Field depuration and biotransformation of paralytic shellfish toxins in scallop<Emphasis Type="Italic"> Chlamys nobilis</Emphasis> and green-lipped mussel<Emphasis Type="Italic"> Perna viridis</Emphasis>

Under laboratory conditions, the scallop Chlamys nobilis and the mussel Perna viridis were exposed to N-sulfocarbamoyl toxins (C2 toxin), a paralytic shellfish toxin (PST), by feeding a local toxic strain of the dinoflagellate Alexandrium tamarense (ATDP) that produced C2 toxin exclusively. The bivalves were subsequently depurated in the field, and their depuration kinetics, biotransformation and toxin distribution were quantified. Depuration was characterized by a rapid loss within the first day, followed by a secondary slower loss of toxins. In the fast depuration phase, scallops detoxified PSTs more quickly than the mussels (depuration rate constants for scallops and mussels were 1.16 day^–1 and 0.87 day^–1, respectively). In contrast, the mussels detoxified PSTs more quickly than the scallops in the slow depuration phase, and the calculated depuration rate constants (mean+SE) from day 2 to day 13 were 0.063+0.009 day^–1 and 0.040+0.019 day^–1 for mussels and scallops, respectively. The differences in the appearances of gonyautoxins, GTX2 and GTX3, and their decarbamoyl derivatives, dcGTX2, dcGTX3 and GTX5, which are all derivatives of C2 toxin, indicated active and species-specific biotransformation of the algal toxins in the two bivalves. In both species of bivalves, the non-viscera tissue contained fewer toxins and lower concentrations than the viscera-containing tissue compartment. In scallops, very little toxin was distributed in the adductor muscle. In mussels, most of the PSTs were found in the digestive gland with significant transport of toxins into the digestive gland from other tissues during the course of depuration. The toxin profiles of scallops and mussels differed from each other and from that of the toxic algae fed. A significant fraction of GTX5 was detected in the mussels but not in the scallops. Our study demonstrates a species specificity in the depuration kinetics, biotransformation and tissue distribution of PSTs among different bivalves.Communicated by T. Ikeda, Hakodate     

Occurrence and detrimental effects of the bivalve-inhabiting hydroid <Emphasis Type="Italic">Eutima japonica</Emphasis> on juveniles of the Japanese scallop <Emphasis Type="Italic">Mizuhopecten yessoensis </Emphasis>in Funka Bay,Japan: relationship to juvenile massive mortality in 2003

In November 2003, we first observed prevalent occurrence of a hydroid, Eutima japonica, on soft body tissues of age zero Japanese scallop (Mizuhopecten yessoensis) juveniles cultured in large areas of Funka Bay, Hokkaido. The occurrence coincided with massive death of juvenile scallops. A major objective was to clarify ecological relationships between the symbionts, and to infer the relationship between symbiosis and the massive mortality. To do this, we investigated distributions of association rates of hydroids with juvenile scallops at 15–34 sites over 3 years (2003–2005), with age one adult scallops at 24 sites in 2003, and with mussels at 13 sites in 2004. We studied seasonal changes in association rates with juvenile scallops, and numbers of polyps per juvenile scallop at three sites from November 2003 to June 2004. We also quantified the hydroid impacts on juvenile scallop shell length growth and triglyceride accumulation in the digestive gland. The association rate of E. japonica polyps with juvenile scallops was high in large areas of Funka Bay in 2003, and overlapped the distribution of mussels bearing polyps. Association rates with age one adult scallops were very low in November 2003, even at the sites where polyps were very common on juvenile scallops. Levels of hydroid occurrence in juvenile scallops varies by year. We found that hydroids presence in juvenile scallops declined drastically in 2004 and 2005. The association rates with juvenile scallops, and numbers of polyps per juvenile scallop declined during winter, until they disappeared completely in the following June. Since polyps were rare in adult scallops, we believe that infection of juvenile scallops was probably initiated from the planulae produced by medusae released from polyps growing on Mytilus spp., especially M. galloprovincialis. Subsequently, the inhabitation spread intraspecifically and interspecifically. In juvenile scallops, inhabitation of polyps reduced shell length growth by 43%, and triglyceride accumulation in digestive glands by 24–47%. Inhabitation of E. japonica on juvenile scallop is best regarded as parasitism, rather than inquilinism or commensalism. Occurrence of polyps was probably not a direct lethal factor for juvenile scallops, because there were some sites where association rates were high, but mortalities were low. Massive mortalities in 2003 may have resulted from simultaneous impacts of heavy polyp load and stresses caused by the way in which the animals were handled (transferred from cages for pre-intermediate culture to cages for intermediate culture), because the massive mortality occurred within a month of the transfer. The presence of polyps in juvenile scallops does not affect the quality of the product in Funka Bay, because market size scallops are hydroid-free.     

