Larval settlement and metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> in response to biofilms

Biofilms were allowed to develop on glass slips immersed 1.0–1.5 m below the sea surface in Tachibana Bay, Nagasaki, Japan, for different periods of time from November 2003 to January 2005. The effects of age, immersion month, dry weight, bacterial and diatom densities of these biofilms on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Furthermore, biofilms were subjected to various treatments to investigate the nature of the settlement and metamorphosis cue in the biofilm. Pediveliger larvae of the mussel settled and metamorphosed in response to biofilms. Settlement and metamorphosis to the post-larval stage significantly increased with the biofilm age. In addition, the biofilm activity varied depending on the immersion month (season), e.g., for biofilms with the same age, those immersed between June and August had higher activities than those immersed between November and March. The activity of the biofilm also positively correlated with the dry weight, bacterial and diatom densities. These three quantitative parameters of the biofilm were significantly affected by the film age but were not affected by the immersion month, suggesting that other parameters (e.g., community structures, extracellular products) also affected the inductive activity of the biofilm. The fixative agents (formalin and glutaraldehyde), heat, ethanol, ultraviolet irradiation and antibiotics treatments of the biofilm resulted in significant reduction or loss of its inductive activity. The survival of bacterial cells in the treated films where activities were either reduced or lost also decreased significantly. No settlement and metamorphosis were obtained when larvae were exposed to the conditioned water of the biofilm. Thus, larvae of M. galloprovincialis settled and metamorphosed in response to a cue produced by living bacteria in the biofilm. The cue may be a bacterial extracellular product which was susceptible to the above treatments.     

Larval metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> in response to <Emphasis Type="Italic">Alteromonas</Emphasis> sp. 1: evidence for two chemical cues?

Bacterial isolates from multi-species biofilms were identified by 16S rDNA gene sequences and investigated for their inductive effects as monospecific biofilms on larval metamorphosis of Mytilus galloprovincialis. Alteromonas sp. 1 biofilm was found to have inductive activity, which increased with increasing cell density. The cue(s) of Alteromonas sp. 1 biofilm responsible for inducing larval metamorphosis was further investigated. Treatment of the biofilm with formalin, ethanol, heat or ultraviolet irradiation resulted in a significant reduction in the inductive activity of Alteromonas sp. 1, and the crude extract of surface-bound products of the biofilm showed no activity. These results indicated that if the cue was a surface-bound chemical cue, it was unstable, or susceptible to the treatments or the extraction process. On the other hand, the inductive activity of treated biofilms had a linear regression to the cell survival of bacteria, indicating a metabolically active biofilm was a requirement for larval metamorphosis. Conditioned water of the biofilm did not induce larvae to metamorphose. However, larval crawling behavior in the conditioned water was the same as that in the biofilm prior to larval metamorphosis, and significantly different to larval behavior in seawater. This indicated that a potential or partial waterborne cue existed, but remained inactive when alone. A synergistic effect of the conditioned water with formalin-fixed Alteromonas sp. 1 biofilm resulted in a significant increase in larval metamorphosis. Heat treatment and fractionation of the conditioned water demonstrated that the waterborne cue was heat-stable and <3,000 Da in molecular weight. Platinum-coating, Lentil Lectin and Wheat Germ Agglutinin treatments of the formalin-fixed biofilm significantly reduced its synergistic effect with the conditioned water, suggesting that a surface-bound cue was present on the biofilm and that the cue might be associated with the bacterial exopolysaccharide or glycoprotein. Evidence presented here suggests that two chemical cues derived from bacteria act synergistically on larval metamorphosis of Mytilus galloprovincialis.     

