José M. F. Babarro

Metabolism of the mussel Mytilus galloprovincialis from two origins in the Ría de Arousa (north-west Spain)

Mussel seed Mytilus galloprovincialis (Bivalvia: Mytilidae) from two origins (rocky shore and collector ropes) was cultivated on a raft in the Ria de Arousa (north-west Spain), from seeding to thinning out, for 226 d (November 1995-July 1996) and two aspects of metabolism, oxygen consumption rate (VO 2 ) and ammonia excretion rate (VNH 4 -N) were studied in situ. The model derived from multiple analysis of oxygen consumption accounted for 91.9% of the variance, based on dry weight of the mussels and the environmental factors quality of food (organic content) and mainly chlorophyll-a. Seed origin also showed significant influence. The seasonal pattern of the oxygen consumption can be attributed mainly to the variation of chlorophyll-a, which showed a higher range of values in the spring months. Origin of seed did not show a homogeneous effect on oxygen consumption throughout the cultivation period. Collector rope mussels showed higher oxygen consumption values at the beginning of the cultivation period and after the first 15 d, but the rocky shore mussels showed a higher oxygen consumption between days 22 and 110. Energy-conserving patterns and lower condition index at the onset of the experiment for rocky shore mussels could explain these initial differences. Multiple analysis on the variation of ammonia excretion rate provided a model that accounted for 72.6% of the variance based on dry weight of mussels, seed origin and the environmental parameters chlorophyll-a and total particulate matter. The rocky shore mussels showed a significantly higher excretion values for most of the cultivation period, although there was no constant tendency throughout. High excretion values were recorded between January and March, whilst for the rest of the cultivation period values were low. The O:N index was higher in collector rope mussels for most of the cultivation period, which may suggest a more favourable energy metabolism and/or a more appropriate nutritional state for these specimens.

Anoxic survival potential of bivalves: (arte)facts

The anoxic survival time of the bivalves Chamelea gallina, Cerastoderma edule and Scapharca inaequivalvis from two different ecosystems and differing anoxia tolerances was studied in static (closed) and flow-through systems. The antibiotics chloramphenicol, penicillin and polymyxin were added, and molybdate (specific inhibitor of the process of sulfate reduction). Survival in (near) anoxic seawater of Chamelea was studied in a static system by comparing untreated seawater with autoclaved seawater and untreated clams with clams incubated in well-aerated seawater, containing the broad-spectrum antibiotic chloramphenicol, prior to the anoxic survival test. With untreated clams and natural seawater (median mortality time 2.4 days) a decrease in pH and exponential accumulation of sulfide and ammonium was observed in the anoxic medium, indicating excessive growth of (sulfate reducing) bacteria. In sterilized seawater LT50 (2.1 days) was not significantly different and again considerable amounts of ammonium and sulfide accumulated. However, pre-treatment of clams with chloramphenicol resulted in an increase of LT50 (11.0 days) by approximately fivefold. Accumulation of ammonium and sulfide was retarded, but was finally even stronger than in the medium containing untreated clams. Median mortality times were 2.5 and 2.4 days for Chamelea and 2.7 and 2.9 days for Cerastoderma for static and flow-through incubations, respectively. Addition of chloramphenicol increased strongly survival time in both systems with corresponding values of 11.0 and 16.3 days for Chamelea, and 6.4 and 6.5 days for Cerastoderma. LT50 of Scapharca in anoxic seawater was 14.4 days. Chloramphenicol and penicillin increased median survival time to 28.5 and 28.7 days, respectively, whereas polymyxin displayed no effect (LT50=13.6 days). Molybdate added to artificial sulfate free seawater blocked biotic sulfide formation, but did not improve survival time (LT50=13.7 days). Overall the results indicate that proliferation of anaerobic pathogenic bacteria, firmly associated with the bivalves, is a main cause of death besides lack of oxygen. Bacterial damage is probably caused by injury of the tissues of the clams and not by the release of noxious compounds to the medium.

Anoxic survival of Macoma balthica: the effect of antibiotics, molybdate and sulphide.

