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The earthworm gut is a unique microzone in aerated soils that has been proposed to selectively stimulate ingested soil microorganisms by its in situ conditions, which include anoxia, high water content, a near-neutral pH, and high concentrations of organic compounds. The central objective of this study was to resolve potential links between in situ conditions and anaerobic microbial activities during the gut passage of Lumbricus terrestris. Both H(2) and N(2)O were emitted by living earthworms, and in situ microsensor analyses revealed both H(2) and N(2)O in the O(2)-free gut center. The highest H(2) concentrations occurred in foregut and midgut regions, whereas the highest N(2)O concentrations occurred in crop/gizzard and hindgut regions. Thus, H(2)-producing fermentations were more localized in the foregut and midgut, whereas denitrification was more localized in the crop/gizzard and hindgut. Moisture content, total carbon, and total nitrogen were highest in the foregut and decreased from the anterior to posterior end of the gut. Nitrite, ammonium, and iron(II) concentrations were highest in the crop/gizzard and decreased from the anterior to posterior end of the alimentary canal. Concentrations of soluble organic compounds were indicative of distinct fermentation processes along the alimentary canal, with maximal concentrations of organic acids (e.g., acetate and butyrate) occurring in the midgut. These findings suggest that earthworms (i) contribute to the terrestrial cycling of carbon and nitrogen via anaerobic microbial activities in the alimentary canal and (ii) constitute a mobile source of reductant (i.e., emitted H(2)) for microbiota in aerated soils.

Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified. Other Cry4Ba binding proteins identified include the putative lipid raft proteins flotillin and prohibitin, V-ATPase B subunit and actin. These identified proteins might play important roles in mediating the toxicity of Cry4Ba due to their location in the gut brush border membrane. Cadherin-type protein was not identified, although previously, we identified a midgut cadherin AgCad1 as a putative Cry4Ba receptor in Anopheles gambiae mosquito larvae [Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101-5110]. Other identified proteins in this study that might have lesser roles include mitochondrial proteins such as ATP synthase subunits, mitochondrial processing peptidase and porin; which are likely contaminants from mitochondria and are not brush border membrane components. Trypsin-like serine protease was also identified as a protein that binds Cry4Ba. Identification of these toxin-binding proteins will lead to a better understanding of the mode of action of this toxin in mosquito.

We identified a serine protease with a molecular mass of 37kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

Digestion of proteins in the midgut of lepidopteran larvae relies on different trypsin and chymotrypsin isoforms. In this study we describe three chymotrypsin-like proteinases (CTLP2-4) from the larval midgut of Manduca sexta, which are closely related to CTLP1 and less closely related to another chymotrypsin (CT), two previously described proteinases present in the larval midgut of M. sexta. CTLP1-4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. When we examined CTLP expression in different tissues, most of the proteinases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. When we examined CTLP expression at different physiological states, we observed that the CTLP mRNA amounts did not differ considerably in feeding and starving larvae except for CTLP2, whose mRNA dropped significantly upon starvation. During moulting, however, the mRNA amounts of all CTLPs dropped significantly. When we immunologically examined CTLP amounts, mature proteinases were only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or moulting larvae, suggesting that CTLP secretion is suspended during starvation or moult.

The purpose of this study was to examine the toxic effects of cadmium-telluride (CdTe) quantum dots on freshwater mussels. Elliption complanata mussels were exposed to increasing concentrations of CdTe (0, 1.6, 4 and 8 mg/L) and cadmium sulfate (CdSO(4), 0.5mg/L) for 24h at 15 degrees C. After the exposure period, they were removed for assessments of immunocompetence, oxidative stress (lipid peroxidation) and genotoxicity (DNA strand breaks). Preliminary experiments revealed that CdTe dissolved in aquarium water tended to aggregate in the particulate phase (85%) while 15% of CdTe was found in the dissolved phase. Immunotoxicity was characterized by a significant decrease in the number of hemocytes capable of ingesting fluorescent beads, and hemocyte viability. The cytotoxic capacity of hemocytes to lyse mammalian K-562 cells was significantly increased, but the number of circulating hemocytes remained unchanged. Lipid peroxidation was significantly increased at a threshold concentration of 5.6 mg/L in gills and significantly reduced in digestive glands at a threshold concentration <1.6 mg/L CdTe. The levels of DNA strand breaks were significantly reduced in gills at <1.6 mg/L CdTe. In digestive glands, a transient but marginal increase in DNA strand breaks occurred at the lowest concentration and dropped significantly at the higher concentrations. A multivariate analysis revealed that the various response patterns differed based on the concentration of CdTe, thus permitting the identification of biomarkers associated with the form (colloidal vs. molecular) of cadmium.

In this study we investigated the effect of kefir on the levels of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) in the liver, stomach, spleen and colon of mice with colonic aberrant crypts formed by azoxymethane (AOM). Thirty 12 weeks old Swiss Albino mice averaging 31.5 g weight were used as experimental animals. The mice were separated into 3 groups. The first group was the control group, second group was the AOM and third group was the AOM+kefir group. We applied AOM to the second and third groups. Mice were fed ad libitum by laboratory rodent chow during the experiment period. Water was given to the first and second groups and third group received only kefir diluted with water (50%). AOM was injected subcutaneously to the second and third groups for 7 weeks (two times a week, 5 mg/kg). Six weeks after the final AOM treatment the animals were sacrificed and liver, stomach, spleen and colon samples were collected from all the groups. MDA level demonstrated an increase only in stomach for the third group (p < 0.001), while an elevation was observed for all of the four organs for the second group (spleen p < 0.001, liver p < 0.001, colon p < 0.01). GSH level showed an increase in the second group at stomach (p < 0.01) and colon (p < 0.001), while in the third group, a small increase was determined only at the colon (p < 0.05). NO level increased at all of the organs in the second group (spleen, liver, colon p < 0.001, stomach p < 0.05), but only at liver and colon in the third group 3 (p < 0.001). In conclusion these results showed that kefir plays an antioxidant role.