<Emphasis Type="Italic">Semibalanus</Emphasis><Emphasis Type="Italic">balanoides</Emphasis> in winter and spring: larval concentration,settlement, and substrate occupancy

This study measured the progression from pelagic larvae to juvenile barnacles, and examined whether recruitment of barnacles, Semibalanus balanoides Linnaeus, at two intertidal sites in contrasting hydrodynamic regimes was determined by pre-settlement or post-settlement processes. The two sites were 1.5 km apart in the vicinity of Woods Hole, Mass., USA. Quantitative plankton samples were taken twice weekly from December 1997 to May 1998 at a nearby site as an estimate of nearshore larval abundance. The presence of S. balanoides nauplii was noted, and cyprids were enumerated and measured. Larval settlement at the two sites [Gansett Point, Buzzards Bay (GP) and Little Harbor, Vineyard Sound (LH)] was estimated from examination of replicate settlement plates exposed for 2 or 3 days throughout the settlement season, and from replicate plots on marked rock quadrats at each site. On both plates and rocks settled cyprids and metamorphs were enumerated. Space occupancy on unmanipulated rock quadrats by all stages from cyprids to adult barnacles was also examined. Settlement occurred from 2 January to 20 May, and major settlement peaks coincided with peaks in pelagic cyprid concentration at LH, but not at GP. Space occupied by juvenile barnacles was close to zero up until late February despite substantial settlement prior to that. At LH, juvenile barnacle cover was zero at the end of the observations; all settlement failed. Almost 100% of settled cyprids failed to metamorphose within 2 days from late January to late March. Then the proportion metamorphosing increased sharply coinciding with a sudden increase of 3°C in water temperature. Observed site differences in space occupancy by juvenile barnacles suggest that while cyprid supply is a necessary condition for barnacle settlement, other factors affecting metamorphosis of settled cyprids and early juvenile mortality determine recruitment.     

Larval settlement and metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> on different macroalgae

Settlement of mussels is commonly associated with macroalgae. The effects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were filamentous in morphology. Macroalgae that were cylindrical, phylloid, flabellate, palmate and pinnate all showed low (<8%) percentages of post-larvae but four other filamentous macroalgae also had low mussel larval settlement, suggesting that chemical factors may also be involved. Seasonal variation had no effect on inductive activities of C. fastigiata and C. tenerrimum. Treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the significant decrease or loss of their inductive activities. Survival of algal cells within treated macroalgae also decreased significantly. Treatment of the two macroalgae with antibiotics and GeO₂ reduced the numbers of bacteria and diatoms on their surface but did not affect their inductive activities, indicating that the cue was produced by macroalgae and not by coexisting bacteria and diatoms. However, conditioned water and crude extracts of these two macroalgae did not induce larval settlement and metamorphosis. Thus, larvae of M. galloprovincialis settled and metamorphosed on specific filamentous macroalgae. The chemical cues produced by C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the conditioned water nor in the extracts. The finding that inactive C. tenerrimum can be produced from culturing its apical segments provides a new tool to elucidate the chemical cue(s) from macroalgae through manipulation of their culture conditions.     

Larval metamorphosis of the sea urchins,<Emphasis Type="Italic"> Pseudocentrotus depressus</Emphasis> and<Emphasis Type="Italic"> Anthocidaris crassispina</Emphasis> in response to microbial films

In Japan, mass-production of sea urchin juveniles involves the culture of periphytic diatom films on plastic plates in 5- to 15-tonne tanks for the induction of larval metamorphosis. This study focused on the larval response of sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina, to natural microbial films in the sea and diatom-based films formed in the tanks. The effect of diatoms and bacteria on larval metamorphosis was also examined using laboratory-cultured diatom-based films in the presence of germanium dioxide and antibiotics during film culture. Furthermore, the nature of the cue of the cultured diatom-based film was also investigated. Results showed that P. depressus and A. crassispina metamorphosed both on natural microbial films and diatom-based films in a tank. In the sea, the metamorphosis (%) of P. depressus increased gradually in accordance with the immersion period of film formed on glass slides, whereas the larval metamorphosis of A. crassispina had a bell-shaped response curve. In the tank, although the diatom-based films showed a low inducing activity for larval metamorphosis of A. crassispina, the metamorphosis of P. depressus larvae increased linearly in accordance with the diatom density. These results suggest that diatom-based films could promote the larval metamorphosis of P. depressus, but are less important in A. crassispina. In a simultaneous larval assay (May), P. depressus showed a higher percentage of metamorphosis than A. crassispina. We concluded that the former is more sensitive to diatom-based film than the latter and that this is due to differences in their natural habitats. For laboratory-cultured diatom-based film, both species of sea urchins showed a similar response, in which reduction in diatom and bacteria density resulted in a decrease in the original inducing activity. There seems to be a synergistic effect between diatom and bacteria in inducing larval metamorphosis. Films subjected to treatment with 0.1 N HCl were no longer inductive for either sea urchin, while those films treated with 40^°C heat or EtOH (5% and 10% EtOH) showed a significant reduction in the inducing activity. Thus the surface-associated cues may be highly susceptible to the above treatments.Communicated by T. Ikeda, Hakodate     