Anterior regeneration in the spionid polychaetes<Emphasis Type="Italic"> Dipolydora quadrilobata</Emphasis> and<Emphasis Type="Italic"> Pygospio elegans</Emphasis>

Like many other benthic infaunal invertebrates, spionid polychaetes often lose portions of their bodies to predators, which affects their activities. Feeding palp loss and tail loss have been studied in several spionids, but the capacity for anterior tissue regeneration has not been compared in different species. The present study examines anterior tissue regeneration in two species, Dipolydora quadrilobata (Jacobi 1883) and Pygospio elegans Claparède 1863, in two laboratory experiments. Tissue removal treatments included removal of palps only, removal of anterior tissue through the first setiger, anterior tissue through the fifth setiger, all anterior tissue through half of the gill-bearing setigers, and all anterior tissue through the last gill-bearing setiger. Regeneration was monitored by capturing images of the worms and digitizing the area of regenerated anterior tissue or counting the number of segments that grew over time. Worms of both species regenerated anterior tissue regardless of the amount removed. Morphogenesis during regeneration followed a similar pattern in these two species regardless of the amount of anterior tissue lost, progressing from wound healing to formation of a recognizable prostomium and peristomium (“head”) by 6 days post-ablation. Palp and setal growth, addition of segments, and formation of nuchal organs and the ciliated food groove followed so that worms appeared to have re-grown “normal,” but smaller, “heads” and palps by 9–12 days following ablation. Over the course of 16 days, worms that lost more segments regenerated less tissue relative to their initial intact size and did so more slowly. There was no significant palp growth during the first 3 days following ablation. Rate of segment addition was directly related to the degree of tissue loss in D. quadrilobata. P. elegans added segments at similar rates whether 50 or 70% of the original segments was removed.     

Altered c-Fos expression demonstrates neuronal stress in mummichog, <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>, exposed to <Emphasis Type="Italic">Pfiesteria shumwayae</Emphasis> and <Emphasis Type="Italic">Chaetoceros concavicornis</Emphasis>

To better understand sublethal effects of harmful algal blooms (HABs) on fish, mummichog, Fundulus heteroclitus (L.), were exposed in the laboratory to varying, environmentally relevant densities of Pfiesteria shumwayae (Glasgow et Burkholder, CCMP 2089, dinoflagellate) and Chaetoceros concavicornis (Mangin, CCMP 169, diatom). Two experiments were conducted during the spring of 2003 and 2004 to quantitatively examine the effects of acute (2 h) P. shumwayae and C. concavicornis algal exposure on mummichog brain activity using c-Fos expression as a marker of altered neuronal activity. Brains from HAB-exposed fish were removed, sectioned, and stained using immunocytochemistry prior to quantifying neuronal c-Fos expression. Fish exposed to P. shumwayae and C. concavicornis showed increased c-Fos expression compared to unexposed control fish. A significant dose-response relationship was observed, with increased labeling in brains of fish exposed to higher cell densities for both HAB species tested (P ≤ 0.01). Increased labeling was found in the telencephalon, optic lobes, midbrain, and portions of the medulla. The greatest increases in expression were observed in the telencephalon of P. shumwayae-exposed fish, and in the telencephalon and optic lobes of C. concavicornis-exposed fish (P ≤ 0.01). These increases in c-Fos expression are consistent with other physical and chemical stress exposures observed in fish. Neuronal stress, evidenced by c-Fos expression, demonstrates a sublethal effect of exposure and changes in brain activity in fish exposed to HAB species.     