In anoxic semi-closed systems, the survival time of the clam Macoma balthica was compared to clams which were incubated in the presence of several antibiotics (chloramphenicol, 5-oxytetracycline hydrochloride, penicillin, streptomycin, a mix of penicillin and streptomycin and a mix of chloramphenicol, polymyxin, neomycin and penicillin), sulphide and chloramphenicol at pH 6.8 and 8.2 and molybdate (specific inhibitor of the process of sulphate reduction). The aim was to detect maximum survival times of this clam and indications for the cause of mortality under the conditions tested. Median survival time (LT(50)) of the clam was 4.8 days (at 19 degrees C) in incubations without any addition. Added sulphide (200 µM) decreased survival time. At pH 8.2, LT(50) decreased by 20.8% and at pH 6.8 by 35.2%. However, added molybdate, which suppressed biotic sulphide formation, did not improve survival time (LT(50)=4.4 days). Biotic sulphide probably did not speed up mortality rate, but indicated excessive growth of sulphate reducing bacteria once mortality started. The presence of different antibiotics increased significantly survival time (LT(50)) from 8.9 to 14.9 days. Qualitative estimations were made of the numbers of bacteria present in the systems. Compared to a seawater control, highest numbers were observed in the incubation of clams without additions and in the presence of molybdate. Nevertheless, due to the presence of molybdate, bacteria numbers were significantly lower. However, very low numbers of bacteria were observed in the incubations of clams in the presence of chloramphenicol. These data demonstrated that the presence and proliferation of bacteria was probably the cause of death of the clams.

Secretion of byssal threads and attachment strength of Mytilus galloprovincialis : the influence of size and food availability

Byssogenesis rate and attachment strength of the mussel Mytilus galloprovincialis were investigated in the laboratory considering different body sizes and feeding conditions. Byssal thread secretion was significantly higher in juveniles as compared to larger mussels of approximately 87 mm shell length. Asymptotic number of threads attached was obtained from approximately 72 hours onwards within a range of 42 -46 and 27-31 for juveniles and larger experimental mussels, respectively (P < 0.05 ANOVA). Absorption efficiency values ofcontrol fed individuals dropped significantly from 0.78 in juveniles to 0.70 in larger mussels (P < 0.001 ANOVA) which pointed out energetic constraints of mussels with regard to their size and its probable effect on byssus secretion rates. Attachment force was 2.8 times higher in larger mussels than in juveniles (2.21 versus 0.78 Nfor both sizes, respectively; P < 0.001 ANOVA) which in turn followed the same order of magnitude than differences in the threads thickness values of both experimental mussel sizes. Tenacity followed a reverse pattern with juveniles presenting two-fold higher values than larger mussels (P < 0.001 ANOVA) based on a lower increment of attachment force (x2.8) as compared to shell area (x6) for the comparison larger versus juvenile mussel size, which in turn might suggest that larger specimens secreted weaker threads. When animals were maintained unfed for a week, a significant drop in both byssus secretion and attachment force were observed in juveniles but was not the case for larger mussels most likely as a consequence of a relatively short period of maintenance under food availability stress for the latter individuals that showed significantly higher initial condition and/or energetic store values. Accordingly, the use of energetic reserves in juveniles kept unfed for a week together with a significant drop in byssus secretion and attachment force might suggest a link, i.e. transfer of energy between soft tissues and byssus under stress. Quantitative values of byssal threads, based on the significance of the byssus versus attachment force relationship, together with its morphometric value, i.e. threads thickness, represented a primary mechanism to explain variability in attachment strength of M. galloprovincialis of the different body sizes studied here.