Quantitative real-time PCR was employed to measure the Solenopsis invicta virus 1 (SINV-1) load in tissues, individuals, and among colonies of the red imported fire ant, S. invicta Buren. Among tissues examined from SINV-1-infected adults and larvae, the alimentary canal (specifically the midgut) consistently had the highest number of SINV-1 genome copies (91.1 and 99.9%, respectively). Negative staining of a supernatant of the gut homogenate demonstrated the presence of spherical virus particles with a diameter of 30-35 nm, consistent with SINV-1. The number of SINV-1 genome copies in infected larvae and workers from the same queenright colonies were similar to each other. In other words, the infection rate was consistent among both developmental stages. No significant differences were observed in SINV-1 genome copy number among infected colonies sampled during the winter and summer. Although the SINV-1 infection rate of summer-collected mounds was previously shown to be six-times higher than winter-collected mounds, the intra-colony infection rate appears to be unaffected by season. Perhaps less inter-mound interaction during the winter months among S. invicta restricts spread of the virus. A positive correlation between intra-colony infection rate and mean SINV-1 genome copy number per ant was also observed. Based on these results, it is likely that SINV-1 replicates in gut epithelia of S. invicta and virus is shed into the gut lumen where it may be transmitted to nestmates by trophallaxis.

High-performance liquid chromatography (HPLC) was used to determine the effects of a patent Schistosoma mansoni infection on certain carboxylic acids in the digestive gland gonad complex (DGG) and hemolymph of Biomphalaria glabrata. An analysis of DGG samples was done using extraction with 50% Locke's solution, cleanup of the extract by anion exchange solid phase extraction (SPE), and ion exclusion HPLC with ultraviolet detection. Hemolymph was applied directly to the SPE column. Acetic, fumaric, malic, and pyruvic acids were detected, confirmed, and quantified at concentrations ranging from 12 to 280 ppm in the DGG and less than 124 to 8,000 microg/dl in the hemolymph. Infection with S. mansoni caused a significant reduction (Student's t test, P < 0.05) in the concentrations of acetic, fumaric, malic, and pyruvic acids in the DGG but not the hemolymph of B. glabrata compared to uninfected cohort snails. The significant reduction of certain carboxylic acids in the DGG of B. glabrata patently infected with S. mansoni suggests that these acids are utilized by the sporocysts and cercariae in the snail tissue, or that the infection stimulates reduced production or increased utilization by the snail tissue.

Two FMRFamide-related peptides (FaRPs) have been isolated and sequenced from the whole gut of Locusta migratoria L. Peptides were extracted from 500 locust whole guts and separated using reversed-phase high performance liquid chromatography (RP-HPLC). Fractions containing FMRFamide-like immunoreactive (FLI) material were identified using radioimmunoassay (RIA). Sequencing of fractions, using tandem mass spectrometry (MALDI-TOF MS/MS), revealed the myosuppressin previously isolated from the locust CNS, SchistoFLRFamide (PDVDHVFLRFamide), and a novel extended RFamide (LWENLRFamide). The isolation of SchistoFLRFamide from midgut tissue supports the hypothesis that this myosuppressin is released locally from FLI processes over the gut and/or from endocrine-like midgut cells to play a role in the regulation of digestion.

Regarding Brazilian coast, industrial and urban developments are concentrated along Central South Brazil Bight. Samples from inshore and offshore species from the concerned area were analyzed, comprising 24 cetaceans (9 species) and 32 squids (2 species). Cadmium was determined by GFAAS and our results were in agreement with certified values (DOLT-2, NRCC). Mean cadmium concentration (in microg/g, wet weight) observed in the digestive gland of sexually mature Argentine short-finned squids (Illex argentinus) was 1002.9. To our knowledge this is the highest cadmium level ever reported for a cephalopod. Concerning cetaceans, our results include one of the highest renal cadmium concentrations described for striped dolphins (71.29 microg/g, wet weight). Anthropogenic action, upwelling and cannibalism of Argentine short-finned squid on the studied area are possible reasons for such remarkable cadmium concentrations.

