Endosymbiotic and Host Proteases in the Digestive Tract of the Invasive Snail Pomacea canaliculata: Diversity,Origin and Characterization

Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen.     

Kinetics of metals in molluscan faecal pellets and mineralized granules,incubated in marine sediments

Marine snails excrete metals via the gut. The sediment-feeding, tower shell Cerithium vulgatum, from a polluted environment, accumulates Cr and Ni to 3500 ppm dry weight in the faecal pellets. Pellets from C. vulgatum and the grazing, top shell Monodonta mutabilis were incubated in surface and deeper anoxic layers of clean and metal-polluted sediment. Pellets retained the original load of metals and in some cases gained Ti, Cr, Mn, Fe and Ni. Effects were modified by sediment properties. Pellets of C. vulgatum are durable, membrane-bound structures and they reduce metal bioavailability to food chains by compartmentalization. Intracellular, phosphate granules bind metals in digestive glands of snails and they are excreted via the gut and faecal pellets. These granules were extracted from digestive glands of both species of snail and incubated in the same sediments. Magnesium phosphate granules from M. mutabilis dissolved but calcium/metal phosphate granules from C. vulgatum remained; they had differentially retained or lost Mn, Fe, Co, Ni and Zn according to the type of sediment.     

Molecular fractionation of zn,cu, cd and pb in the midgut gland of Helix pomatia l.

1. Specimens of Helix pomatia were fed lettuce enriched with Zn, Cu, Cd and Pb for 30–35 days, after which time the distribution of the four metals in the midgut gland was studied by centrifugation, ion exchange and gel chromatography.2. In control snails the distribution between the soluble and particulate fraction of the midgut gland is relatively uniform, with a ratio of approximately 3:1 for Cu, Cd and Pb, and of 1:1 for Zn. However, after metal-loading about two-thirds of total Zn and Pb appears in the particulate fraction whereas little change in relative distribution is observed with Cu and Cd.3. After ion exchange chromatography of the supernatant both Cd and Zn eluted in one distinct peak, Cu in two peaks, whereas Pb seemed to be distributed unspecifically throughout the elution volume.4. If ion exchange is followed by gel chromatography, Zn, Cu and Cd are found associated with from one to four molecular species, ranging in mol. wt from 1000 to > 20,000, whereas no complex was found for Pb. The molecular patterns are specific for each of the three metals and there are some specific shifts in abundance, usually involving one molecular fraction, if control snails are compared with metal-loaded snails.     

Molecular fractionation of Zn, Cu, Cd and Pb in the midgut gland of Helix pomatia L

Specimens of Helix pomatia were fed lettuce enriched with Zn, Cu, Cd and Pb for 30-35 days, after which time the distribution of the four metals in the midgut gland was studied by centrifugation, ion exchange and gel chromatography. In control snails the distribution between the soluble and particulate fraction of the midgut gland is relatively uniform, with a ratio of approximately 3:1 for Cu, Cd and Pb, and of 1:1 for Zn. However, after metal-loading about two-thirds of total Zn and Pb appears in the particulate fraction whereas little change in relative distribution is observed with Cu and Cd. After ion exchange chromatography of the supernatant both Cd and Zn eluted in one distinct peak, Cu in two peaks, whereas Pb seemed to be distributed unspecifically throughout the elution volume. If ion exchange is followed by gel chromatography, Zn, Cu and Cd are found associated with from one to four molecular species, ranging in mol. wt from 1000 to greater than 20,000, whereas no complex was found for Pb. The molecular patterns are specific for each of the three metals and there are some specific shifts in abundance, usually involving one molecular fraction, if control snails are compared with metal-loaded snails.     

Comparative study of the bioaccumulation and elimination of trace metals (Cd,Pb, Zn,Mn and Fe) in the digestive gland,gills and muscle of bivalve Pinna nobilis during a field transplant experiment

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.     

Trace metal transfer in a soil–plant–snail microcosm field experiment and biomarker responses in snails

A microcosm experiment was performed to investigate temporal (up to 16 weeks) and spatial variation in metal transfer in a soil–food (nettle)–snail (Cepaea nemoralis) food chain and biomarker responses in the digestive gland of the same snails. Adult snails were sampled from an uncontaminated site and transferred to five sites located 0.5, 2.5, 3, 5, and 10 km from a historically polluted point source. All sites were park areas where grasslands interfered with patches of deciduous forest. Soil physicochemical properties (pH, clay%, OC%) significantly explained the uptake of metals by nettle. Concentrations of metals in the digestive gland (DG) of snails were significantly related to those in nettle, but rarely to soil physicochemical properties. In general metal concentrations in the DG fluctuated while As, Ni, Pb and Zn showed a site dependent increase with time. Despite the long term exposure, biomarker concentrations (lipid, glycogen, proteins, glutathione-S-transferases), and shell morphology, were not related to DG metal concentrations. Our investigation emphasizes the need for controlled long-term studies on the transfer and effects of metals in food chains since short term studies might only show temporary physiological changes due to experimental acclimation.     