On the consortium of the tintinnid <Emphasis Type="Italic">Eutintinnus</Emphasis> and the diatom <Emphasis Type="Italic">Chaetoceros</Emphasis> in the Pacific Ocean

The morphology and distribution of the diatoms Chaetoceros tetrastichon and Ch. dadayi as epiphytes on the loricae of the tintinnids Eutintinnus apertus and E. pinguis investigated in the open waters of the Pacific Ocean. The Eutintinnus–Chaetoceros consortia was encountered in 38 of the 52 sampling stations from 34°N to 33°S, and together were among represented the most wide-spread species. The abundance was low with a maximum of 32 consortia l⁻¹ and E. apertus was often the most abundant species of the genus. The free-living Eutintinnus congeneric species showed a wider vertical distribution, whereas E. apertus–Chaetoceros tended to be near the surface. The success of E. apertus in consortium with Chaetoceros may be due to increase of the clearance rate and/or the lower susceptibility to predation. Chaetoceros modifies its morphology to adapt the epiphytic life, especially Ch. dadayi. The shorter curved setae may facilitate the transfer to the lorica of the daughter tintinnid after the cell division. The free-living Ch. tetrastichon and Ch. dadayi are very rare and Chaetoceros remained attached to empty loricae or encysted tintinnid cells. This suggests that the Eutintinnus–Chaetoceros consortium is obligate for the success of the diatom and renders the tintinnid more competitive versus congeneric species.     

Evidence of a spawning area of <Emphasis Type="Italic">Anguilla</Emphasis><Emphasis Type="Italic">marmorata</Emphasis> in the western North Pacific

Leptocephali of the tropical eel Anguilla marmorata have been consistently collected in the same area of the North Equatorial Current (NEC) in the western North Pacific during three consecutive cruises in June and July of 1991 (N=28) and 1994 (N=20), and July and September of 1995 (N=27), indicating that this is a spawning area of this species. These leptocephali were collected from 130°E to 142°E and 12°N to 20°N, to the west of the Mariana Islands, in 20 tows in 1991, in 13 tows in 1994 and in 17 tows in 1995, indicating a widespread presence, but a relatively low abundance. Six of these specimens (16.3-36.0 mm total length) from the 1995 cruise, which were of the typical size range of these leptocephali, were genetically confirmed to be A. marmorata in a previous study. The consistent presence of recently spawned A. marmorata leptocephali (9-20 mm) in all 3 years, suggests that the western region of the NEC is the spawning area of the northern population of A. marmorata that was identified in a recent population genetics study. These leptocephali would thus be transported westward by the NEC and then transported north into the Kuroshio Current and toward Taiwan and Japan, or south toward the southern Philippines and into the Celebes Sea by the Mindanao Current. Available evidence indicates that A. marmorata may have potentially year-round spawning, and the presence of a spawning area of this species in the same region as that of Anguilla japonica suggests that the northern population of A. marmorata has evolved a spawning migration from East Asia, the Philippines and the Celebes Sea region to the NEC area, but differs from A. japonica by having some individuals that recruit to more southern areas.     