The aim of this study was to investigate the effects of polycyclic aromatic hydrocarbons (PAHs) on the physiological status of the bivalve Mya arenaria. Specimens were exposed to four different sources of PAHs: aluminium smelter soot, sediment from an industrial discharge pound, charcoal fine particles and dietary PAHs assessed by feeding clams with phytoplankton freshly impregnated with dissolved PAHs. The exposure period lasted 30 days and bivalves were let to recover for an additional 20 days. At days 8, 15, 30 and 50, immune parameters (phagocytic activity and efficiency) were monitored in haemocytes. Oxidative stress measures such as catalase and lipid peroxidation were quantified in digestive gland as well as concentrations of bioaccumulated PAHs. In a second experiment, clams were exposed to [(14)C]-pyrene via the phytoplankton, and the tissue distribution of radiolabelled compound was studied. Glycogen levels in gonad and digestive gland were also measured and gametogenesis stages were investigated. Results showed a high bioaccumulation in clams exposed to dietary PAHs and contaminated sediments. Tissue distribution of [(14)C]-pyrene revealed that the radiolabelled compound persisted mainly in the gonad during 14 days. A decrease of phagocytosis was observed in contaminated male clams. The lipid peroxidation (malondialdehyde) was found to increase in the digestive gland tissues of clams exposed to dietary PAHs, smelter soot and discharge, but no differences were observed in the catalase activity. A delay in gametogenesis occurred in all exposed males and in females contaminated with coke dust and dietary PAHs. Males were more sensitive than females to PAH exposure. A dysfunction in steroid synthesis is suspected to occur due to the exposure to all sources of PAHs.

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the beta-cells of the islets.

Podocalyxin (PC) was initially identified as a major sialoprotein on the apical surface of glomerular podocytes to perform the filtration barrier function. Later, it was reported to be expressed in endothelial cells, megakaryotes/platelets, and hemangioblasts, the common progenitor cells of the hematopoietic and endothelial cells. Recently, increasing numbers of reports have indicated that PC is not merely a molecule restricted at renal glomerulus, angiogenic or hematopoietic system. To further elucidate the expression pattern and address the possible physiological role of PC in adult mammals, we conducted an extensive study by immunohistochemistry and immunofluorescence staining on various tissues of healthy adult beagle dogs. By combinatory usage of two different anti-podocalyxin antibodies recognizing distinct epitopes in PC, we have demonstrated that (1) PC is expressed in renal tubules, mesothelium, myocardium, striated muscles in tongue, esophagus and extraocular region, myoepithelial cells in esophagus and salivary glands, neurons, and ependyma, etc.; (2) there are at least three forms of PC proteins, depending upon the accessibility of two different PC antibodies, expressed in different organs/systems; and (3) a particular form of PC is distributed in a vesicle-like compartment in certain organs/systems, such as the central nervous system.

Two of the questions raised in the validation process of biomarkers are their relevance in the identification and discrimination of environmental perturbations, and the influence of seasonal factors on these biological endpoints. Determining the advantages and restrictions associated with the use of native or transplanted animals and comparing their responses is also needed. To obtain this information, a 2-year integrative field study was conducted in the vicinity of a nuclear power plant in northeastern France. A station was located in the reservoir receiving the cooling waters of the plant, and two other sites were studied 2 km upstream and 5 km downstream from the reservoir's discharge in the Moselle river. Elevated temperatures, copper contamination and a 1.4-fold-concentration factor of dissolved salts affected water quality of the reservoir. Native and transplanted zebra mussels (Dreissena polymorpha) were collected monthly and their digestive glands were processed for histochemical determinations of the lysosomal and peroxisomal systems and of the lipofuscin and neutral lipid contents. The responses were quantified using automated image analysis and stereology. Apart from neutral lipid contents, there were no systematic seasonal patterns in mussel populations or from 1 year to another. Principal Component Analyses showed a general higher discrimination potential of biological responses in transplanted organisms compared to native ones. They also pointed out the relationships between the cellular and physiological markers and abiotic factors. The present multiple biomarker integrative approach in transplanted D. polymorpha brings promising elements in their validation process as relevant biomonitoring tools.

Background: In atopic dermatitis (AD) patients, the intestinal mucosal barrier function is weakened, permiting frequent invasion by antigens. Polyamines and short-chain fatty acids (SCFA) produced by intestinal bacteria are involved in the promotion of intestinal mucosal barrier functions., Aim: Our aim was to investigate the effect of pro-biotic yogurt containing Bifidobacterium animalis subsp. lactis LKM512 (LKM512 yogurt) on subjective symptoms, intestinal microbiota, intestinal bacterial metabolites (polyamines and SCFA), and T-helper type 1 (Th1)/Th2 balance in intractable AD patients., Methods: In a double-blind, placebo-controlled, crossover study, LKM512 yogurt was given for 4 weeks to 10 adult AD patients who were diagnosed with moderate AD (four males and six females; average age, 22.1 years). The subjective symptoms were recorded after each intervention. The dynamics of fecal microbiota were analysed by the terminal-restriction fragment length polymorphism method. The effects of LKM512 yogurt on fecal polyamines, SCFA, and serum cytokines were evaluated., Results: Scores of itch and burning tended to improve to a greater extent by LKM512 yogurt consumption than by placebo consumption. LKM512 yogurt (P<0.005) and placebo consumption (P<0.05) significantly increased the serum IFN-gamma concentration by six- and threefold, respectively. Fecal microbiota was altered dynamically by LKM512 yogurt consumption, in particular, the bacterial species and phylotypes of Bifidobacterium, Clostridium cluster IV and subcluster XIVa were increased in number. In addition, fecal spermidine concentration was significantly (P<0.05) increased, while fecal butyrate also tended to be increased by LKM512 yogurt consumption., Conclusion: We conclude that LKM512 yogurt consumption may be effective against intractable adult-type AD and this effect depends on the recovery of the intestinal mucosal barrier function and the induction of the Th1-type cytokine by polyamines and SCFA, particularly, butyrate, produced by the altered intestinal microbiota.

