Pedro E. Saucedo

Effect of temperature on oxygen consumption and ammonia excretion in the Calafia mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856)

Abstract The effect of temperature on oxygen consumption and ammonia excretion in the pearl oyster Pinctada mazatlanica was studied as a strategy to define the optimum thermal range for experiments on reproductive conditioning of broodstock of this species. Oysters were taken to the laboratory, acclimated for 2 weeks at four temperatures (18, 23, 28, and 33 °C), and transferred to respiration chambers for individual measurements of oxygen consumption and ammonia excretion at the same temperatures. Respiration and excretion rates, as well as routine respiratory/excreted energy and Q 10 coefficients were calculated. One-way ANOVA was applied to determine differences in these parameters as a function of temperature. All physiological parameters yielded highly significant differences with increasing temperature, suggesting that an adequate metabolic temperature range for the species may lie between 23 and 28 °C. Within this range, a combination of active respiration and low ammonia excretion, together with Q 10 coefficients near 2, suggest the existence of compensatory mechanisms that allow the species to perform seasonal regulation during moderately warm temperature changes. In contrast, 18 and 33 °C represented marginal temperature conditions close to the tolerance limits of the species. In particular, 33 °C was a stressful temperature for P. mazatlanica because of high ammonia excretion. More studies under conditions of satiation and hypoxia, as well as experiments testing lower and higher lethal temperatures are needed to understand the energy budget of the species.

Reproduction, Development and Growth

Pearl oysters are typical marine bivalves in many features of their reproductive biology, embryological and larval development, and growth. They are protandrous hermaphrodites with various sex reversals during their lifetime in response to complex interactions of endogenous and environmental factors. They develop first as males and retain this condition for one or several reproductive cycles until changing sex. Pearl oysters have diffuse gonadial tissue, which is composed of small granular bags, acini. The acini contain stem cells, which may develop into oocytes or spermatocytes, the gametogenic processes being largely supported by energy and metabolites from the digestive gland and adductor muscle. The overall pattern of reproduction in pearl oyster populations is synchronous, with male and females undergoing sequential processes that lead to a simultaneous breeding period. Pearl oyster populations may spawn once or multiple times during a year. Spawning in pearl oysters is usually triggered by a change in the environment or presence of water-borne gametes. Early development follows the typical marine bivalve pattern of trochophore, D-stage veliger, umbo stage, eye-spot stage, pediveliger, metamorphosis, and newly settled spat. It takes in the order of 3–4 weeks. Development rates are particularly influenced by food availability and stored lipid is probably the primary energy reserve used during metamorphosis.

FACTORS INFLUENCING RECRUITMENT OF HATCHERY REARED PEARL OYSTER (PINCTADA MAZATLANICA; HANLEY 1856) SPAT

Abstract We evaluated experimental collection of Pinctada mazatlanica spat on substrates of different textures and colors placed at different depths within settlement tanks. When larvae reached the pediveliger stage (day 25), black-colored spat collectors (“envelope” type) composed of the following materials were offered as settlement substrates: onion bag, mosquito net, fishing net, and 63% shade-cloth. Spat collectors measured 30 × 30 cm and were composed of an outer bag and inner substrate made of the same material. The influence of the color of spat collector material on recruitment was investigated using a second type of spat collector composed of onion bags as the outer bag and inner substrate (“bag” type). Bags were prepared in 3 different outer/inner (O/I) color combinations of green/green, green/black, and red/black (O/I). Both types of collectors were deployed at different depths within settlement tanks. Approximately 2.7% of the initial larval population survived, resulting in 35,583 spat. Of these, 71.3% recruited to bag type collectors, 21.9% to envelope type collectors, and 6.8% to the surfaces of the culture tanks. Spat recruitment was significantly affected by collector material (P < 0.01), with fishing net and shade-cloth yielding highest and lowest density of spat, respectively. Similarly, spat collector color combination significantly influenced spat collection (P < 0.01), with higher recruitment to red and green substrates. For both spat collector types, there was significantly greater (P < 0.01) recruitment of spat to collectors in the middle of the water column (60–90-cm depth). This study showed that both the type and color of material used for spat collectors, as well as depth of deployment, influenced recruitment of P. mazatlanica spat. These results will help fine-tune current hatchery techniques for this species.