High reproduction of <Emphasis Type="Italic">Calanus finmarchicus</Emphasis> during a diatom-dominated spring bloom

Feeding, egg production, hatching success and early naupliar development of Calanus finmarchicus were measured in three north Norwegian fjords during a spring bloom dominated by diatoms and the haptophyte Phaeocystis pouchetii. Majority of the copepod diet consisted of diatoms, mainly Thalassiosira spp. and Chaetoceros spp., with clearance rates up to 10 ml ind⁻¹ h⁻¹ for individual algae species/groups. Egg production rates were high, ranging from ca 40 up to 90 eggs f⁻¹ d⁻¹, with a hatching success of 70–85%, and fast naupliar development through the first non-feeding stages. There was no correlation between the egg or nauplii production and diatom abundance, but the hatching success was slightly negatively correlated with diatom biomass. However, the overall high reproductive rates suggested that the main food items were not harmful for C. finmarchicus reproduction in the area, although direct chemical measurements were not conducted. The high population egg production (>1,20,000 eggs m⁻² d⁻¹) indicated that a large part of the annual reproduction took place during the investigation, which stresses the importance of diatom-dominated spring phytoplankton bloom for population recruitment of C. finmarchicus in these northern ecosystems.     

Influence of bacteria and diatoms in biofilms on metamorphosis of the marine slipper limpet <Emphasis Type="Italic">Crepidula onyx</Emphasis>

Larvae of the slipper limpet Crepidula onyx metamorphose in response to marine biofilms. In this study, we investigated how the percentage of larval metamorphosis in this species was affected by biofilms that differed in certain attributes. To manipulate bacterial and diatom cell densities and community composition, we developed biofilms in the laboratory (1) at different temperatures (16, 23 and 30°C) and salinities (20, 27 and 34‰), (2) with or without addition of antibiotics, and (3) in the light or in the dark. We also allowed biofilms to develop at three field sites with different prevailing environmental conditions so as to generate biofilms with different, but natural, attributes. Bacterial and diatom community composition in biofilms were determined using a DNA fingerprinting technique and microscopic examination, respectively. The effects of biofilms on metamorphosis were investigated in laboratory assays. The percentage of larval metamorphosis correlated with bacterial and diatom cell densities in only one of the three experiments conducted, but was substantially affected by differences in bacterial and diatom community composition in all three experiments. It also appears that metamorphosis of C. onyx depends on the simultaneous presence of both bacterial and diatom communities in biofilms.     

Genetic evidence for the cryptic species pair, <Emphasis Type="Italic">Lottia digitalis </Emphasis>and <Emphasis Type="Italic">Lottia austrodigitalis</Emphasis> and microhabitat partitioning in sympatry

It has been proposed that the common West Coast limpet, Lottia digitalis, is actually the northern counterpart of a cryptic species duo including, Lottia austrodigitalis. Allele frequency differences between southern and northern populations at two polymorphic enzyme loci provided the basis for this claim. Due to lack of further evidence, L. austrodigitalis is still largely unrecognized in the literature. Seven additional enzyme loci were examined from populations in proposed zones of allopatry and sympatry to determine the existence of L. austrodigitalis as a sibling species to L. digitalis. Significant allele frequency differences were found at five enzyme loci between populations in Laguna Beach, southern California, and Bodega Bay, northern California; strongly supporting the existence of separate species. Both species exhibit two microhabitat morphotypes, a gooseneck barnacle morph in the mid-intertidal zone and a rock morph in the high-intertidal zone. In sympatry, L. austrodigitalis was more abundant higher in the intertidal on rocks, whereas L. digitalis was more abundant lower in the intertidal on barnacles. This finding supports earlier claims of microhabitat partitioning in this sibling species pair. In addition to this finding, the transition zone between the species was found to have shifted substantially northward in only two decades, from Monterey Peninsula, CA to near Pigeon Point, CA, where L. digitalis previously dominated.     