Accumulation of paralytic shellfish poisoning toxins in planktonic copepods during a bloom of the toxic dinoflagellate<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> in Hiroshima Bay,western Japan

Concentrations of paralytic shellfish poisoning (PSP) toxins in toxic dinoflagellate cells and in marine planktonic copepods were monitored during the bloom of Alexandrium tamarense in Hiroshima Bay, western Japan. Concentration of the toxins retained by copepods was a function of the ambient toxin concentration, i.e. the product of A. tamarense cell density and cellular toxicity. The toxin concentration in copepods increased with the increase of toxicants in the seawater then leveled off, but decreased significantly at higher concentrations. In the field, the maximum toxin concentration was 1.2 pmol ind^-1, whereas in the laboratory, the copepod Acartia omorii accumulated a much higher concentration of PSP toxins (24 pmol ind^-1). Feeding avoidance against Alexandrium tamarense and a shift to alternative food sources such as diatoms in the field might keep their toxin levels lower than their potentially maximum level. The copepod toxin levels in the field were not so high as to cause an instantaneous lethal effect on their predator fishes but may reach possibly lethal levels after a few days' continuous feeding. Overall toxin retention by copepods after 12 h feeding and 2 h starvation was only 2.5% of total ingested toxins, which suggested that a significant amount of toxins was released into the seawater. Measurements of toxin reduction and gut evacuation suggested that the toxins were removed through both fecal evacuation and metabolism (e.g. excretion, decomposition and transformation). The results, as a whole, imply that copepods can be a link for PSP toxin flux in both pelagic and benthic food webs and can also be a sink for toxins by metabolizing and removing them from the environment.Communicated by T. Ikeda, Hakodate     

Monitoring acoustically tagged king prawns <Emphasis Type="Italic">Penaeus</Emphasis> (<Emphasis Type="Italic">Melicertus</Emphasis>) <Emphasis Type="Italic">plebejus</Emphasis> in an estuarine lagoon

Fine-scale movement patterns in penaeid prawns are rarely observed in situ, but are essential in understanding habitat use, foraging, and anti-predator behaviour. Acoustic telemetry was applied to examine the activity, space utilization, and habitat use of the eastern king prawn Penaeus (Melicertus) plebejus, at small temporal and spatial scales. Tracking of sub-adult P. plebejus (n = 9) in Wallagoot Lake (36.789°S, 149.959°E; 23 April–12 May 2009) and calculation of a minimum activity index (MAI) revealed high variation in activity rates across diel periods and in different habitats. Elevated activity rates and movement indicated foraging in unvegetated habitats during the night. Areas within the 95 and 50% space utilization contours averaged 2,654.1 ± 502.0 and 379.9 ± 103.9 m², respectively, and there was a significant negative relationship between these areas and prawn activity rates in unvegetated habitats. This study provides the first estimates of prawn activity rates and space utilization in the field. Application of acoustic telemetry can increase knowledge of prawn movements and their interactions with other marine species in different habitats.     

Effects of substrate type on growth and mortality of blue mussels (<Emphasis Type="Italic">Mytilus</Emphasis><Emphasis Type="Italic">edulis</Emphasis>) exposed to the predator <Emphasis Type="Italic">Carcinus</Emphasis><Emphasis Type="Italic">maenas</Emphasis>

Structure and complexity of the substrate are important habitat characteristics for benthic epifauna. The specific growth and mortality rates and inducible defence characters on medium-sized blue mussels (Mytilus edulis L.) exposed to shore crabs (Carcinus maenas L.) were examined on three different substrate types in combined field and laboratory experiments. The experiments showed that complexity of the substrate increased blue mussel survival significantly, through a decrease in predation pressure. However, increased intraspecific competition for food on the complex substrate resulted in significantly lower growth rates of the mussels. Inducible defence characters were also influenced by substrate type. Blue mussels were more affected by predators on the structurally simple substrate, where they developed thicker shells and a larger posterior adductor muscle.     

Temporary cyst enables long-term dark survival of <Emphasis Type="Italic">Scrippsiella hangoei</Emphasis> (Dinophyceae)

We examined the dark survival strategy of the cold-water dinoflagellate Scrippsiella hangoei from the Baltic Sea. Cultures of S. hangoei were placed in dark and light and we followed the morphological developmentand, determined respiration rate and activity of extracellular leucine aminopeptidase (LAP). S. hangoei had LAP activity in the light, but not in the dark, suggesting that the degradation and use of organic substrates is not part of the dark survival strategy. After prolonged time in darkness (>5 days), S. hangoei started to shed flagella and theca, and produced a previously undescribed temporary cyst. The transformation from vegetative cell into the temporary cyst initially increased respiration rate tenfold, but after the transformation the respiration rate decreased to almost undetectable levels. The presented temporary cyst enables survival through long periods in dark by reducing the respiration rate.     