Bottom culture of the tropical scallop Lyropecten (Nodipecten) nodosus (L.) in the Golfo de Cariaco, Venezuela

Growth and survival of scallop Lyropecten nodosus were studied fromJuly to November 1997 using three bottom culture methods, (1) in corrals,(2) in pockets, and (3) in anchored sleeves. All size parameters studied (dryweight of the muscle, gonad, remaining tissues and shell, and shell length)showed significant differences due to culture method. The body componentswere larger for scallops in corrals than for those in pockets and greater forthose in pockets than in sleeves. In contrast, survival did not vary withculture method. Tissue components increased rapidly during the first 2months, when temperatures were lower and phytoplankton abundant(upwelling and transition periods). Subsequently values leveled off, or insome cases (muscle) decreased, and this coincided with stratification of thewater column and associated high temperatures and scarce food resources(and possible energetic demands for gonadal development). In contrast,shell weight and length showed no apparent affect of the environmentalchanges. The increased growth in the corrals was possibly because thecorral walls permitted the scallops to raise themselves off the bottom whichcould have provided greater access to food resources (suspendedparticles), or to better quality food.

Anaerobic survival potential of four bivalves from different habitats. A comparative survey.

A comparative survey of the anaerobic survival potential of four different bivalve species and the interference of associated bacteria has been carried out. Individuals from both subtidal and intertidal environments were considered by selecting the following species: Mytilus edulis (subtidal epifaunal), Spisula subtruncata (subtidal infaunal), Macoma balthica (intertidal infaunal) and Cerastoderma edule (intertidal infaunal). Anaerobiosis was simulated in the laboratory by subjecting individuals to the following conditions: nitrogen atmosphere, air atmosphere and anoxic seawater incubation. Moreover, the effect of the antibiotic CA (chloramphenicol) was investigated, either as a pre-treatment of individuals kept under normoxic conditions for a week or directly added to the anoxic incubation media. According to survival performances of the individuals, intertidal animals that use to cope with tidal fluctuations in the coastline (emersion processes) had an extraordinary greater capacity to survive aerial exposure as compared to both nitrogen gas and anoxic seawater incubations most likely due to their capacity to perform aerobiosis at certain rate from atmospheric oxygen availability. Specifically, Macoma balthica enlarged its survival potential up to 24.8 days (LT(50)) under air exposure at 12 degrees C as compared to other specific treatments used here (4.9 days). The latter pattern was also observed, although in a much lower magnitude, for the other intertidal species Cerastoderma edule that survived 3.7 and 4.6 days (LT(50)) under nitrogen atmosphere and anoxic seawater incubation, respectively as compared to 9.5 days for emersed individuals. In contrast to the subtidal species, aerial exposure of both intertidal species led to a much higher survival performances than incubation of individuals in anoxic media with the presence of antibiotic. Survival capacity of the subtidal species Mytilus edulis and Spisula subtruncata was statistically similar under air and nitrogen atmospheres and anoxic seawater incubation. Then, subtidal species have a limited ability to air breathing as a conclusion of a similar survival in atmospheric and anoxic seawater incubations. Remarkably, M. edulis represented the only exception when considering longer-term survival capacity compared to the LT(50) values. Indeed, differences in LT(90) values for M. edulis were statistically different, values decreasing significantly from 19.7-19.9 days (under both nitrogen and air atmospheres) to 16.7 days when individuals are incubated in anoxic seawater. This may be due to the adverse effects of anaerobic bacteria that spontaneously proliferate within the static seawater incubations. As well as for S. subtruncata, possible aerobic processes under aerial exposure of mussels seemed to be not significant for the enlargement of its survival potential, since results obtained for both air and nitrogen atmospheres are similar. Pre-treatment with the antibiotic chloramphenicol caused survival capacity to increase by a factor of approx. 2 (M. edulis) and 34-44% (S. subtruncata). In contrast to intertidal species, the direct addition of the antibiotic to the incubation media caused the highest survival performances in both subtidal species. Habitat differences and species-dependent variability must be considered as significant sources of variation when studying the anaerobic performance of individuals using the most common experimental anaerobic techniques to test survival potential.

From broad-spectrum biocides to quorum sensing disruptors and mussel repellents: antifouling profile of alkyl triphenylphosphonium salts.

