Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Glucose reabsorption by the prosobranch gastropod Littorina littorea (L.)

The renopericardial complex of Littorina is capable of glucose reabsorption in vivo, as the glucose concentration of the urine may be markedly lower than that of the haemolymph. Glucose influx in vitro was estimated for the kidney (dorsal wall and nephridial gland) and heart, using [³H]glucose as tracer. Glucose influx to both dorsal wall and nephridial gland of the kidney was sensitive to phloridzin, and appeared to reflect net inward transport by a saturable, Na⁺-dependent carrier mechanism believed to be responsible for reabsorption in vivo. There was some intrarenal metabolism of transported glucose. The mechanism and rôle of glucose reabsorption in Littorina are discussed, and the limitations of the investigative techniques are considered.     

Effects of barnacle epibionts on the periwinkle Littorina littorea (L.)

In a sandy bay with mussel beds in the Wadden Sea (Island of Sylt, eastern North Sea), periwinkles Littorina littorea (L.) were often strongly overgrown with the barnacle Balanus crenatus Bruguière in the lower intertidal zone. Consequences of this epibiosis on mobility, reproduction and mortalityof the snail were examined. B. crenatus growing on L. littorea increased snail volume up to 4-fold and weight up to 3.5-fold and crawling speed of fouled L. littorea was significantly slowed down. The epibiotic structure also caused a decrease in reproductive output. In laboratory experiments, egg production of fouled L. littorea was significantly lower than in snails free of barnacles. Presumably, copulation of the periwinkles is hampered by the voluminous and prickly cover of barnacles. Field studies demonstrated an increased mortality of overgrown L. littorea. A decrease in reproductive output and a lower survival of snails with a cover of barnacles suggest that B. crenatus epibionts may have a significant impact on the population of L. littorea. Received: 20 June 1998 / Accepted: 2 October 1998     

Histochemistry and elemental composition of the stomach cells in Littorina littorea (L.)

The present work deals with the histochemical, histoenzymological and micro-analytical study of the stomach of Littorina Littorea (L.). The results suggest the existence of intracellular digestion within the stomach cells. Thus, the occurrence of lysosomes in the mid-cytoplasm of stomach cells has been revealed by demonstration beta-glucuronidase activity in cryostat sections of frozen tissues. Other enzymatic activities have been proved with the same tissues. Accordingly, the results on lipid and pigment histochemistry indicate a certain digestion in this cell type. Mucocytes show a protective role for the epithelium, their main secretions being acid MPS. The elemental composition of electron dense concretions from the stomach cells indicate the presence of very high amounts of iron associated to lower peaks for sulphur, phosphorous and other cations found as calcium, copper and zinc.     

Age-dependent zonation of the periwinkle Littorina littorea (L.) in the Wadden Sea

On sedimentary tidal flats near the island of Sylt (German Bight, North Sea) abundance and size distribution of periwinkles, Littorina littorea L., were studied in low intertidal and in shallow and deep subtidal mussel beds (Mytilus edulis L.). In low intertidal mussel beds, surveys revealed that high densities (1,369±571 m^–2) of juvenile snails (≤13 mm) were positively correlated with strong barnacle epigrowth (Semibalanus balanoides L. and Balanus crenatus Bruguière) on mussels. A subsequent field experiment showed that recruitment of L. littorea was restricted to the intertidal zone. Abundances of periwinkles (213±114 m^–2) and barnacles abruptly decreased in the adjacent shallow subtidal zone, which served as a habitat for older snails (>13 mm). L. littorea was completely absent from disjunct deep (5 m) subtidal mussel beds. Snail abundance varied seasonally with maxima of >4,000 m^–2 in low intertidal mussel beds in October and minima in July, just before the onset of new recruitment. I suggest that the presence of cracks and crevices among the dense barnacle overgrowth in intertidal mussel beds favoured recruitment and survival of juvenile snails. Larger (older) specimens are assumed to actively migrate to the less favourable adjacent subtidal. Therefore, intertidal mussel beds are considered as nurseries for the population of L. littorea in the Wadden Sea. Received in revised form: 25 September 2000 Electronic Publication     

Mate discrimination in Littorina littorea (L.) and L. saxatilis (Olivi) (Mollusca:Prosobranchia)

The ability of males of Littorina littorea and L. saxatilis to discriminate between mates of different sex, species and size was examined. In partner choice experiments males of L. littorea had the possibility to initiate a copulation with either a female or a male. The males did not show a preference for either sex. There was therefore no evidence that they could determine the sex of a conspecific prior to copulation. The duration of intrasexual copulation was considerably shorter than for intersexual copulation, both in the field and in laboratory experiments. For the two species, intersexual copulations were far more frequent than intrasexual ones. This can partly be explained by the difference in copulation time.Few interspecific copulating pairs were found on the shore. This may reflect a low interspecific encounter rate rather than a mechanism of species recognition. On all of these occasions, however, the active male was of L. saxatilis. It is argued that selection against precopulatory species and sex recognition is a more likely explanation than an hypothesis that states that the required mutations for precopulatory mate identification has not yet occurred. L. littorea males copulated longer with large than with small females. Copulation time was short with parasitized females, which are sterile or of low fecundity. The allocation of mating effort by males is discussed.     