Energy Storage and Allocation During Reproduction of Pacific Winged Pearl Oyster Pteria sterna at Bahía de la Paz, Baja California Sur, Mexico

Abstract Seasonal variations in storage, partitioning, and allocation of energy reserves (proteins, carbohydrates, lipids, and triglycerides) between germinal and somatic tissues (gonad, digestive gland, mantle tissue, and adductor muscle), were investigated related to reproduction of Pacific winged pearl oyster Pteria sterna. Tissue samples were collected every three months and analyzed with histological and biochemical techniques. Energy coefficients were also calculated with data from chemical composition of tissues. Gonad samples in almost all developmental stages occurred throughout the year, suggesting that P. sterna is a multispawning species. The evidence indicates that the main reproductive season runs from January through April (21°C to 22°C) and was identified by higher frequency of ripe gonads, more and larger postvitellogenic oocytes and higher protein, lipid, and triglyceride levels in gonad tissue. Within this study period, there were two spawning peaks, July 2003 and January 2004. Gametogenesis was sustained from energy mainly obtained from the digestive gland and secondly from the adductor muscle. Only proteins from these two tissues were mobilized to the gonad for maturation of sex organs, because carbohydrates were stored despite the progress of gametogenesis. The role of mantle tissue was negligible. P. sterna appears to use a combination of stored reserves (conservative strategy) and food supply (opportunistic strategy) as an overall strategy to regulate reproduction.

Changes in lipid content of oocytes of the penshell Atrina maura as a criterion of gamete development and quality: a study of histochemistry and digital image analysis.

ABSTRACT Tools of histochemistry and digital image analysis were used to quantify changes in the coverage area of lipid droplets (lipid content) of oocytes of the penshell Atrina maura during oogenesis and to determine its relation to changes in water temperature and seston content. These data led to calculating a lipid index as a criterion of gamete development and quality. Gonads were collected monthly for 18 mo and prepared for histochcmical processing with Sudan Black B for identification of lipids. Finished slides were digitized for determining stages of oogenesis and variations in the size of oocytes. Two periods of greatest reproductive activity occurred during the study, with a lower peak from November through January (∼15°C; 26 mg/g) and a major peak from April through June (∼20°C; 25–40 mg/g). Oocyte area significantly varied during the stages of active development (516–2,743 µm2), ripeness (1,073–2,930 µm2), spawning (145–2,939 µm2), and atresia (331–2,001 µm2). Lipid incorporation into oocyte cytoplasm followed a clear seasonal pattern, peaking again in winter and spring. Temporal variations in the lipid index and its relation to oocyte diameter were irregular, but also peaked in winter and spring. Histochemistry and digital image analysis resulted in reliable methods for estimating oocyte development and quality in this species, and can certainly be applied in studies of reproduction of other bivalve, invertebrate, and vertebrate species.

DIGITAL IMAGE ANALYSIS OF LIPID AND PROTEIN HISTOCHEMICAL MARKERS FOR MEASURING OOCYTE DEVELOPMENT AND QUALITY IN PEARL OYSTER PINCTADA MAZATLANICA (HANLEY, 1856)

Abstract We applied quantitative histochemical techniques and digital image analysis to study seasonal cycles of use of lipid and protein reserves during vitellogenesis in the pearl oyster Pinctada mazatlanica. Female gonad samples were collected seasonally during an annual cycle and processed histologically to characterize the gametogenic cycle, analyze variations in the frequency and size of vitellogenic and postvitellogenic oocytes and calculate the ooplasm:nucleoplasm ratio for both types of oocytes. Lipid and protein inclusions in each type of oocyte were identified using Sudan Black B and Schiffs ninhydrin stains. In both cases, quantification of lipid and protein components was performed through measuring variations in the color coverage area of the oocyte with a digital image analysis system. With this procedure, we calculated a lipid index and a protein index to refer oocyte quality. The lipid index was higher in winter, suggesting a strategy towards storage in the gonad. The protein index was highest during spring in vitellogenic oocytes and during winter in postvitellogenic oocytes, indicating that proteins are actively used during oocyte growth. These results, together with data of the ooplasm:nucleoplasm ratio, suggest differential accumulation of lipid and protein components within the ooplasm during oocyte development. Quantitative histochemistry and digital image analysis represent a combination of reliable techniques for evaluating reproductive processes and oocyte growth and quality.