Effects of elevated temperature on larval settlement and post-settlement survival in scleractinian corals, <Emphasis Type="Italic">Acropora solitaryensis</Emphasis> and <Emphasis Type="Italic">Favites chinensis</Emphasis>

We examined the effects of elevated temperature under different exposure periods on larval settlement and post-settlement survival in scleractinian corals, Acropora solitaryensis and Favites chinensis. In the first experiment with the subtropical coral, A. solitaryensis, the numbers of larvae settling and those dead were examined daily for 5 days at 20, 23 (ambient), 26 and 29°C conditions. Larval settlement of A. solitaryensis was initially greater at higher temperature conditions, but the peak in number of settled larvae shifted from 29 to 26°C by day 5, due to ca. 90% post-settlement mortality at 29°C condition. In order to determine the effects under short-term exposure, larvae of F. chinensis were exposed to 27 (ambient), 31 or 34°C only for one hour in the second experiment. The number of larvae settling for 24 h after the exposure and their survivorship over subsequent week was monitored in the ambient temperature condition. Larvae of F. chinensis exhibited greater settlement at higher temperature treatments and constantly low post-settlement mortalities (< ca. 17%) in all temperature treatments, resulting in the highest number of settled larvae at 34°C treatment. These results suggested two different effects of elevated temperature on the early stages of recruitment process of scleractinian corals; (1) the positive effect on larval settlement and (2) the negative effect on post-settlement survival under prolonged exposure.     

Settlement of <Emphasis Type="Italic">Macoma balthica</Emphasis> larvae in response to benthic diatom films

The role of multi-species benthic diatom films (BDF) in the settlement of late pediveliger larvae of the bivalve Macoma balthica was investigated in still-water bioassays and multiple choice flume experiments. Axenic diatom cultures that were isolated from a tidal mudflat inhabited by M. balthica were selected to develop BDF sediment treatments characterized by a different community structure, biomass, and amount of extracellular polymeric substances (EPS). Control sediments had no added diatoms. Although all larvae settled and initiated burrowing within the first minute after their addition in still water, regardless of treatment, only 48–52% had completely penetrated the high diatom biomass treatments after 5 min, while on average 80 and 69% of the larvae had settled and burrowed into the control sediments and BDF with a low diatom biomass (<3.5 μg Chl a g⁻¹ dry sediment), respectively. The percentage of larvae settling and burrowing into the sediment was negatively correlated with the concentration of Chl a and EPS of the BDF. This suggests higher physical resistance to bivalve penetration by the BDF with higher diatom biomass and more associated sugar and protein compounds. The larval settlement rate in annular flume experiments at flow velocities of 5 and 15 cm s⁻¹ was distinctly lower compared to the still-water assays. Only 4.6–5.8% of the larvae were recovered from BDF and control sediments after 3 h. Nonetheless, a clear settlement preference was observed for BDF in the flume experiments; i.e., larvae settled significantly more in BDF compared to control sediments irrespective of flow speed. Comparison with the settlement of polystyrene mimics and freeze-killed larvae led to the conclusion that active selection, active secondary dispersal and, at low flow velocities (5 cm s⁻¹), passive adhesion to the sediment are important mechanisms determining the settlement of M. balthica larvae in estuarine biofilms.     

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.     

Cardiac output during exposure to <Emphasis Type="Italic">Chattonella</Emphasis><Emphasis Type="Italic">marina</Emphasis> and environmental hypoxia in yellowtail (<Emphasis Type="Italic">Seriola</Emphasis><Emphasis Type="Italic">quinqueradiata</Emphasis>)

The experiments were undertaken to measure, for the first time, cardiac output in yellowtail (Seriola quinqueradiata) during exposure to a harmful red tide flagellate (Chattonella marina). The responses were compared with those during exposure to environmental hypoxia to evaluate the significance of the drop of arterial oxygen partial pressure (PaO₂) in the fish-kill mechanisms by C. marina. PaO₂ immediately decreased, whereas heart rate (HR) was maintained until shortly before death during exposure to C. marina. Suffocation developing during the exposure resulted from a decrease in blood oxygen content, but not from lowered blood flow to the tissue. Although exposure to both C. marina and hypoxia immediately decreased PaO₂, arterial oxygen content (CaO₂) and pH (pHa) were significantly lower, whereas HR and cardiac output (&Qdot;) remained significantly higher, for the C. marina-exposed fish than for hypoxia-exposed fish. Although the drop in PaO₂ appears to play a pivotal role in the mechanisms of fish death by C. marina, other physiological response(s) should also be considered.     

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.     