Possible xenoestrogenic effects were investigated, for the first time, in two bivalve species from the Lagoon of Venice (Italy): the clam Tapes philippinarum and the cockle Cerastoderma glaucum. Bivalves were collected far from their reproductive phase at the very early stage of gametogenesis (January), and in the pre-spawning period (June) in six sites. Vitellogenin (Vg)-like proteins (a biomarker of exposure to estrogenic compounds) were measured by the alkali-labile phosphate method (ALP), in both haemolymph and digestive gland from males and females (when it was possible to distinguish sex by microscopic observation of gonadal tissue). Haemolymph calcium concentrations (a parameter considered closely related to the presence of Vg-like proteins) and the bivalve condition index (CI) were also measured. In both seasons, bivalves collected at Campalto (near a sewage treatment plant) and Marghera (a highly contaminated area) had higher Vg-like protein levels, particularly in haemolymph, than animals from the other sampling sites. Interestingly, CI had high values in these polluted sites. In June only, Vg-like proteins and Ca(2+) levels in haemolymph exhibited similar trends in both bivalve species at most sampling sites. The responsiveness of bivalves to environmental xenoestrogens was higher in June, allowing better discrimination among sites. The present study demonstrates that animals from highly polluted areas have increased Vg-like protein levels. As endocrine disruption due to exposure to estrogenic compounds may cause fertility reduction, alterations in the sex ratio, and a decrease in reproductive rate, a condition of potential risk for bivalve populations in estuarine areas is highlighted.

Objective of this study was to investigate the effect of using pre-packed Si (Si), manually packed silica hydrated with water (Si-H(2)O) and pre-packed aminopropyl-bonded silica (NH(2)), at various mass ratios of lipid to sorbent, on the recovery of polar lipids following the solid-phase extraction (SPE) of a standard mixture of lipids. We also applied SPE using these sorbents to the separation of lipids from oyster tissues and compared the fatty acid (FA) composition of each fraction. Recoveries of phospholipids after SPE using Si increased with an increasing ratio of lipid to sorbent. Although the use of Si-H(2)O improved the recovery of polar lipid compared to that obtained on Si, the neutral lipid from gills and muscles of oyster showed distorted FA compositions presumably due to a leakage of polar lipids. Finally, NH(2) eluted with methanol provided good recoveries of phospholipids from the standard mixture; although polar lipids of oyster tissues showed a reduction in 20:4n-6 and MUFA likely due to the selective retention of acidic phospholipids.

The autometallographic approach was applied to three filter-feeder bivalves from differently polluted sites of the Lagoon of Venice. The area density (AD) values of black silver deposits (BSDs) were therefore seasonally quantified by image analysis in digestive cell lysosomes. Sediments were characterised in terms of grain size, organic content and metal concentrations (Cd, Cu, Ni, Pb, Zn). The physiological status of the bivalves was also evaluated by applying the "survival in air" test as a generic stress index. Chemical results showed that Zn contributed to the total metal load for more than 60% in sediments and for 85%-94% in mussel and clam digestive glands, respectively (data processed from published results obtained on the same samples). Regression analyses between extent of BSDs and both total metal and Zn concentrations in digestive gland followed a linear pattern in mussels and clams, but not in arks. Similarly, in mussels and clams only, BSD amount was significantly correlated with total metal concentrations in sediments according to a logarithmic model. Consequently, results obtained from autometallography can discriminate sites on the basis of their metal pollution levels, like chemical analyses on both sediments and digestive glands, evaluations being supported by the results of the physiological stress index. As a conclusion, it is suggested that autometallography in bivalves may provide overall estimates of metal fractions moving from sediments towards organisms through the ingestion of metal-enriched particles. Future work is needed to define the bioaccumulation model in arks and to clarify their possible use as metal biomonitors.

Thin layer chromatography was used to analyze the glucose and maltose concentrations of the digestive gland-gonad complex (DGG) of uninfected-estivated Biomphalaria glabrata snails and estivated B. glabrata patently infected with Schistosoma mansoni. All snails were estivated in a most chamber at a relative humidity of 98+/-1% and a temperature of 23+/-1 degrees C for 14 days. Carbohydrates were extracted from the DGG with 70% aqueous ethanol, and extracts were analyzed on silica gel preadsorbent plates using ethyl acetate-glacial acetic acid-methanol-water (60:15:15:10) mobile phase, alpha-naphthol-sulfuric acid detection reagent, and quantification by densitometry. The concentrations of glucose and maltose were significantly reduced in both uninfected-estivated snails and infected-estivated snails.

Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring are the most appropriate sampling seasons for application of translation activity as a possible biomarker in biomonitoring studies.

Glutathione S-transferase is involved in the detoxication of many chemical compounds. Northern blot analysis of mRNA GST gene from the mussel Mytilus galloprovincialis shows the presence of a transcript of 850 bp (GenBank accession no. Gi:22094808 and AF527010). The cDNA cloned sequence is constituted by an ORF of 621 bp encoding for a protein of 23,700 Da present in the gills and digestive gland of M galloprovincialis. The sequence, called Mg (M. galloprovincialis) GST-p, is clearly related to the pi class GST. M. galloprovincialis treated with Cd (200 microg/L) and BaP (100 microg/L) or co-treated with Cd and BaP take up cadmium in the gills (16.2 +/- 4.2 and 12.6 +/- 1.2 microg Cd/g dry wt. after exposure to Cd and Cd + BaP, respectively) and in the digestive gland. The transcription of GST-pi gene, by semi-quantitative RT-PCR, shows the lowest value in the digestive glands of mussels exposed to BaP, whereas the treatment with cadmium and the co-treatment with cadmium and BaP evoke GST-pi gene expression higher than controls. Mussels collected from six sites along the south coast of Portugal show different GST-pi transcription levels, some of which are related to their pollutant content.

