Dietary metal toxicity to the marine sea hare, Aplysia californica.

Metal pollution from anthropogenic inputs is a concern in many marine environments. Metals accumulate in tissue and in excess cause toxicity in marine organisms. This study investigated the accumulation and effects of dietary metals in a macroinvertebrate. The green seaweed, Ulva lactuca and the red seaweed, Agardhiella subulata were each concurrently exposed to two concentrations (100 or 1000 µg/L) of five metals (Cu, Ni, Pb, Cd, and Zn). Additionally, U. lactuca was exposed to 10 µg/L of the metal mixture as well as 10 or 100 µg/L of each metal individually for 48 h. The seaweeds were then used as food for the sea hare, Aplysia californica for two to three weeks depending on the exposure concentration. Body mass of A. californica was measured weekly, and at the end of the exposure duration, metal concentrations were quantified in dissected organs (mouth, esophagus, crop, gizzard, ovotestis, heart, hepatopancreas, gill, and the carcass). Metal distribution and accumulation in the organs of A. californica varied with the metal. A. californica fed the metal-exposed diets had significantly reduced body weight by the end of the exposure periods, as compared to controls; however, differences were observed in the extent of growth reductions, dependent on exposure concentration, duration, and exposure regime (metal mixture versus individual metal-exposed diet). Metal mixture diets decreased A. californica growth more so than comparable individual metal diets, despite more metal accumulating in the individual metal diets. Additionally, Zn- and Cu-contaminated algal diets decreased control-normalized growth of A. californica significantly more than comparable Cd-, Pb-, or Ni-contaminated diets. The seaweed diets in this study contained environmentally relevant tissue metal burdens. Therefore, these results have implications for metals in marine systems.

Comparative analysis of early ontogeny in Bursatella leachii and Aplysia californica.

Opisthobranch molluscs exhibit fascinating body plans associated with the evolution of shell loss in multiple lineages. Sea hares in particular are interesting because Aplysia californica is a well-studied model organism that offers a large suite of genetic tools. Bursatella leachii is a related tropical sea hare that lacks a shell as an adult and therefore lends itself to comparative analysis with A. californica. We have established an enhanced culturing procedure for B. leachii in husbandry that enabled the study of shell formation and loss in this lineage with respect to A. californica life staging.

Decreased response to acetylcholine during aging of aplysia neuron R15.

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.

Juvenile growth of the tropical sea urchin Lytechinus variegatus exposed to near-future ocean acidification scenarios.

To evaluate the effect of elevated pCO(2) exposure on the juvenile growth of the sea urchin Lytechinus variegatus, we reared individuals for three months in one of three target pCO(2) levels: ambient seawater (380 µatm) and two scenarios that are projected to occur by the middle (560 µatm) and end (800 µatm) of this century. At the end of 89 days, urchins reared at ambient pCO(2) weighed 12% more than those reared at 560 µatm and 28% more than those reared at 800 µatm. Skeletons were analyzed using scanning electron miscroscopy, revealing degradation of spines in urchins reared at elevated pCO(2) (800 µatm). Our results indicate that elevated pCO(2) levels projected to occur this century may adversely affect the development of juvenile sea urchins. Acidification-induced changes to juvenile urchin development would likely impair performance and functioning of juvenile stages with implications for adult populations.

Changes in D-aspartate ion currents in the Aplysia nervous system with aging.

D-Aspartate (D-Asp) can substitute for L-glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of -70 mV, where molluscan NMDA receptors open free of constitutive block by Mg(2+). Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.

Larval growth, development, and survival of laboratory-reared Aplysia californica: effects of diet and veliger density.

Over the last three decades, the California sea hare, Aplysia californica, has played an increasingly important role as a model organism in the neurosciences. Since 1995, the National Resource for Aplysia has supported a growing research community by providing a consistent supply of laboratory-reared individuals of known age, reproductive status, and environmental history. The purpose of the present study was to resolve the key biological factors necessary for successful culture of large numbers of high quality larval Aplysia. Data from a sequence of five experiments demonstrated that algal diet, food concentration, and veliger density significantly affected growth, attainment of metamorphic competency, and survival of Aplysia larvae. The highest growth and survival were achieved with a mixed algal diet of 1:1 Isochrysis sp (TISO) and Chaetoceros muelleri (CHGRA) at a total concentration of 250 x 10(3) cells/mL and a larval density of 0.5-1.0 per mL. Rapid growth was always correlated with faster attainment of developmental milestones and increased survival, indicating that the more rapidly growing larvae were healthier. Trials conducted with our improved protocol resulted in larval growth rates of >14 microm/day, which yielded metamorphically competent animals within 21 days with survival rates in excess of 90%. These data indicate the important effects of biotic factors on the critical larval growth period in the laboratory and show the advantages of developing optimized protocols for culture of such marine invertebrates.