Very slow development in two Antarctic bivalve molluscs,the infaunal clam <Emphasis Type="Italic">Laternula elliptica</Emphasis> and the scallop <Emphasis Type="Italic">Adamussium colbecki</Emphasis>

Embryos of the large infaunal clam Laternula elliptica and the scallop Adamussium colbecki, from Antarctica, were cultured over an 18-month period. Their development rates were extremely slow, taking 240 and 177 h, respectively, to reach the trochophore stage. This is ×4 to ×18 slower than related clams and scallops from temperate latitudes. The relationship between temperature and development rate for bivalve molluscs shows the expected slowing with reduced temperature (Q ₁₀ in the range 2–4) for temperate and tropical species. However, the slowing at polar latitudes is much stronger than at warmer waters, and all of the limited data for Antarctic species are well above the Arrhenius plot for the overall bivalve data, and the Q ₁₀ value for Antarctic to cool temperate species is 11.8, well outside the expected range for biological systems. Either the relationships describing the effects of temperature on the kinetics of biological systems do not apply to Antarctic bivalve molluscs, or some other factor that cannot be compensated for becomes important at low temperature. In the laboratory, L. elliptica embryos stayed viable in very sticky egg capsules for up to 18 months without hatching. However, even the disturbance of removing eggs using a pipette ruptured some egg capsules allowing embryo release. Gametogenesis in Antarctic marine invertebrates is almost universally slowed compared to temperate species, with nearly all cases documented requiring more than 1 year to complete oogenesis. The only exception so far appears to be A. colbecki, which has a 1-year gametogenic cycle. The data here indicate that it has been unable to adapt embryonic development in a similar way, and we are not aware of any exceptions to the markedly slowed development at low temperature rule.     

Intraguild predatory interactions between the jellyfish <Emphasis Type="Italic">Cyanea capillata</Emphasis> and <Emphasis Type="Italic">Aurelia aurita</Emphasis>

While qualitative observations of jellyfish intraguild predation abound in the literature, there are only few rate measurements of these interactions. We quantified predation rates among two common jellyfish in northern boreal waters, Cyanea capillata and its prey Aurelia aurita, both of which also feed on crustacean zooplankton and fish larvae. A series of incubation experiments using a wide range of prey concentrations (0.38–3.8 m⁻³) in large containers (2.6 m³) was carried out. By replenishing the prey continuously as they were captured we maintained a nearly constant prey concentrations. Ingestion rates increased linearly up to prey concentrations of 1.92 m⁻³, yielding maximum clearance rates of ∼2.37 ± 0.39 m³ predator⁻¹ h⁻¹ for C. capillata predators 16 ± 2.3 cm in diameter. Mean ingestion rate at saturated prey concentrations (1.92–3.85 m⁻³) was 4.01 ± 0.78 prey predator⁻¹ h⁻¹. Behavioral observations suggested that predators did not alter their swimming behavior during meals, and thus that feeding rates were generally handling limited rather than encounter limited. Predators captured more prey than needed, and semi-digested prey was often discarded when fresh prey was encountered.     

Foraging responses in the butterflies <Emphasis Type="Italic">Inachis io</Emphasis>, <Emphasis Type="Italic">Aglais urticae</Emphasis> (Nymphalidae), and <Emphasis Type="Italic">Gonepteryx rhamni</Emphasis> (Pieridae) to floral scents