In the present work, mussels (Mytilus edulis) were transplanted into a copper (Cu) gradient in Visnes (Norway) for a period of 3 weeks during November 2003. Sites 1 and 2 showed similar low levels of Cu, site 3 had intermediate Cu levels and site 4 was the most polluted with Cu as confirmed by AAS of digestive gland tissue. Values of lysosomal membrane labilization period were significantly lower at sites 3 and 4 compared to sites 1 and 2. The volume density and size of lysosomes was significantly decreased at site 4. The volume density of neutral lipids was also significantly lower at site 4 compared with the rest of sites. The volume density of lipofuscins showed significantly higher values at sites 2 and 3 compared to the reference site 1. Similar results were obtained regarding bioavailable metal levels measured by autometallography. All together, results are indicative of exocytosis of metal-containing lysosomes and lipofuscins to the digestive tubule lumen in mussels from site 4. In fact, autometallographic metal deposits were detected in digestive tubule lumen, brown cells and stomach in site 4 mussels. In agreement, there was a loss of digestive cells in mussels from site 4 (atrophy of the digestive epithelium) and cell type replacement (diminished volume density of digestive cells and increased volume density of basophilic cells). In conclusion, selected biomarkers indicated that mussels transplanted to sites closest to the Cu mine showed significant differences in metal accumulation pattern and in organization of the digestive gland tissue. Finally, female mussels closest to the Cu mine showed advanced gametogenesis with higher gonad index and vitellogenin-like protein levels than mussels at sites 1 and 2.

In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as "pilot basin" within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1+/-7.6) than in transplanted (9.3+/-4.7) and reference ones (5.8+/-5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.

The present study was undertaken to elucidate the cellular mechanisms, which govern metal sequestration and detoxification in gastropods. For this purpose the pond snail, Lymnaea stagnalis was exposed to environmentally relevant concentrations of three species of metals (Al, Zn and Cd) for 30 days and the localization and fate of these metals were followed in different tissues of the snails. The measurement of relative distribution of metals between tissues revealed that the digestive gland and kidney account for most of the accumulated metals. Al and Cd (non-essential metals) were redistributed to the digestive gland, possibly because of the presence of specific binding entities in the digestive glands of the herein species. This study focuses on the role of intracellular metal-containing granules on metal sequestration. Three main types of granules were identified in the digestive gland cells namely small, green and yellow granules. The morphological examination and the progressive accumulation of elements within these granules revealed that they are developmental stages with the yellow granule being the mature one. The total number of these granules was found to be significantly increased upon exposure of the snails to Al only. This increase may be a response to the large amount of Al that is accumulated through feeding route of this grazing snail. X-ray microanalysis (XRMA) revealed that metals were localized in all three types of digestive gland granules. The increased amount of ligands (P and S) in the granules may give evidence for their role in metal sequestration. Levels of Al and P were positively correlated in the digestive gland granules. It is possible that aluminium is bound to phosphorus to render it insoluble and so to both immobilize it within the lysosome and to be excreted in a highly insoluble form. On the other hand, both Zn and Cd induced marked upregulation of S in mature (yellow) granules by 26- and 11-folds, respectively. The lysosomal codeposition of S and either Cd or Zn in the lysosomal granules in addition to the increase in RER cisternae may indicate that the exposure to these metals could induce metallothionein synthesis in the cells. The microscopical examinations in the present study revealed that metal detoxification from the digestive gland cells may occur via faeces or via basal exocytosis towards hemocytes dispersed by the connective tissue in the visceral mass. In the kidney, one type of granules, the excretory concretions, was identified in the nephrocytes. The significant increase in the number of these concretions in the snail L. stagnalis upon exposure to metals may give further evidence for their role in metal excretion.

Seasonal and age-specific variations of cadmium (Cd) concentration in the digestive gland were investigated in the Japanese scallop Patinopecten yessoensis from Peter the Great Bay, Sea of Japan, with different degrees of Cd pollution. The seasonal changes in Cd concentrations of the digestive gland were inversely proportional to the dry weight of the gland. Concentrations of Cd and total Cd content (mug Cd per organ) increased with age (age-specific) to the same extent in contaminated and uncontaminated areas. There was also a strong positive correlation between Cd content in the whole digestive gland and shell weight and it is proposed that this relationship can be used as a new criterion for comparative evaluation of Cd levels in scallops from different areas We hypothesize that Cd is uptaken into scallops in proportion to the amount of calcium that absorbed through ion channels, and in addition, Cd in the digestive gland is in immobile forms (e.g. metal-rich granules) that accumulate with age. Moderate environmental pollution has no effect on the relationship between Cd content and shell size and the observed decrease in growth performance of the scallops from polluted areas may be due to other factors.

Serotonin is an important neuromessenger used in a variety of signaling pathways throughout the animal kingdom. In insects, serotonin has been demonstrated to mediate feeding and feeding-related behaviors. In this study, serotonin antibody was localized in cells and processes throughout the central nervous system (CNS) and midgut of female horse fly Tabanus nigrovittatus Macquart. In the CNS, immunoreactivity was localized in cells and processes throughout the brain and ventral nerve cord. In the midgut, a fine network of immunoreactive processes was observed running along the outer surface of the midgut, with a decrease in innervation toward the posterior region of the midgut.