Temperature effects on growth, maturation, and lifespan of the california sea hare (Aplysia californica).

We conducted a hatchery growth study to describe the variability in growth rates, spawning, and mortality of Aplysia californica in regard to rearing temperature. Animals were housed at a standard hatchery density of five animals per cage, at temperatures of 13, 15, 18, and 21 degrees Celsius. Animals reared at 13 or 15 degrees C grew as much as four times as large, lived twice as long, matured later, and spawned longer than did animals reared at 18 or 21 degrees C. At age 170 to 205 days the fastest growth rates occurred at 18 and 21 degrees C, and the slowest at 13 degrees C. As animals at 18 and 21 degrees C reached sexual maturity at ages 190 to 197 days, or approximately 60% through their lifespans, their growth rates slowed such that by age 260 days, the fastest growth rate was at 13 degrees C, and the slowest was at 21 degrees C. Animals reared at 13 and 15 degrees C reached sexual maturity at 242 and 208 days, respectively, or at approximately 40% of their life spans. Lifespan and maximum average animal weight were significantly inversely correlated with temperature (P

Reproductive output in the hatchery-reared california sea hare at different stocking densities.

Although the California sea hare (Aplysia californica) is well known from neurobiological studies and is raised in the laboratory for this purpose, the life history of this species in the laboratory is less well studied. Therefore we conducted a study of the reproductive period of hatchery-born and -raised A. californica in which sibling animals were reared at stocking densities of 2, 5, 10, 15, and 20 animals per 16-liter cage. Temperature was controlled at 13 to 15 degrees, and the photoperiod was a 14:10-h light:dark cycle. Seawater O2 concentration, pH, and salinity were optimized by seawater flow through the animal cages. Compared with scheduled feedings, an ad libitum algal diet produced early sexual maturity. Despite different growth rates at different animal densities per cage, the age at first sexual maturity (defined as the first egg mass) did not differ significantly among the different densities and averaged 210 +/- 15 days of age. Although there was no difference in the total spawn weight per cage as a function of animal density from 2 to 20 animals per cage, the number of spawning episodes per animal decreased as the cage density increased. The average weight of spawn per animal over its reproductive lifetime decreased as cage density increased. When expressed per day of reproductive maturity, spawn weight per animal decreased as the cage density increased. Finally, larger animals produced more eggs during the 10 spawns at the peak of the spawning period. The largest animals corresponded to the cages containing the fewest animals. Stocking densities between 2 and 20 per cage had little influence on time to maturity and on average number of days of reproductive life. However, stocking density (and thus animal size) strongly influenced so-called lifetime fecundity.

The effect of stocking density on growth rate and maturation time in laboratory-reared california sea hares.

California sea hares (Aplysia californica) were reared from the late juvenile period (approximately day 100 posthatch) to senescence in a laboratory study of growth and maturation at different stocking densities. Temperature, light, and food were controlled, and other seawater parameters such as O2 concentration, pH, and salinity, although not controlled, were optimized by the flow-through design of seawater through the cages. Stocking densities evaluated were 1, 2, 5, 10, 15, and 20 animals per 16-liter cage. Food availability is likely to be a limiting factor to growth in wild populations of A. californica, but in our experiments, algal diet was ad libitum at all densities and presumably was not a controlling factor. The animals maintained at each of the various densities grew at different rates but reached sexual maturity (defined as the age at the appearance of the first egg mass) at approximately the same age, 204 +/- 4 days (mean +/- standard error), for densities higher than 2 animals per cage. Age at sexual maturity for 2 animals per cage was 274 days. Growth rates were highest in cages with the lowest stocking densities and lowest in high-density cages, ranging from 3.72 g live weight/day in animals housed individually to 1.06 g live weight/day for those housed 20 per cage during the period 100 to 200 days of age. Growth differed significantly among the various stocking densities beginning at 9 weeks of growth (age, 167 to 174 days). In summary, we show that stocking density has an important influence on growth and is a key factor for consistently rearing Aplysia as an animal model under hatchery conditions.