Summary. For butterflies to be efficient foragers, they need to be able to recognize rewarding flowers. Flower signals such as colours and scents assist this recognition process. For plant species to attract and keep butterflies as pollinators, species-specific floral signals are crucial. The aim of this study is to investigate foraging responses to floral scents in three temperate butterfly species, Inachis io L. (Nymphalidae), Aglais urticae L. (Nymphalidae), and Gonepteryx rhamni L. (Pieridae), in behavioural choice bioassays. The butterflies were allowed to choose bet-ween flower models varying in scent and colour (mauve or green). Flowers or vegetative parts from the plants Centaurea scabiosa L. (Asteraceae), Cirsium arvense (L.) (Asteraceae), Knautia arvensis (L.) (Dipsacaceae), Buddleja davidii Franchet (Loganicaeae), Origanum vulgareL. (Lamiaceae), Achillea millefolium L. (Asteraceae), and Philadelphus coronarius L. (Hydrangiaceae) were used as scent sources. All visits to the models — those that included probing and those that did not — were counted, as was the duration of these behaviours. Both flower-naive and flower-experienced (conditioned to sugar-water rewards, the colour mauve, and specific floral scents) butterflies were tested for their preference for floral versus vegetative scents, and to floral scent versus colour. The butterflies were also tested for their ability to switch floral scent preferences in response to rewards. Flower-naive butterflies demonstrated a preference for the floral scent of the butterfly-favourable plants C. arvense and K. arvensis over the floral scent of the non-favourable plants Achillea millefolium (Asteraceae), and Philadelphus coronarius cv. (Hydrangiaceae). Most of the butterflies that were conditioned to floral scents of either C. arvense, K. arvensis, or B. davidii readily switched theirfloral scent preferences to the one most recently associated with reward, thus demonstrating that floral scent constancy is a result from learning. These findings suggest that these butterflies use floral scent as an important cue signal to initially identify and subsequently recognize and distinguish among rewarding plants. Received 2 September 2001; accepted 9 September 2002.     

Inter-polyp genetic and physiological characterisation of <Emphasis Type="Italic">Symbiodinium</Emphasis> in an <Emphasis Type="Italic">Acropora valida</Emphasis> colony

Corals harbouring genetically mixed communities of endosymbiotic algae (Symbiodinium) often show distribution patterns in accordance with differences in light climate across an individual colony. However, the physiology of these genetically characterised communities is not well understood. Single stranded conformation polymorphism (SSCP) and real time quantitative polymerase chain reaction (qPCR) analyses were used to examine the genetic diversity of the Symbiodinium community in hospite across an individual colony of Acropora valida at the spatial scale of single polyps. The physiological characteristics of the polyps were examined prior to sampling with a combined O₂ microelectrode with a fibre-optic microprobe (combined sensor diameter 50–100 μm) enabling simultaneous measurements of O₂ concentration, gross photosynthesis rate and photosystem II (PSII) quantum yield at the coral surface as a function of increasing irradiances. Both sun- and shade-adapted polyps were found to harbour either Symbiodinium clade C types alone or clades A and C simultaneously. Polyps were grouped in two categories according to (1) their orientation towardps light, or (2) their symbiont community composition. Physiological differences were not detected between sun- and shade-adapted polyps, but O₂ concentration at 1,100 μmol photons m⁻² s⁻¹ was higher in polyps that harboured both clades A and C symbionts than in polyps that harboured clade C only. These results suggest that the acclimatisation of zooxanthellae of individual polyps of an A. valida colony to ambient light levels may not be the only determinant of the photosynthetic capacity of zooxanthellae. Here, we found that photosynthetic capacity is also likely to have a strong genetic basis and differs between genetically distinct Symbiodinium types.     

Seasonal variations in the growth rates of euphausiids (<Emphasis Type="Italic">Thysanoessa inermis</Emphasis>, <Emphasis Type="Italic">T. spinifera</Emphasis>, and <Emphasis Type="Italic">Euphausia pacifica</Emphasis>) from the northern Gulf of Alaska

The euphausiids Thysanoessa inermis (Kroyer 1846), Thysanoessa spinifera (Holmes 1900), and Euphausia pacifica (Hansen 1911) are key pelagic grazers and also important prey for many commercial fish species in the Gulf of Alaska (GOA). To understand the role of the euphausiids in material flows in this ecosystem their growth rates were examined using the instantaneous growth rate (IGR) technique on the northern GOA shelf from March through October in 2001–2004. The highest mean molting increments (over 5% of uropod length increase per molt) were observed during the phytoplankton bloom on the inner shelf in late spring for coastal T. inermis, and on the outer shelf in summer for T. spinifera and more oceanic E. pacifica, suggesting tight coupling with food availability. The molting rates were higher in summer and lower in spring, for all species and were strongly influenced by temperature. Mean inter-molt periods calculated from the molting rates, ranged from 11 days at 5°C to 6 days at 8°C, and were in agreement with those measured directly during long-term laboratory incubations. Growth rate estimates depended on euphausiid size, and were close to 0 in early spring, reaching maximum values in May (0.123 mm day⁻¹ or 0.023 day⁻¹ for T. inermis) and July (0.091 mm day⁻¹ or 0.031 day⁻¹ for T. spinifera). The growth rates for E. pacifica remained below 0.07 mm day⁻¹ (0.016 day⁻¹) throughout the season. The relationship between T. inermis weight specific growth rate (adjusted to 5°C) and ambient chlorophyll-a concentration fit a Michaelis–Menten curve (r ² = 0.48) with food saturated growth rate of 0.032 day⁻¹ with half saturation occurring at 1.65 mg chl-a m⁻³, but such relationships were not significant for T. spinifera or E. pacifica.     