Monthly collections of the clam Paphia malabarica were made for period of 1 year from May 2002 to April 2003 from Verem, Mandovi estuary, Goa. Clams were categorised into two groups, 25-35 mm (small size) and 35-40 mm (big size). Significant difference was not observed in the accumulation of metals in the whole soft tissue between the two size groups. Irrespective of size, annual mean, metal content recorded as microg/g dry weight in the whole soft tissue was 3.8, 30.3, 13.5, 36.6 and 105.7, respectively, for Cd, Pb, Cu, Zn and Fe. Of the two toxic elements Cd and Pb, Cd content was almost uniform throughout the year except for a rise in September for the small size and October for the big size. Pb, on the other hand, was low from the beginning of the monsoon and exhibited distinct accumulation from December onwards up to April, May and, to some extent, June. The pattern was similar in both the size groups, the values being higher for the bigger size group. The three essential elements Cu, Fe and Zn exhibit trends similar to one another with peaks in September and December-January in both the size groups. Cadmium accumulation was highest in the mantle and adductor muscle, Lead, in foot, Copper, in digestive gland and gonad, and Zn and Fe, in gills. Correlation coefficient between different metal couplings as tested statistically revealed significant coupling for Zn-Fe (r 0.92) in the bigger size group, the same was observed between Cu-Fe (r 0.62) and Cd-Zn (r 0.94). Seasonal difference in Pb accumulation was highly significant.

Sulfated glycosaminoglycans (GAGs) were isolated and characterized in thoracic muscle, fat body, whole digestive tract (stomach+intestine) and reproductive tract of adult male cockroaches, Periplaneta americana. Heparan sulfate (HS) was the predominant sulfated GAG species in the tissues analyzed, corresponding to more than 90% of the total sulfated GAG content. In both the thoracic muscle and fat body it was the only sulfated GAG species detected. We also determined the location of sulfated GAGs in most of these organs by histochemical analysis using 1,9-dimethylmethylene blue. In the thoracic muscle, sulfated GAG metachromatic staining was detected only in the connective tissue that surrounds the muscle bundles or fascicles. In the intestinal tract, metachromatic staining was observed in both epithelial and lining columnar cells. Only spermatozoa presented metachromatic material in the male reproductive tract. Since, HS corresponds to 90-100% of total sulfated GAGs in these tissues, the metachromatic staining specifically reflects the location of this particular sulfated GAG in these organs. In conclusion, the present study extends previous observations on the GAG composition in cockroaches providing new information on the tissue distribution and location of HS in several internal organs of adult males of the cockroach P. americana.

Several Cry1Ac binding proteins from midgut of Helicoverpa armigera were purified using toxin-affinity chromatography. Enzyme assays showed that the purified proteins had strong aminopeptidase activity. The N-terminal sequences confidently identified a 124-kDa binding protein as an aminopeptidase N (APN), and some similarity suggests that a 162-kDa binding protein may also be an APN. Two minor binding proteins were not characterized.

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRalpha, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.

This study assesses the potential of the scallop Chlamys varia as a biomonitor of metal contamination in the field. Cd, Cu, and Zn concentrations were determined in the soft tissues and organs of individuals from the French Atlantic coast sampled over a 1 year period and covering a wide range of size. All metals were selectively distributed among the different body compartments considered, and their concentrations were influenced by the size of the specimens or the sampling-season. The present work shows the importance of considering the body compartment, the sampling period and the size in studies aiming at using this scallop as a biomonitor species. Among tissues, the digestive gland and kidneys exhibited the highest metal concentrations whatever the season or the size. The digestive gland contained 65 and 48% of the whole Cd and Cu body burdens, respectively, and kidneys accounted for 85% of the Zn load. Those tissues are therefore particularly recommended for use in biomonitoring programs.

Sandflies are vectors of several pathogens, constituting serious health problems. Lutzomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named peritrophic matrix (PM). One of the major roles of PM in blood-fed insects includes protection against ingested pathogens by providing a defensive barrier to their development. We used traditional and modern morphological methods as well as biochemical and immunolabeling tools to define details of the PM structure of the Lu. longipalpis sandfly, including composition, synthesis, and degradation. The kinetics of PM formation and degradation was found to be related to the ingestion and time of digestion of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during the course of digestion. A striking morphological modification takes place in the midgut epithelium after the stretching caused by the bloodmeal, revealing a population of cells that was not observed in the unfed midgut. The transmission and scanning electron microscopies were used to reveal several morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an alpha-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least five protein bands with molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was produced against PMs induced by protein-free meal and used in Western blotting, which revealed at least three associated proteins.

Enlargement and membrane destabilisation in digestive cell lysosomes of mussels are biomarkers of pollution effect. Cytochemical methods are currently applied to determine lysosomal membrane stability (LMS) and lysosomal structural changes (LSC). LMS, determined after grading N-acetyl-beta-hexosaminidase activity on cryotome sections of digestive gland, is measured as labilisation period (LP). LSC, determined after image analysis of cryotome sections where beta-glucuronidase activity is revealed, are measured as lysosomal volume (Vv), surface (Sv), numerical (Nv) densities and surface-to-volume ratio (S/V). Both methods have now been compared in a field study. Mussels were collected from Biscay Bay (Plentzia, reference; Muskiz, moderately polluted) and North Aegean Sea (Olympiada, reference; Limani, heavily polluted). Higher Vv and Sv and lower S/V and LP were recorded in polluted sites than in reference sites. Significant correlations with LP were found for Vv and S/V. The cost/effectiveness and environmental significance of both methods are discussed.