Trace metal ingestion and assimilation by the green mussel <Emphasis Type="Italic">Perna viridis </Emphasis>in a phytoplankton and sediment mixture

We examined trace metal ingestion and assimilation (Cd, Se, Zn) by the green mussel Perna viridis in a mixture of diatoms and sediment at concentrations below and above the pseudofeces production levels. Dual gamma radiotracers (¹⁰⁹Cd, ⁶⁵Zn) were used to investigate particle selection on marine diatoms and sediments. The diatom (Thalassiosira weissflogii) was radiolabeled with ¹⁰⁹Cd, and the natural sediment was radiolabeled with ⁶⁵Zn. By comparing the ratios of ¹⁰⁹Cd:⁶⁵Zn in the particle mixture, mussel tissues and pseudofeces within a short-term exposure period (35 min), the results demonstrated that the green mussels were able to selectively ingest the diatom particles at a high particle load. Efficiency of selection for nutritious particles (e.g. diatoms) increased with increasing ratio of sediments in the particle mixture. Pseudofeces contained a higher ratio of sediments relative to that in the feeding suspension. No major particle selection was observed at concentrations below the level for pseudofeces production. The assimilation of Cd, Se and Zn by the green mussels was quantified using a pulse-chase feeding technique. The assimilation of Se and Zn by the green mussels from ingested diatoms was reduced with the presence of sediment within the mussel gut, presumably due to the resorption of metals onto the sediment, leading to a quicker passage of metals through the digestive tract and a lower proportion of metals subjected to intensive digestion. In contrast, the presence of diatoms did not significantly affect metal assimilation from ingested sediment. A significant correlation between metal assimilation efficiency and metal gut passage time was also observed. Metal assimilation by the green mussels appeared to be little dependent on the particle concentration in seawater. Our study suggests that particle selection may potentially alter metal influx from ingested food sources, particularly at high particle concentrations. Selective feeding of nutritious particles, coupled with a high assimilation efficiency from these ingested particles, may increase metal influx into mussels from the dietary phase.     

Settlement of the wreckfish (<Emphasis Type="Italic">Polyprion</Emphasis><Emphasis Type="Italic">americanus</Emphasis>)

The wreckfish (Polyprion americanus) is a panoceanic, long-lived, deep-water demersal species, characterized by an extended pelagic juvenile phase associated with floating objects. In the present study morphometric data from 435 specimens collected from Greece (Crete), Italy (Ionian Sea) and the Atlantic coast of France from September 1999 to March 2001 were analyzed to estimate the settlement size of the species and associated changes in morphology. The fishery sample included specimens from both the pelagic and the demersal stage. Length-at-settlement (TL₅₀) and 95% confidence intervals were estimated by fitting a logistic function. Eleven morphometric characters were analyzed, and the existing inflection points, reflecting alterations in body shape, were defined: (1) iteratively, as the transition point, splitting the data set into two groups, for which the reduced major axis functions, between a character and total length, of the successive groups best fit (minimum sum of squares of residuals) the combined data set and (2) where the second derivative of the fitted third-order polynomial functions to morphometric ratios equaled zero. The main size range within which wreckfish settled was 56-65 cm, and the means of the inflection points defined by the two methods were 61 and 64 cm. These results indicate changes in functional morphology associated with settlement. The monitoring of wreckfish juveniles caught in the wild and kept individually in captivity showed changes in growth and food intake at approximately the estimated settlement length and suggested that temperature was the most likely factor triggering settlement.     

Dynamics of pigment degradation by the copepodite stage of <Emphasis Type="Italic">Pseudodiaptomus</Emphasis><Emphasis Type="Italic">euryhalinus</Emphasis> feeding on <Emphasis Type="Italic">Tetraselmis</Emphasis><Emphasis Type="Italic">suecica</Emphasis>