Fine structure of the cephalic sensory organ in veliger larvae of Littorina littorea, (L.) (Mesogastropoda,Littorinidae)

The cephalic sensory organ (CSO) in planktonic veliger larvae of Littorina littorea is situated dorsally between the velar lobes at the level of the shell aperture. It consists of ciliated primary sensory cells, adjacent accessory cells and supporting epithelial cells. Cell bodies of the ciliated cells originate in the cerebral commissure and their dendrites pass to the epidermis. The flask-shaped sensory cells are characterized by a deep invaginated lumen with modified cilia arising from the cell surface in the lumen. These cilia are presumed to be non-motile because they lack striated rootlets and show a modified microtubular pattern (6 + 2, 7 + 2 and 8 + 2). The adjacent accessory cells never possess an invaginated lumen; occasionally cilia and branched microvilli arise from the apical surface. These cells may be sensory, but there is no obvious direct connection with the nervous system. The supporting epithelial cells are part of the epidermis and flank the apical necks of the sensory and accessory cells. Morphological evidence suggests that the CSO may function in chemoreception related to substrate selection at settlement, feeding or other behaviour.     

Effects of trematode parasitism on the behaviour and ecology of a common marine snail (Littorina littorea (L.))

Cryptocotyle lingua (Creplin) is a digenean trematode parasite of the littoral prosobranch gastropod Littorina littorea (L.). The literature suggests the snails become infected by grazing guano of the final host, the herring gull, Larus argentatus Pontoppidan. The parasite emerges from the snail as free-swimming cercariae. Interactions between the snail and the parasite at cellular and life-history levels are well established, but little is known of the influences the interaction has on the behaviour and the ecology of the snail. We tested the response of the snail to encounters with cercariae, examined the longevity of the guano on-shore and tested the responses of the snail to encounters with guano. Over half the L. littorea tested were able to detect both cercariae and a filtered homogenate of cercariae in conspecific mucus trails, approximately one-third of animals refusing to cross the treatments. Chemoreception by the mouth or foot is considered the most likely means of detection. Guano samples (mean weight 3.22 g) naturally deposited at approximately mid-tide level were completely washed away by one tidal inundation. We consider this period too brief to allow for ingestion of eggs in guano by the snail. Further, snails would not cross guano placed in conspecific trails. Most snails would not cross guano diluted by 10³×(10 mg ml⁻¹) and some snails could still detect guano diluted by 10⁶×(10 μg ml⁻¹), though all were prepared to cross it. Detection of guano is again believed to be by chemoreception by the mouth or foot. These results are discussed in terms of the mating and aggregating behaviour of L. littorea. Ingestion of the parasite by L. littorea is likely to take place once the guano has washed away as the eggs are negatively buoyant in seawater and may adhere to rock (biofilm) or algal fronds which may be grazed by the snail.     

Direct and indirect effects of Littorina littorea (L.) on barnacles growing on mussel beds in the Wadden Sea

On the extensive sedimentary tidal flats of the Wadden Sea, beds of the blue mussel Mytilus edulis represent the only major hard substratum and attachment surface for sessile organisms. On this substratum, the barnacle Semibalanus balanoides is the most frequent epibiont. In summer 1998, it occurred on over 90% of the large mussels (>45 mm shell length) and the dry weight of barnacles reached 65% of mussel dry weight. However, the extent of barnacle overgrowth is not constant and differs widely between years. Periwinkles (Littorina littorea) may reach densities >2000 m^–2 on intertidal mussel beds. Field experiments were conducted to test the effect of periwinkle grazing on barnacle densities. An experimental reduction of grazing and bulldozing pressure by periwinkles resulted in increased recruitment of barnacles, while barnacle numbers decreased with increasing snail density. The highest numbers of barnacles survived in the absence of L. littorea. However, a lack of periwinkle grazing activity also facilitated settlement of ephemeral algae which settled later in the year. Field experiments showed that the growth rate of barnacles decreased in the presence of these ephemeral algae. Thus, L. littorea may reduce initial barnacle settlement, but later may indirectly increase barnacle growth rate by reducing ephemeral algae. It is suggested that periwinkle density may be a key factor in the population dynamics of S. balanoides on intertidal mussel beds in the Wadden Sea.     

Periphyton Architecture and Susceptibility to Grazing by Periwinkles (Littorina littorea,Gastropoda)

Periphyton was grown on transparent plastic substrata in the Kiel Fjord and used for short-term laboratory experiments to study the feeding selectivity of the periwinkle Littorina littorea in response to the vertical structure of the periphyton. The susceptibility of algae to periwinkle grazing was assessed by comparing the species-specific biomass within the grazing tracks of the snails to the biomass outside the tracks. After 3 weeks of incubation, the periphyton consisted of a scattered monolayer of algal cells without vertical structure. No apparent grazing could be found. After 6 weeks of incubation, periphyton consisted of a tightly attached undergrowth (mainly Cocconeis scutellum, Bacillariophyceae, and Myrionema sp., Phaeophyceae) and canopy of filamentous (Melosira moniliformis, Bacillariophyceae) and stalked forms (Achnanthes longipes, Bacillariophyceae). The unicellular diatoms Fragilaria tabulata and Stauroneis constricta grew partly on the primary substratum and partly as epiphytes on the canopy species. The canopy species and the epiphytes were decimated inside the grazing tracks, while the tightly attached undergrowth species appeared ungrazed.     