Mate preferences in <Emphasis Type="Italic">Drosophila</Emphasis> infected with <Emphasis Type="Italic">Wolbachia</Emphasis>?

The maternally inherited bacterium Wolbachia pipientis generates strong reproductive incompatibilities between uninfected females and infected males (cytoplasmic incompatibility), significantly reducing both female and male reproductive success. Such fitness costs are thought to place selective pressure on hosts to evolve pre-copulatory preferences for mating with compatible mates, thereby enabling them to avoid the reproductive incompatibilities associated with Wolbachia. Therefore, uninfected females are predicted to prefer mating with uninfected males, whereas infected males are predicted to prefer mating with infected females. Despite these predictions, previous investigations of pre-copulatory mate preferences in Wolbachia-manipulated Drosophila have not found evidence of female preference for uninfected or compatible males. However, none of these studies utilised a design where focal individuals are provided with a simple choice in a relatively non-competitive situation. We examined both female and male pre-copulatory mate preference based on mate infection status in Drosophila simulans and D. melanogaster using simple choice assays involving between 30–50 replicates per treatment. Although we found no evidence of female pre-copulatory mate preferences in either species, male D. simulans exhibited some preference for mating with females of the same infection status. However, this preference was not evident when we repeated the experiment to confirm this finding. Consequently, we conclude that neither male nor female D. melanogaster and D. simulans exhibit significant Wolbachia-associated pre-copulatory mate preferences.     

Particle sorting and formation and elimination of pseudofaeces in the bivalves <Emphasis Type="Italic">Mulinia edulis</Emphasis> (siphonate) and <Emphasis Type="Italic">Mytilus chilensis</Emphasis> (asiphonate)

Mulinia edulis and Mytilus chilensis are suspension-feeding bivalves with homorhabdic gills that live in different sedimentary habitats in the lower and upper intertidal, respectively, in Yaldad Bay, Chile. They are faced with different suspended particle size distributions when feeding, and both eliminate most of the inorganic particles by pseudofaeces production. This study used histology, scanning and transmission electron microscopy, and video endoscopy to compare particle processing on the labial palps and the mechanisms of particle sorting, acceptance, and rejection in the two species. In both species, disaggregation of mucus-bound particles occurs on the plicate surface of the palps. Particles destined for ingestion pass anteriorly from crest to crest and reach the mouth via the palp acceptance tract. Those destined for rejection enter the troughs between the plicae and move ventrally to the palp rejection tract. The palps manipulate the pseudofaeces into a mucous ball, which is transferred to the mantle rejection tract. In Mulinia edulis (an infaunal, siphonate mactrid), the pseudofaeces are stored in a chamber at the base of the inhalant siphon until expelled by intermittent contraction of the siphon wall. In contrast, Mytilus chilensis (an epifaunal, non-siphonate mytilid) releases pseudofaeces continuously when submerged.     

Cryopreservation of the dinoflagellate symbiont of the octocoral <Emphasis Type="Italic">Pseudopterogorgia elisabethae</Emphasis>

