne Cd) on carbonic anhydrase (CA), its distribution in various body compartments and subcellular fractions, and its correlation to Cd and Ca concentrations in the freshwater mussel Anodonta anatina were investigated. CA activity in the hemolymph (HML) and extrapallial fluid were strongly affected in both directions, i.e. inhibition and then induction depending on the length of exposure and depuration. Cd also lowered total and subcellular CA activity in all tested tissues, especially in the digestive gland and gills. These effects were confirmed by negative correlation of both total and cytoplasmic CA activity relative to newly incorporated Cd. The specific activity of total CA was correlated to Ca in the HML and in the mantle but not in other compartments. These observations suggest that Cd exerted indirect effects on ion regulation and Ca homeostasis through its effects on CA, perhaps constituting the physiological basis of Cd-toxicity in this freshwater bivalve.

Bioinformatics tools help molecular characterization of Perkinsus olseni differentially expressed genes.

In the 80ies, in Southern Europe and in particular in Ria Formosa there was an episode of heavy mortality of the economically relevant clam Ruditapes (R.) decussatus associated with a debilitating disease (Perkinsosis) caused by Perkinsus olseni. This protozoan parasite was poorly known concerning its' differential transcriptome in response to its host, R. decussatus. This laboratory available protozoan system was used to identify parasite genes related to host interaction. Beyond the application of molecular biology technologies and methodologies, only the help of Bioinformatics tools allowed to analyze the results of the study. The strategy started with SSH technique, allowing the identification of parasite up-regulated genes in response to its natural host, then a macroarray was constructed and hybridized to characterize the parasite genes expression when exposed to bivalves hemolymph from permissive host (R. decussatus), resistant host (R. philippinarum) and non permissive bivalve (Donax trunculus) that cohabit in the same or adjacent habitats in Southern Portugal. Genes and respective peptides full molecular characterization depended on several Bioinformatic tools application. Also a new Bioinformatic tool was developed.

Evaluation of scorpion hemolymph (blood) proteinic pattern by SDS-PAGE

Evaluation of scorpion hemolymph (blood) proteinic pattern by SDS-PAGE

Shaping Cells and Organs in Drosophila by Opposing Roles of Fat Body-Secreted Collagen IV and Perlecan

Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study invivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form.

Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane

Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date. C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH 2PO 4 supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH 2PO 4, pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities. Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes. After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K + ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E rev) of 47.7 mV, which indicates K +-selectivity of this protein. The success of the purification of coronatin-1 will allow further characterization of the mode of action of this molecule, including ability of coronatin-1 to form potassium channels in immunocompetent hemocytes.

Cellular Immune Response of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) during Mycosis of the Entomopathogenic Fungus Beauveria bassiana (Balsamo) Vuillemin

Beauveria bassiana one of the most common and ubiquitous entomopathogens, is known to be a highly potential bioagent for the control of insects belonging to various orders. The cellular immune response was examined in Spodoptera litura larvae infected with B. bassiana . Third instar larvae were treated with a fungal suspension @ 2.03x10 8 spores ml -1 . Haemolymph of treated and untreated larvae were taken after 2, 12, 24,48,72, 96 and 120 hours after inoculation. Plasmatocytes and granulocytes swelled 2 hours after inoculation (HAI) followed by lysis of plasma membrane. Within 24 HAI, apposition was observed. The encapsulation process started after 48 hours and continued for up to 72 hours. After 96 hours of inoculation the granulocytes and plasmatocytes were observed to form nodules. With increasing inoculation time, the size of clump increased, forming multiple layers at 120 HAI. However, despite these insect defense responses, growth of fungal hyphae was observed after 120 hours of inoculation which ultimately resulted in the death of the host.

Effects of temperature and photoperiod on glucose and vitellogenin hemolymph concentrations in crayfish Cherax quadricarinatus (van Martens 1868)

The freshwater crayfish Cherax quaricarinatus has been recently introduced in several countries world-wide. From a practical point of view there is a growing interest in the biology of this species. Nevertheless, there is a paucity of scientific information concerning the reproduction of this species and the effects of environmental factors which occur during ovarian maturation (Sagi et al., 1996). In tropical crustacean species, the reproduction cycle is very short compared to the European crayfish native species and it occurs several times per year...