Short-term (3 h) changes in concentration of chlorophylls and their derivatives in stage V Pseudodiaptomus euryhalinus and their fecal material were followed by HPLC during a 24 h experiment. Copepodites were fed with the prasinophyte Tetraselmis suecica. Intact chlorophyll a and b were found in animals and fecal material and had similar dynamics of accumulation over time. The extent of transformation of chlorophyll a and b to colorless compounds was different with chlorophyll a being more extensively degraded. Additionally, several chlorophyll derivatives (pheophytin and pyropheophytin-like pigments) were found. Pyropheophytin a was the most abundant followed by pheophytin b, pheophytin a, and pheophorbide a. Relative amounts of pheopigments were different in copepodites and fecal material, and pheophytin a, pheophorbide a, and pheophytin b concentrations were low and variable. The amount of ingested chlorophyll recovered as chlorophyll a and its derivatives in fecal and copepodite pools was generally low (<5%), with one exception occurring after 9 h, when it accounted for >70%. These data suggest individual pheopigments are produced at different rates and that chemical or enzymatic mechanisms in the gut of copepodites act on the two chlorophylls in different ways.     

Feeding aggregations of sea stars (<Emphasis Type="Italic">Asterias</Emphasis> spp. and <Emphasis Type="Italic">Henricia sanguinolenta</Emphasis>) associated with sea urchin (<Emphasis Type="Italic">Strongylocentrotus droebachiensis</Emphasis>) grazing fronts in Nova Scotia

Migrating feeding aggregations (or fronts) of sea urchins can dramatically alter subtidal seascapes by destructively grazing macrophytes. While direct effects of urchin fronts on macrophytes (particularly kelps) are well documented, indirect effects on associated fauna are largely unknown. Secondary aggregations of predators and scavengers form around fronts of Strongylocentrotus droebachiensis in Nova Scotia. We recorded mean densities of the sea stars Asterias spp. (mainly A. rubens) and Henricia sanguinolenta of up to 11.6 and 1.7 individuals 0.25 m⁻² along an urchin front over 1 year. For Asterias, mean density at the front was 7 and 15 times greater than in the kelp bed and adjacent barrens, respectively. There was strong concordance between locations of peak density of urchins and sea stars (Asterias r = 0.98; H. sanguinolenta r = 0.97) along transects across the kelp–barrens interface, indicating that sea star aggregations migrated along with the urchin front at rates of up to 2.5 m per month. Size–frequency distributions suggest that Asterias at the front were drawn from both the barrens (smaller individuals) and the kelp bed (larger individuals). These sea stars fed intensively on mussels on kelp holdfasts and in adjacent patches. Urchin grazing may precipitate aggregations of sea stars and other predators or scavengers by incidentally consuming or damaging mussels and other small invertebrates, and thereby releasing a strong odor cue. Consumption of protective holdfasts and turf algae by urchins could facilitate feeding by these consumers, which may obtain a substantial energy subsidy during destructive grazing events.     

How do dietary diatoms cause the sex reversal of the shrimp <Emphasis Type="Italic">Hippolyte inermis</Emphasis> Leach (Crustacea,Decapoda)

Hippolyte inermis Leach 1915 is a protandric shrimp largely distributed in Posidonia oceanica meadows and other Mediterranean seagrasses. Previous studies demonstrated several physiological peculiarities, such as absence of female gonadic buds in adult males (the new female gonad is produced starting from few undifferentiated cells), the consequent absence of an ovotestis, 2 yearly periods of reproduction with different population structures (a spring outburst producing both males and primary females, and a fall reproduction producing mainly males), and a process of sex reversal influenced by the diatom food ingested. We performed several laboratory analyses to compare the effects of various species of benthic diatoms, in order to test the effect of different diatoms and provide information on the mechanism of action of the ingested compounds. In addition, we performed molecular tests (TUNEL) and TEM observations, to check the hypothesis that the effect of benthic diatoms may be mediated by a process of apoptosis acting on the male gonad. The results obtained allowed for a ranking of a series of benthic diatoms according to their effects on sex reversal, and a confirmation of the striking effect of Cocconeis sp. diatoms, which are able to trigger the appearance of primary females. We also demonstrated the presence of apoptosis both in the male gonad and in the androgenic glands of postlarvae. The effect is species specific, strictly localized to the male gonad and androgenic gland, and limited to a very short period of time, from the 5th to the 12th day of postlarval development.