In this paper we describe a cryopreservation protocol followed by the culture of Symbiodinium sp. isolated from the Caribbean gorgonian Pseudopterogorgia elisabethae as a potential renewable source of the dinoflagellate symbiont. Four different freezing protocols were designed: a controlled cooling device designed to cool at 1°C/min, a three-step protocol (−20°C for 2 h, −70°C for 2 h, liquid nitrogen-LN₂), a two-step protocol (−70°C for 2 h, LN₂), and a one-step protocol (LN₂). All cells were stored in LN₂ after cryopreservation. The cryoprotective agents (CPA) used were ethanol (EtOH) and methanol (MeOH) at 10 and 20%, and seawater (FSW) was used as a control. Viability measurements using cell counts showed that all cryopreservation protocols were relatively successful, and no trends were observed regarding freezing protocol or CPA used. After 19 weeks in culture the viability of samples which had high biomass was determined by the fluorescent assay CellTiter Blue™. The most viable cultures were those cryopreserved by a two-step protocol using 20% MeOH or 20% EtOH as a CPA. A genetic examination of the DNA of these samples using Symbiodinium-specific PCR primers confirmed that the composition of the culture had not changed. For the first time, we report that Symbiodinium sp. isolated from a gorgonian can be cryopreserved and subsequently cultured successfully. Lory Z. Santiago-Vázquez and Nealie C. Newberger contributed equally to this publication.     

Compressibility and shell failure in the European Atlantic <Emphasis Type="Italic">Patella</Emphasis> limpets

The specimens of Patella intermedia, Patella rustica, Patella ulyssiponensis and Patella vulgata were analysed for morphological and morphometric characters, and for the resistance to compression and crushing to a force applied at the apex. Shell shape in these species ranged from the high symmetrical cone, with a rounded base of P. rustica, to the flat, asymmetrical, narrow-pear-shaped base of P. ulyssiponensis. P. intermedia and P. vulgata showed intermediate morphologies. Shell thickness increased linearly with age, but differed in the four limpets. P. rustica had the thickest shells, and P. ulyssiponensis and P. vulgata had the thinnest shells. P. intermedia displayed intermediate shell thickness. Considering deformability and toughness, P. intermedia shells usually needed the highest force to compress in height, the highest pressure to collapse, and were appreciably deformed at collapse. On the opposite side, P. ulyssiponensis shells usually needed the lowest force to compress in height, the lowest pressure to collapse, and were much less deformed at collapse. P. intermedia shells were therefore the most deformable and tough, and P. ulyssiponensis, the most stiff and fragile. P. rustica and P. vulgata shells displayed intermediate behaviour. However, numerical simulations based on the finite element method using the experimentally determined shells’ geometry and thickness, but considering similar shells’ material and structure in the four species, predicted that shell toughness should be decreased in the order P. rustica >> P. intermedia > P. vulgata >> P. ulyssiponensis. P. rustica shells’ geometry (a high and centred cone) and thickness (very thick) were therefore, theoretically, the most fitted for shells to resist crushing by compression. Yet, in the experimental tests, they were not the most resistant shells. It was concluded that resistance to crushing was not a direct function of shell morphology and morphometry, but appeared to be mainly determined by shell deformability. This is most probably related to differences in the internal composition and architecture of the shell in the four species. By comparison with data reported in the literature on the pressures normally exerted by ocean waves, it was concluded that these limpets have resistances to crushing in far excess to pressures normally endured in nature, being therefore unlikely that these species are crushed by the action of even very strong waves on shores. Hard objects, like logs and boulders, can be hurled onto the shore by waves, and constitute a much greater threat to limpets than the waves themselves. The high toughness of the limpet shell can be related to resistance to the impact of incidental hard objects. There was no direct relationship between the habit preferences of these limpets and the resistance to crushing. Other factors are involved in the distribution of these species in the shores.     

Lack of sequestration of host plant glucosinolates in <Emphasis Type="Italic">Pieris rapae</Emphasis> and <Emphasis Type="Italic">P. grarricae</Emphasis>

Summary. Sequestration of plant toxins in herbivores is often correlated with aposematic coloration and gregarious behaviour. Larvae of Pieris brassicae show these conspicuous morphological and behavioural characteristics and were thus suggested to sequester glucosinolates that are characteristic secondary metabolites of their host plants. P. rapaeare camouflaged and solitary, and are thus not expected to sequester. To test this hypothesis and to check the repeatabi-lity of a study that did report the presence of the glucosinolate sinigrin in P. brassicae, larvae were reared on three species of Brassicaceae (Sinapis alba, Brassica nigra and Barbarea stricta), and different leaf and insect samples were taken for glucosinolate analysis. The major host plant glucosinolates could only be found in traces or not at all in larval haemolymph, bled or starved larvae, faeces or pupae of both species or P. brassicae regurgitant. Haemolymph of both Pieris spp. was not rejected by the ant Myrmica rubra in dual-choice assays; the regurgitant of P. brassicae was rejected. This suggests the presence of compounds other than glucosinolates that might be sequestered in or produced by P. brassicae only. In faeces of both Pieris spp. a compound which yielded 4-hydroxybenzylcyanide (HBC) upon incubation with sulfatase was detected in high concentrations when larvae had been reared on S. alba. This compound may be derived from hydrolysis of sinalbin, the main glucosinolate of that plant. The unidentified HBC progenitor was apparently not sequestered in the two Pieris spp., and was not detected in faeces of larvae reared on B. nigra or B. stricta. Received 18 July 2002; accepted 11 September 2002.     