A long-term biomonitoring study was carried out in the Lagoon of Venice (North-East Italy) with the aim of evaluating variations in biological responses to environmental stress in estuarine bivalves. Two different species, the mussel Mytilus galloprovincialis and the clam Tapes philippinarum, both widespread in the Lagoon, were studied in several sites. Two cellular biomarkers: lysosomal membrane stability in digestive cells and thickness of digestive epithelia, were evaluated in native organisms (on a seasonal basis), and in organisms which have been transferred from a reference site to several differently influenced ones. Results indicate that, to some extent, both test and organisms were able to highlight site-specific differences, but the effects of pollution were generally more easily detected by reduction in lysosomal stability than by reduction in digestive tubule epithelium. Further findings show that the inherent variability of a number of natural parameters, particularly in the reference sites, produced less effective results when biological responses in the reference organisms were compared with the polluted ones. The assessment of the two conditions was most valuable when they referred to the 25-75% range of values comprised within the seasonal medians, recorded respectively in control and polluted sites. Impaired from steady states were most effectively distinguished when the control values were medians from two reference locations. Lately, the overall results indicate that both biomarkers are more suitably deployed through the translocation approach, revealing it to be more sensitive than traditional biomonitoring, at least in the sense that it may overcome problems related to the adaptation of native organisms to sub lethal chronic pollution levels.

This study was undertaken to improve the understanding of arsenic species and their pathways of formation in marine animals: fish (Odax cyanomelas), abalone (Haliotis rubra), and sea urchins (Heliocidaris erythrogramma and Centrostephanus rodgersii) that are directly exposed through their diets to dimethyl arsenoriboses in macroalgae (Phyllospora comosa and Halopteris platycena). The identification of dimethyl arsenoriboses (phosphate, sulfonate, and glycerol) in both dominant macroalgae species, and especially digestive tissues of marine animals that consume them, suggests these arsenic species, are to some degree accumulated directly from their diets without degradation or conversion. An unknown arsenic species in H. rubra intestinal tissue was identified using tandem mass spectrometry as 2',3'-dihydroxypropyl 5-deoxy-5-trimethyl arsonioriboside (trimethyl glycerol arsenoribose). The concentration of trimethyl glycerol arsenoribose in H. rubra intestinal tissue was estimated to account for 28% (5.0 microg g(-1) dry mass) of the methanol-water-soluble arsenic fraction. The presence of a trimethyl glycerol arsenoribose in marine animal tissues may be due to microbial-mediated processes that promote the reduction and methylation of dimethyl arsenoriboses released during the breakdown of macroalgae in their diets. Arsenobetaine formation may then occur in the lumen of the digestive tract (i.e., mediated by microorganisms) or in the liver catalyzed by enzymes. The identification of a large amount of trimethyl glycerol arsenoribose in H. rubra intestinal tissue suggests this species is a main constituent in the pathway for arsenic in this marine animal.

The fine structure of the midgut and the Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) specimens was described. We observed the presence of electron-dense granules (EDGs) in the midgut epithelial cells, similar in genesis, structure and aspect to the type A spherocrystals described in the midgut epithelium of Collembola and Diplopoda. Energy-dispersive X-ray microanalysis was used to detect the chemical composition of the granules and to relate it to the concentrations of some potential toxic heavy metals (Pb, Cu, Zn) in soil and litter. Chemical composition of the granules seems strongly influenced by the presence and bioavailability of heavy metals in the external environment. Specimens from a contaminated abandoned mining and smelting area (Colline Metallifere, southern Tuscany) were able to accumulate Fe, Mn, Zn, Pb and Cu in their midgut EDGs. In addition, we observed that C. (M.) quilisi was able to excrete the metal-containing granules into the external medium by the moulting of the intestinal epithelium. This confirms that the process of ionic retention of midgut cells is particularly significant in animals lacking Malpighian tubules.

The concentrations of V, Mn, Fe, Ni, Cu, Zn, As, Se, Mo, and Pb were measured in digestive gland (DG), branchial hearts (BH), gill (G), and muscle (M) of Octopus vulgaris Cuvier, 1797 caught in three fishing areas of the Portuguese coast, Viana do Castelo, Cascais, and Santa Luzia, for 2 years. The elemental concentrations measured for the different tissues were in accordance with values reported in the literature. The digestive gland presented high concentration levels of Fe, Cu, and Zn, while the branchial hearts showed elevated levels of V, Ni, Mo, as well as Fe and Cu. Significant variations in As, V, Cu, Mo, and Pb tissue concentrations were observed for animals originated from different sampling sites. Pb and As determined in the digestive gland and branchial hearts of animals from Cascais and Santa Luzia, can reflect local environmental characteristics. The variability observed in the elemental concentrations may be useful to further assess the species susceptibility to environmental conditions.