Gelechiidae Moths Are Capable of Chemically Dissolving the Pollen of Their Host Plants

Background: Many insects feed on pollen surface lipids and contents accessible through the germination pores. Pollen walls, however, are not broken down because they consist of sporopollenin and are highly resistant to physical and enzymatic damage. Here we report that certain Microlepidoptera chemically dissolve pollen grains with exudates from their mouthparts. Methodology/Principal Findings: Field observations and experiments in tropical China revealed that two species of Deltophora (Gelechioidea) are the exclusive pollinators of two species of Phyllanthus (Phyllanthaceae) on which their larvae develop and from which the adults take pollen and nectar. DNA sequences placed the moths and plants phylogenetically and confirmed that larvae were those of the pollinating moths; molecular clock dating suggests that the moth clade is younger than the plant clade. Captive moths with pollen on their mouthparts after 2-3 days of starvation no longer carried intact grains, and SEM photographs showed exine fragments on their proboscises. GC-MS revealed cis-b-ocimene as the dominant volatile in leaves and flowers, but GC-MS analyses of proboscis extracts failed to reveal an obvious sporopollenindissolving compound. A candidate is ethanolamine, which occurs in insect hemolymphs and is used to dissolve sporopollenin by palynologists. Conclusions/Significance: This is the first report of any insect and indeed any animal chemically dissolving pollen.

The use of cholinergic biomarker, cholinesterase activity of blue mussel Mytilus edulis to detect the effects of organophosphorous pesticides

The aim of the study was to investigate the effects of organophosphorous pesticide on thecholinesterase activity of different organs of Mytilus edulis. The mussels were exposed to serial dilutions of the pesticides (0, 50, 100, 200, 500, and 1000 µg/l) for 96 h. A significant inhibition of the cholinesterase activity from gill occurred at the lowest concentration, which indicated that gill was the most sensitive organ. The moderate sensitive organs were foot and mantle, which wereinhibited by trichlorfon at 200 μg/l. The least sensitive organs were hemolymph, posterior adductor muscle and digestive gland were inhibited at 1000 μg/l. After incubation of the trichlorfon-exposed mussels for seven days in clean media, the cholinesterase activities from different organs of mussels were not cured completely. The cholinesterase activities from hemolymph, gill, posterior adductor muscle and digestive gland recovered, while the persistent inhibition of cholinesterase activity from foot and mantle were observed. Key words: Biomarker, cholinesterase, mussel, trichlorfon.

Biochemical evaluation of aestivation and starvation in two snail species

There is resurgence in incidence of schistosomiasis in Nigeria with attendant socio-economic and health impact. The agents transmitting this disease are theBulinus snails which employ aestivation to survive conditions of unfavourable weather such as lack of food and water. The mechanism of aestivation under aridity and drought is not clear. This study therefore investigated the effects of aestivation and starvation on endogenous metabolic reserves in haemolymph of two snail species namely: Bulinus globosus (Morelet) and Bulinus rohlfsi (Clessin). Aestivation, starvation and control experiments were set up for 30 days in the laboratory by placing three groups of snails collected from Oyan dam, Abeokuta in standard aestivation slope (30 B. globosus and 19 B. rohlfsi), aquarium (30 B. globosus and 23 B. rohlfsi) and control slope which had 20 B. globosus and 15 B. rohlfsi. Aestivation and control slopes contained water and mixture of sand and clay (3:1), while aquarium contained water only for starvation. All the snails were fed on lettuce ad libitum for 28 days during which water was completely drained out in the aestivation slope. The aestivation slope and aquarium were left for another 30 days without lettuce. Snails were thereafter sacrificed and haemolymph biochemical parameters were assayed. In aestivating and starving B. globosus, haemolymph creatinine, urea, total protein, glucose, alanine transferases (ALT) and aspartate transferases (AST) were significantly decreased, while haemolymph total cholesterol, triglyceride and a-amylase concentrations and activity increased significantly (p < 0.05). In B. rohlfsi, creatinine, urea, ALT and AST were significantly decreased when compared with controls (p < 0.05). B. globosus and B. rohlfsipossess ability to survive unfavourable conditions by economical utilization of stored metabolites, thus enabling them to carry infection from one season to the next. Our findings suggest that B. globosus is a better aestivator than B. rohlfsi. Key words: Aestivation, enzymes, Bulinus globosus, Bulinus rohlfsi, schistosomiasis.

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Insulin-degrading enzyme antagonizes insulin-dependent tissue growth and Aβ-induced neurotoxicity in Drosophila

Insulin-degrading enzyme (IDE) is implicated in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer’s disease (AD). Here we provide genetic evidence that Drosophila Ide (dIde) antagonizes the insulin signaling pathway and human Aβ-induced neurotoxicity in Drosophila. In this study, we also generated a dIde knockout mutant (dIdeKO) by gene targeting, and found that loss of IDE increases the content of the major insect blood sugar, trehalose, thus suggesting a conserved role of IDE in sugar metabolism. Using dIdeKO as a model, further investigations into the biological functions of IDE and its role in the pathogenesis of DM2 and AD can be made.

Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

BackgroundPhotorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts.ResultsA comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora.ConclusionsWe studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

Intracardiac Hemolymph Sampling Technique in the Domestic Tarantula

Exotic animals, including tarantulas, have become more and more popular among enthusiasts nowadays. Under these circumstances, the practicing veterinarian may be occasionally faced with the task of examining, diagnosing and treating such an unusual pet. In order to successfully achieve this, some special knowledge about their husbandry, nutrition, anatomy, restraint and specific pathology is absolutely necessary. Because of their limited space and high humidity and temperature, tarantulas are prone to opportunistic bacterial and/or fungal infections. This paper would like to help fellow veterinarians by presenting a practical method of intracardiac hemolymph collection. This procedure is used for diagnostic purposes, when a systemic infection is suspected, or for therapeutic ones, when dehydration or hemolymph loss are noted.