Relationship between filtration activity and food availability in the Mediterranean mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>

The filtration activity of the Mediterranean mussel, Mytilus galloprovincialis, was assessed under different concentrations and compositions of seston by using a new automated image acquisition and analysis system. This approach allowed for frequent and simultaneous measurements of valve gape and exhalant siphon area. Filtration rates were measured through clearance measurements whereas pumping rates were measured using hot-film probes. The average filtration rate (17.5 l g h⁻¹ DW⁻¹ for a 0.36 g DW mussel) recorded during the present study was higher than those available for Mytilus edulis when standardized to flesh dry weight but almost equivalent (17.5 l h⁻¹ g DW⁻¹ for a 53 mm shell length mussel) to those rates when standardized to shell length. Immediately after the addition of algal cells (Isochrysis galbana; 4.5 μm in size), valve gape, exhalant siphon area and filtration rate increased quickly as mussels reached their maximum filtration activity. These three parameters then gradually decreased until complete closure of the shell. The algal cell concentration inducing this transition was close to 800 cells ml⁻¹ and 0.5 μg Chl a l⁻¹. When algal concentration was maintained above this threshold by successive algal additions, both valve gape and exhalant siphon area remained maximal. Temporal changes in the exhalant siphon area were continuous as opposed to those of valve gape. Therefore, despite the significant correlation between these two parameters, valves and siphon were sometimes dissociated due to a reduction of the area or even a closure of the exhalant siphon while the valves remained open. The velocity of exhaled water tended to be constant irrespective of exhalant siphon area and thus pumping rates were a linear function of exhalant siphon area. Consequently, reductions in exhalant siphon area and pumping rate were almost similar in M. galloprovincialis. Our results thus clearly support the hypothesis that exhalant siphon area constitutes a better proxy of pumping rate than valve gape as already suggested for Mytilus edulis. Finally, the high filtration rates measured during the present study together with the high concentrations of inorganic matter (> 40 mg DW l⁻¹) requested to alter those rates suggest that the studied mussels were well adapted to oligotrophic waters featuring strong hydrodynamism and frequent sediment resuspension events.     

Seasonal variation in the degree of heterospecific association of two syntopic hyraxes (<Emphasis Type="Italic">Heterohyrax</Emphasis><Emphasis Type="Italic">brucei </Emphasis>and <Emphasis Type="Italic">Procavia capensis</Emphasis>) exhibiting synchronous parturition

Adult rock hyrax (Procavia capensis) and bush hyrax (Heterohyrax brucei) allocate much of their surface activity to being vigilant for vertebrate predators. Individuals of the two species associate for thermoregulatory basking on rock outcrops (koppies) and, in the Matobo National Park, Zimbabwe, frequently constitute heterospecific groups that produce offspring synchronously. Exposure to predators during basking is thought to put a high premium on vigilance, particularly during the presence of dependent offspring in nurseries. We predicted, therefore, an increase in the association of the two species of hyraxes when offspring were present because this would lead to larger groups, thus enhancing the ability to detect predators. Koppie aggregations of hyraxes were classified as homospecific or heterospecific according to basking group composition. Heterospecific association increased when offspring were present, and heterospecific groups were larger than homospecific ones. Fifty-one percent of nurseries were heterospecific for adults/subadults. During the pre-weaning phase (<2 months after birth), 85% of P. capensis and 65% of H. brucei juveniles were in nurseries of heterospecific basking groups. Heterospecific groups had more offspring than homospecific groups. Juvenile mortality is most evident after young are weaned and heterospecific associations break down. Heterospecific aggregation, through increased group size, may result in a higher probability of avoiding predators, thereby reducing the vulnerability of young.