Anthocyanins are present in human diet due to their wide occurrence in fruits and beverages. They possess antioxidant activities and could be involved in several health effects. The aim of this study was to investigate anthocyanin metabolism and distribution in the digestive area organs (stomach, jejunum and liver) and kidney, as well as a target tissue (brain) in rats fed with a blackberry (Rubus fruticosus L.) anthocyanin-enriched diet for 15 days. Identification and quantification of anthocyanin metabolites was carried out by HPLC-ESI-MS-MS and HPLC-DAD, respectively. The stomach exhibited only native blackberry anthocyanins (cyanidin 3-O-glucoside and cyanidin 3-O-pentose), while in other organs (jejunum, liver, and kidney) native and methylated anthocyanins as well as conjugated anthocyanidins (cyanidin and peonidin monoglucuronides) were identified. Proportions of anthocyanin derivatives differed according to the organ considered, with the liver presenting the highest proportion of methylated forms. Jejunum and plasma also contained aglycone forms. In the brain, total anthocyanin content (blackberry anthocyanins and peonidin 3-O-glucoside) reached 0.25 +/- 0.05 nmol/g of tissue (n = 6). The urinary excretion of total anthocyanins was low (0.19 +/- 0.02% of the ingested amount). Thus, organs of the digestive area indicated a metabolic pathway of anthocyanins with enzymatic conversions (methylation and/or glucurono-conjugation). Moreover, following consumption of an anthocyanin-rich diet, anthocyanins enter the brain.

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.

Cadmium concentrations were measured in the flesh and hepatopancreas (digestive gland) of 1,392 specimens of different species of cephalopod molluscs (broadtail squid, spider octopus, curled octopus, horned octopus, elegant cuttlefish, and pink cuttlefish) to determine whether maximum levels fixed by the European Commission were exceeded. In all species, mean cadmium concentrations were higher in hepatopancreas than in flesh. Large differences among the different species were also observed. Pink cuttlefish and spider octopus had the highest concentrations for both flesh (spider octopus, 0.77 microg g(-1); pink cuttlefish, 0.87 microg g(-1)) and hepatopancreas (spider octopus, 9.65 microg g(-1); pink cuttlefish, 18.03 microg g(-1)), and the lowest concentrations were encountered in broadtail squid (flesh, 0.13 microg g(-1); hepatopancreas, 2.48 microg g(-1)). The other species had intermediate concentrations of 0.20 to 0.30 microg g(-1) in flesh and 5.46 to 8.01 microg g(-1) in hepatopancreas. Concentrations exceeding the limit proposed by the European Commission (1.00 microg g(-1)) were observed in 44.4 and 40.0% of flesh samples of spider octopus and pink cuttlefish, respectively. The estimated weekly intake, 0.09 to 0.66 microg/kg body weigh, was below the provisional tolerable weekly intake set by the World Health Organization.

Molecular detection systems used to analyse the gut contents of invertebrate predators have enhanced our understanding of trophic interactions, but do not distinguish between the methods of consumption. Many predators regularly scavenge, which could have profound implications for quantitative analyses of the dynamics of predation. We report the first quantified assessment of the potential error caused by scavenging in post-mortem measurements of predation in a slug-carabid system. An anti-slug monoclonal antibody was able to detect antigens from decayed slugs after surprisingly long periods, significantly longer on relatively sterile peat than on natural soil. On soil the half-life of antibody-detectable slug proteins was 8.2 days while on peat it was 11.5 days. When slugs that had decayed on soil for 100 h were fed to the carabid predator Pterostichus melanarius, slug proteins could still be identified after 6 h (but not 12 h) digestion. Fresh and decayed slug was eaten in equal quantities by the beetles suggesting no aversion to the latter. The results suggest that significant errors may be caused by scavenging leading to inaccurate interpretation of predation rates in the field.

Seventeen elements were analysed in the organs and tissues of the variegated scallop Chlamys varia, from the Atlantic coast of France. Concentration levels were determined in scallops of different sizes sampled in contaminated (La Rochelle Bay) and clean (Re Island) sites. Greater concentrations of Ag, Al, Ce, Cr, La, Mo, Nd, Ti, and V were found in the digestive gland while As, Cd, Co, Cu, Mn, Ni, Pb, and Zn were the highest in the kidneys. In the digestive gland, most of the metals were found in the insoluble fraction while As, Co, Cd, Mo, Ni, and V appeared to be mostly bound to soluble compounds. Among tissues, the adductor muscle always displayed the lowest trace element concentrations. According to size, Ag and Cd showed significantly higher concentrations in larger individuals, while Co and Zn were higher in the smallest ones. According to the sampling area, most of the metals, Ag, Al, Ce, Co, Cu, La, Mn, Nd, Pb, and Zn, showed significantly higher concentrations in La Rochelle Bay compared to the Re Island, reflecting differing inputs from industrial, domestic and harbour activities. However, Cr, Mo, Ni, Ti, and V concentrations did not display significant differences between sites and As and Cd were significantly higher at the Re Island. This study highlighted the ability of the variegated scallop C. varia to concentrate numerous trace elements to high levels, even those reported as poorly bioavailable for marine biota, such as rare earth elements.

The origin of the peritrophic membrane (PM) in bees is still a matter of debate. It is either of type I (synthesized by the entire midgut epithelium) or of type II (released from the anterior midgut end). The present study identified secretory sites of peritrophin-55 kDa, a PM protein in larvae of Melipona quadrifasciata anthidioides. Peritrophin-55 was isolated from PMs and was used for the production of a polyclonal antibody. Our study demonstrates the presence of peritrophin-55 in vesicles and on microvilli of digestive cells and in the PM. It suggests that the PM is of type-I, being specific for the larval phase of this stingless bee.

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut., (Copyright 2004 Wiley-Liss, Inc.)