Accumulation and excretion of aluminium and iron by the terrestrial snail Helix aspersa

1. The snail Helix aspersa was fed one 24 hr meal containing Al, Fe or both together in barley flour pellets. Accumulation and distribution within the digestive gland, kidney, crop and remaining soft tissues were examined over the subsequent 30 days using atomic absorption spectroscopy (A.A.S.).2. The digestive gland contained significantly (P < 0.05) elevated levels of Al and Fe for 8 and 12 days. The digestive gland is the major sink for both Al and Fe in Helix.3. The kidney rapidly accumulated Al and Fe but the increase was short-lived. The kidney may therefore be involved in the elimination of metal not incorporated into the digestive gland.4. Iron was absorbed by the crop but Al was not. This may indicate a route of uptake of Fe into the digestive gland not shared with Al.5. No obvious pattern of accumulation of Al and Fe were seen in the remaining soft tissues or the blood of Helix.6. Aluminium is present in the faeces for 12 days suggesting that Al is released relatively slowly.7. Presence of both Al and Fe in the feed induced a change in the pattern of accumulation in the digestive gland but not in the kidney, crop and remaining soft tissues.8. The distribution of Al is discussed in relation to the suggestion that Al follows the ferretin pathway during accumulation.     

Storage excretion in the Indian apple snail,Pila globosa (Swainson), during aestivation

Uric acid accumulates in several tissues of the Indian apple snail, P. globosa, during aestivation. This accumulation is particularly high in the foot muscle and reproductive organs. Since the kidney, a tiny organ in the snail, has only a limited capacity to store uric acid, extrarenal tissues are used as storage depots of uric acid during aestivation. It is suggested that the aestivating snail, faced with a cleidoic situation, resorts to storage excretion, a phenomenon well documented in insects.     

Localization and fate of aluminium in the digestive gland of the freshwater snail Lymnaea stagnalis

The digestive gland of the freshwater snail Lymnaea stagnalis, exposed to water containing an elevated concentration of aluminium at neutral pH for up to 30 days, followed by a 20 day recovery period, was examined by light and electron microscopy and X-ray microanalysis. Aluminium was localized in the yellow granules present in the digestive and excretory cells and in the green and small granules present in the digestive cells. More aluminium, silicon, phosphorus and sulphur were present in all three granule types from aluminium exposed snails. The number of yellow and green granules from the digestive gland of aluminium exposed snails showed a progressive increase over the experimental period compared to controls. The number and aluminium content of the granules is likely to reflect the role of the digestive gland as a 'sink' for accumulated aluminium. We propose that intracellular monomeric silica is involved in the detoxification of aqueous aluminium which at neutral pH is largely in the form of an insoluble polyhydroxide. The increased amounts of sulphur and phosphorus in the granules are likely to be part of a broad response to metal loading but probably do not play a significant role in the storage and detoxification of aluminium.     

Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland

In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails (Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 µmol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd²⁺ ions, whereas at the cellular level, structural alterations turned into pathological deterioration.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.     

Excretion, accumulation and site of synthesis of urea in the snail Pila globosa during active and dormant periods.

The pattern of urea excretion in active and dormant phases of the amphibious Indian apple snail, Pila globosa, as well as the possible site and mechanism of urea synthesis in this animal have been studied. 1. Urea may be synthesized in the hepatopancreas of this snail and excreted through the nephridia with the nephridial fluid during the active period of the animals life. In dormant snails, this metabolic excretory waste accumulates in its various organs and body fluids. 2. The possibility of the presence of an ornithine cycle for the synthesis of urea in the hepatopancreas is indicated in this snail.     

Metal accumulation and sublethal effects in the sea anemone, Aiptasia pallida, after waterborne exposure to metal mixtures

The marine environment is subjected to contamination by a complex mixture of metals from various anthropogenic sources. Measuring the biological responses of organisms to a complex mixture of metals allows for examination of metal-specific responses in an environmentally realistic exposure scenario. To address this issue, the sea anemone, Aiptasia pallida was exposed to a control and a metal mixture (copper, zinc, nickel, and cadmium) at three exposure levels (10, 50, and 100 μg/L) for 7 days. Anemones were then transferred to metal-free seawater for an additional 7 days after the metal exposure to assess metal depuration and recovery. Metal accumulation, activity of the enzymes catalase, glutathione reductase, and carbonic anhydrase, as well as, cell density of the symbiotic zooxanthellae were measured over 14 days. Metal accumulation in A. pallida occurred in a concentration dependent manner over the 7-day exposure period. Altered enzyme activity and tentacle retraction of the host, as well as decreased zooxanthellae cell density were observed responses over the 7 days, after exposure to a metal concentration as low as 10 μg/L. Metal depuration and physiological recovery were dependent on both the metal and the exposure concentration. Understanding how A. pallida and their symbionts are affected by metal exposures in the laboratory may allow better understanding about the responses of symbiotic cnidarians in metal polluted aquatic environments.     

Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co²⁺, Ni²⁺, or Zn²⁺ for 1 to 4 days was studied by incubating leaf discs in [¹⁴C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7-millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni²⁺ and Zn²⁺. The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions.     

Structural and ultrastructural analysis of gills and gut of Hexagenia rigida nymphs (ephemeroptera) in relation to contamination mechanisms

In the context of an experimental approach to organic and inorganic mercury bio-accumulation by burrowing mayfly nymphs-Hexagenia rigida-, gills and different parts of the gut were investigated by light and electron microscopes. In the gut, two regions were studied: (1) mesenteron (midgut) characterized by cells with microvilli and a peritrophic membrane throughout this part, a lot of fungal hyphae being found on it; (2) proctodeum (hindgut) characterized by macrovilli, the apical face of cells being covered by a thick layer of chitin; many bacteria were observed at the bottom of macrovilli. The stomodeum (foregut) is extremely short. The gills are made up of six pairs of lamellae fringed with long filaments which are arranged on both sides of the long axis. Gills are constituted by tracheae and tracheoles, with a large canal for haemolymph circulation; muscle masses and nerve bundles were also observed. This structural approach shows that the nymph interface with the external environment, at gill and gut barrier levels, are quite different. Links between this structural and ultrastructural analysis and contamination mechanisms, via the trophic or direct route, are discussed; gills and gut could be transfer routes for mercury absorption, but also target organs for metal accumulation.     

The influence of starvation and copper exposure on the composition of the dorsal carapace and distribution of trace metals in the shore crab Carcinus maenas (L.)

1. The influence of starvation and copper exposure on the composition of the carapace of the shore crab, Carcinus maenas has been investigated. The effects of the concentrations of selected trace metals in the carapace and midgut gland have also been examined.2. Differences in nutritional state did not affect the concentrations or relative proportions of the principle carapace components (calcium, chitin and protein), but starvation was associated with a reduction in carapace copper concentration. Copper concentration in the midgut gland increased in “starved” crabs, while midgut gland zinc and calcium concentrations remained unchanged.3. Starvation in combination with copper exposure (0.5 mg Cu/1) resulted in alterations in both carapace composition and trace metal loads. Carapace calcium concentrations were reduced significantly following copper exposure irrespective of the nutritional state of the crabs. However, the reduction was more marked in “fed” crabs than in “starved” animals.4. Copper-exposed crabs exhibited raised carapace copper concentrations while carapace zinc concentrations were depressed. Such changes were most pronounced in copper-exposed “starved” crabs.5. Calcium concentrations in the midgut glands of “starved” copper-exposed animals were significantly elevated compared with those in either control or “fed”, copper-exposed crabs.6. The findings are discussed with regard to their ecotoxicological significance.     

Possible toxic effects of the marine blue-green alga, Spirulina subsalsa, on the blue shrimp, Penaeus stylirostris.

Blooms of a marine species of blue-green algae identified as Spirulina subsalsa (Cyanophyta, Oscillatoriacae) were found to be related to a particular disease syndrome in raceway-reared blue shrimp, Penaeus stylirostris. The disease was characterized by necrosis of the lining epithelium of the midgut, dorsal cecum, and hindgut gland, and a consequent hemocytic enteritis. Bacterial infections due predominately to Vibrio alginolyticus were common in affected shrimp and presumed to be a secondary condition resulting from necrosis of the gut epithelium. These bacterial infections were expressed as local abscesses near or on the gut or as fulminating septicemias.     

Assessment of the adaptation capabilities of the bivalves Modiolus modiolus (Linnaeus, 1758) and Crenomytilus grayanus (Dunker, 1853) under increased levels of heavy metals in the environment

The active biomonitoring method was used to examine the changes in heavy-metal (Fe, Zn, Cu, Cd, Mn, Pb, and Ni) contents in the organs (muscle, gonads, mantle, gills, digestive gland, and kidney) of the mussels Crenomytilus grayanus and Modiolus modiolus. The dynamics of trace element concentrations in mussel organs during the experiment were compared with their subcellular distribution. The defense strategy of M. modiolus consisted of the threshold accumulation of toxic metals in all organs followed by their excretion, whereas the strategy of C. grayanus involved short-term isolation from adverse environmental influence. Under chronic pollution, in C. grayanus the main loads occurred in the digestive gland and kidney. Under acute changes in environmental conditions, the processes of regulation (detoxification/excretion) of Fe, Mn, and Pb in this species were impaired substantially.