Specific dynamic action demonstrated in the herbivorous marine periwinkles,LIttorina littorea L. and Littorina obtusata L. (Mollusca,Gastropoda)

• 1.1. Respiration rates of starved periwinkles, Liltorina littorea and Littorina obtusata, increased by 40–60% when fed their preferred algal food for 1 hr, or after having been exposed for the same period to an aqueous extract of the alga.
• 2.2. The stimulus causing the rise in metabolic rate by algal extracts is therefore thought to be chemosensory in nature, and possibly composed primarily of dissolved free amino acids.
• 3.3. The respiration rates of L. littorea responded only to the green alga Enteromorpha intestinalis while L. obtusata demonstrated an increase in respiration rates when fed the ubiquitous brown alga, Ascophyllum nodosum, and to a lesser degree to Enteromorpha.
• 4.4. These results compare very well to postprandial increases in oxygen consumption demonstrated in vertebrates and marine bivalves where the components of specific dynamic action (SDA) are thought to be chiefly biosynthetic costs of digestion and assimilation.

     

Elemental composition of bacterial metachromatic inclusions determined by electron microprobe X-ray analysis.

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.     

Brackish-water submergence of the common periwinkle,Littorina littorea,and its digenean parasites in the Baltic Sea and in the Kattegat

North Sea and Baltic Sea populations ofLittorina littorea differ with respect to their vertical distribution. In the North SeaL. littorea is strictly intertidal while in the Baltic Sea maximum population densities occur in the sublittoral. Levels of infestation with larval digenetic trematodes diminish qualitatively (number of species recorded) and quantitatively (number of hosts infested) with decreasing salinity. Both the host and two parasite species —Cryptocotyle lingua andMicrophallus pygmaeus — display brackish-water submergence under conditions of reduced surface salinity.     

Genetic variability and large scale differentiation in two species of littorinid gastropods with planktotrophic development, Littorina littorea (L.) and Melarhaphe (Littorina) neritoides (L.) (Prosobranchia: Littorinacea), with notes on a mass occurrence of M. neritoides in Sweden

A planktotrophic larval development suggests a relatively high gene flow over long distances. Both Littorina littorea and Melarhaphe neritoides have an egg capsule and planktotrophic larvae which taken together are pelagic over a period of 4–8 weeks. In the absence of differential selection one would expect low levels of genetic differentiation over large distances in these two species. In this study of allozyme variation, low levels of differentiation were found over thousands of kilometres (Norway to Spain for L. littorea and Sweden to Greece for M. neritoides). This supports the hypothesis of two species with a high dispersal potential. A second expectation from neutral theory is that effective population size is positively correlated with average levels of genetic variation within species. In light of the generally high densities of local populations of these species and the high interpopulation migration rate, both L. littorea and M. neritoides may be considered as having high N_e s. Contrary to neutral expectation, L. littorea revealed very low levels of heterozygosity over its whole European range (mean H_exp= 0.015, 15 loci), while average heterozygosity of M. neritoides (H_exp= 0.084, 11 loci) was no more than in other littorinid species. This paper also reports the occurrence of M. neritoides on the Swedish west coast in 1988, 89 and 90, and two factors which may have promoted this unusually large invasion are discussed.     

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd pool strongly bound to cytosolic Cd-metallothionein complexes; only free Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (Vv_BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, Vv_BSD values were lower at days 7 and 21 than at day 1. This relative decrease in Vv_BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (Vv_BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.     

Occurrence of epifaunshers on the periwinkle,Littorina littorea (L.), and interactions with the the polychaete Polydora ciliata (Johnston)

A population of Littorina littorea on the northshore of Southampton Water, UK, has been examined forthe occurrence of epifauna. Epifaunal cover was notaffected by shore level below MTL but was greatest onlarger shells, which were commoner on the lower shoreclose to MLWS. Epifaunal components includedbarnacles, serpulids and bryozoans. The polychaetePolydora ciliata occurred most frequently on thelower shore where it excavates galleries in periwinkleshells. Heaviest infestations were found in largershells. P. ciliata settles preferentially in orbeside irregularities on the shell surface. Settlementof barnacles close to the shell aperture, where newshell growth takes place, produces shell distortionwhich creates sites for settlement of P. ciliata. Theangle between serpulid tubes and theshell surface also attracts P. ciliata. It issuggested that colonisation by barnacles and serpulidsfacilitates settlement by P. ciliata, and thatthe latter decreases survival of the host byprogressively destroying the shell.