Density of Vibrios in Hemolymph and Hepatopancreas of Diseased Pacific White Shrimp, Litopenaeus vannamei, from Northwestern Mexico

Vibriosis is one of the most widespread infectious diseases of cultured shrimp in Mexico; as a result, the Research Centre for Food and Development (CIAD, for the Spanish wording) offers a diagnostic service to the shrimp farmers where they bring samples of diseased shrimp as judged by themselves. Hemolymph (HL) and hepatopancreas (HP) of diseased Litopenaeus vannamei were analyzed from five regions of Northwestern Mexico (from 2001 to 2006) in tiosulphate citrate bile sacharose (TCBS) agar; a total of 5912 shrimp were examined. The results showed a mean vibrios density of 6.45 × 103, SD = 6.90 × 104 CFU/mL for HL and 1.40 × 105, SD = 3.20 × 105 CFU/g for HP (n = 5912). HL samples had 12% (SD = 26%) of green colonies (GCs) in TCBS, and 23% (SD = 34%) of GCs in HP samples. Of the total shrimp analyzed, 46% were not found to possess vibrios in HL, whereas 60% of the shrimp examined contained more than 105 CFU/g of HP. More than 60 and 40% of the shrimp did not have GC in HL and HP, respectively. The highest Pearson coefficient correlation was observed between vibrios HL and GCs in HL for 2006 (r = 0.611, n = 712, P &lt; 0.001).

First Case Report of Louse – Borne Relapsing Fever in Saudi Arabia

Relapsing fever is endemic in some parts of the world such as Africa, South America, and Far East.1-3 Epidemics have occurred among refugees and war victims in some parts of Africa.4,5 There is very little information about this infection in Saudi Arabia. The prevalence or incidence in Saudi Arabia is unknown. Its apparent rarity may be due to under diagnosis and under reporting. A high index of suspicion in appropriate clinical situations will lead to its early recognition and treatment. Relapsing fever is an acute febrile illness caused by blood spirochetes belonging to genus borrelia. This spirochete can be acquired through the bite of an infected soft tick (Ornithodoros species) or contact with the hemolymph of an infected human body louse (Pediculus humanus) and thus constituting two types of relapsing fever (RF) depending upon the responsible vector i.e. Tick – borne relapsing fever (TBRF) and Louse – borne relapsing fever (LBRF). It is characterized by recurrent cycles of pyrexia which are separated by intervals of apparent recovery. During pyrexia, organisms can be found in blood and that is the only way of reliable diagnosis in many situations.6,7 While during afebrile period, organisms hide themselves in spleen, liver and even central nervous system.

Host–Pathogen Interactions: Proline Gives Insect Pathogens the Green Light

How does a quiescent symbiont of a nematode worm know when to turn nasty? Metabolic analysis and genetic knockouts confirm that model insect pathogens can sense L-proline in insect blood. This not only serves as a wake-up call, activating secondary metabolite virulence factors, but also provides an energy source for a metabolic shift appropriate for adaptation to the host environment.

Effect of Lead on the Expression of Nutritional Content in Edible Lobster, Thenus Orientalis (Lund, 1793)

The edible lobster, Thenus orientalis is an important food of commercial interest in the parts of South India. Evaluation of toxic effect of lead on the chosen animal for the LC 50 value and effect of heavy metal lead on the nutritional status viz. protein, carbohydrate and lipid in ovary, spermatheca, hepatopancreas, muscle and haemolymph was made. The results assume greater interest as most water bodies are increasingly subjected to environmental pressure due to pollution.

The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae

BackgroundThere is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection.ResultsUsing quantitative proteomics, we compared the hemolymph (insect blood) of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO), lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection.ConclusionWe propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct defensive measures, such as the massive enhancement of PO activity.

Effect of stocking density on the growth and haemolymph biochemical value of Archachatina marginata

The growth performance and haemolymph properties of the African giant land snailArchachatina marginata fed ad libitun with pawpaw leaves and reared under different stocking densities were investigated in an eight week experiment. Four different stocking densities of 5, 10, 15 and 20 snails per cage (0.5 m x 0.5 m x 0.2 m) were used with each group replicated. The cage with 15 snails per cage recorded the best growth performance in relation to weight gain, shell length gain as well shell circumference gain, while the cage stocked with 20 snails recorded the least growth performance. There is a strong relationship between stocking density and the concentration of the haemolymph protein, glucose and lipids. However, there was no difference in the concentration of the mineral elements across the stocking densities. Key words: Archachatina marginata, stocking density, haemolymph biochemical values.