‘Onium’ compounds, including ammonium and phosphonium salts, have been employed as antiseptics and disinfectants. These cationic biocides have been incorporated into multiple materials, principally to avoid bacterial attachment. In this work, we selected 20 alkyl-triphenylphosphonium salts, differing mainly in the length and functionalization of their alkyl chains, in fulfilment of two main objectives: 1) to provide a comprehensive evaluation of the antifouling profile of these molecules with relevant marine fouling organisms; and 2) to shed new light on their potential applications, beyond their classic use as broad-spectrum biocides. In this regard, we demonstrate for the first time that these compounds are also able to act as non-toxic quorum sensing disruptors in two different bacterial models (Chromobacterium violaceum and Vibrio harveyi) as well as repellents in the mussel Mytilus galloprovincialis. In addition, their inhibitory activity on a fouling-relevant enzymatic model (tyrosinase) is characterized. An analysis of the structure-activity relationships of these compounds for antifouling purposes is provided, which may result useful in the design of targeted antifouling solutions with these molecules. Altogether, the findings reported herein provide a different perspective on the biological activities of phosphonium compounds that is particularly focused on, but, as the reader will realize, is not limited to their use as antifouling agents.

Energy metabolism and performance of Mytilus galloprovincialis under anaerobiosis

Intertidal individuals of Mytilus galloprovincialis were exposed to anaerobiosis in laboratory at 22°C and a set of biochemical metabolites and survival potential determined. Differences in survival potential between individuals emersed or kept in oxygen-free seawater were residual according to ST 50 values (survival time, P≈0.05) but emersed individuals survived significantly longer when considering ST 90–100 values (P<0.05). Anaerobiosis was similarly activated under both emersion and incubation in anoxic seawater after 6 h according to a sevenfold increase in succinate. Longer exposure of individuals (up to 48 h) caused succinate (and propionate) to increase but in a higher magnitude under incubation with anoxic seawater. Propionate appeared in soft tissues after 24 h of incubation in anoxic seawater and after 48 h when individuals were emersed. Glycogen was not utilized after 6 h in any case, but was progressively used with longer exposure times and in a higher magnitude under incubation in anoxic seawater (48 h). Adenylate energy charge (AEC) was highly affected by both exposure time (P<0.001) and anaerobic treatment (P<0.01). Rapid breakdown of ATP and phospho-Larginine (PLA) did occur during the first 24 h of anaerobiosis, the latter ATP drop was accompanied by slight increase of ADP but strong increase of AMP that accumulated in a higher magnitude under incubation in anoxic seawater. Biochemical results of the present study suggested a certain degree of aerobiosis for emersed M. galloprovincialis that in turn is linked to a slight but significant longer survival performance. Most significant biochemical changes occurred during the first 24 h of oxygen deprivation, but significant differences between treatments were observed after 24–48 h. These lag differences in biochemical metabolites together with more accurate survival analyses have to be considered when investigating the energy metabolism linked to the anaerobic performance of M. galloprovincialis.

Factors involved in the (near) anoxic survival time of Cerastoderma edule: associated bacteria vs. endogenous fuel

The effect of several antibiotics, molybdate and hydrogen sulfide was tested on anoxic tolerance of the cockle Cerastoderma edule, as well as utilisation of glycogen. The aim was to evaluate the role of fuel depletion and growth of bacteria as a cause of mortality. The exponential increase of sulfide and ammonium occurred in anoxic natural seawater incubations and to a lesser extend in artificial, sulfate free, seawater. This could be strongly decreased by antibacterial agents, which led to improved survival time by approximately two-fold. Molybdate suppressed sulfide formation also, but did not affect survival time. Exogenous sulfide showed a negative effect on survival time at pH 6.8 and induced stronger accumulation of free glucose, D-lactate and L-alanine. This was not the case at pH 8.2. Fifty percent (LT50) of cockles in anoxic seawater died after 3.5 days still with half the initial glycogen concentration present. However, in the presence of chloramphenicol (LT50 7.9 days), the cockles utilised their endogenous fuel almost completely. In both incubations there was initially a strong increase of D-lactate and L-alanine. The D-lactate levels subsequently decreased again, probably due to bacterial consumption. This study strongly indicates that in anoxic closed systems, infection by pathogenic bacteria is the first cause of death and not exhaustion of endogenous fuel depots.

Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.