Gonadic conditioning of the calafia mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856), under two temperature regimes.

Abstract Gonadic conditioning of pearl oyster Pinctada mazatlanica was studied under two water temperature regimes using cultured specimens. Before starting conditioning, specimens were induced to spawn with thermal shock. Two different regimes were evaluated, controlled temperatures (CT) and gradually increasing temperatures (IT). In both trials, specimens were fed daily with Isochrysis galbana and Chaetoceros gracilis in a proportion of 7:3. Gonad and muscle samples were excised from the oysters every 15 days and processed for histological analysis. Condition Index and Muscle Condition Index were calculated. Similarly, oocyte frequency and oocyte diameter measurements were made. ANOVA analyses were used to test significant differences in the frequency and diameter of postvitellogenic oocytes, and values of both condition indexes between temperature regimes, and over time. The IT showed more suitable conditions for gonad conditioning than CT, although the time needed for reaching maturity was higher in the former regime. For CT, a temperature of 24°C was the best for conditioning, since there were no traces of atretic oocytes, partially spawned specimens, and there were less previtellogenic oocytes and more postvitellogenic oocytes filling the follicles. At 20°C, no positive results were noticed. Condition indexes appear to be reliable indicators of gonad activity during conditioning in both trials. The ANOVA analysis detected significant differences in almost all the statistical treatments run for oocyte frequency, oocyte diameter and condition indexes according to temperature regime and over time.

Annual Reproductive Effort of Pacific Winged Pearl Oyster Pteria sterna and Its Relation with the Timing for Planning Pearl Seeding Operations

ABSTRACT Using a combination of stereological and calorimetric methods, we studied reproductive effort of Pacific winged pearl oyster Pleria sterna during an annual cycle in Bahia de La Paz, B.C.S., Mexico. The relationship between changes in the volumetric fraction of germinal and somatic tissues (gonad, digestive gland, adductor muscle, and mantle tissue) and changes in their energy content was analyzed. These data were also correlated with changes in water temperature and availability of food (seston). Because P. sterna spawns several times a year, reproductive effort was estimated ∼400% in terms of energy increase from early development in October 2006 to the spawning occurring in January to February 2007. During this period, when water temperature was decreasing and seston concentration was increasing, P. sterna followed a conservative strategy for allocating energy from reserves previously stored in somatic tissues. In contrast, when productivity dropped in spring, the species followed an opportunistic strategy for sustaining gametogenesis from food energy. In decreasing order, total energy channeled for reproduction came from the digestive gland (23 KJ g 1), adductor muscle (19 kJ g 1), and mantle tissue (16 KJ g 1). Based on these results, we recommend that commercial pearl culture practices be conducted from mid-autumn (October) through early spring (April), when P. sterna is energetically more resistant to manipulation. An additional recommendation is to avoid grafting during the summer (June through September), when the species is energetically exhausted and highly vulnerable to manipulation.

Influence of Environmental Factors on the Reproductive Cycle of the Eared Ark Anadara notabilis (Röding, 1798) In Northeastern Venezuela

ABSTRACT The reproductive cycle of the eared ark Anadara notabilis and its relationship with environmental factors was evaluated every 15 days between March 2004 and February 2005 at the northern part of the Peninsula de Araya, Venezuela. Environmental factors measured included temperature, salinity, chlorophyll a, and total seston, including the particulate organic matter and particulate inorganic matter fractions. Adult specimens were collected using a net drag and then randomly selected to estimate wet live biomass and dry biomass of soft body tissues. Gonad samples were processed with histological analysis to determine sex ratio, developmental stages, and variations of follicle size. Reproduction in A. nolabilis was continuous throughout the year, with spawning peaks occurring in June and October, coinciding with the lowest water temperatures. In March, September, and November 2004, specimens attained the highest dry biomass values, whereas lowest dry biomass occurred in June and October 2004. Stepwise regression analyses demonstrated that temperature and particulate organic matter values are the main modulators of reproductive events. When temperature decreased, dribble spawning was detected in this species, possibly inducing a survival strategy whereby the spawning period is extended to increase reproductive success.

Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture.

Aim: This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes. Materials and Methods: Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains. Results: Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4) among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively. Conclusion: Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world.