Carmen Rodríguez-Jaramillo

Gonadal Development and Histochemistry of the Tropical Oyster, Crassostrea corteziensis (Hertlein, 1951) during an Annual Reproductive Cycle

Abstract We describe the quantitative and qualitative histology and histochemistry for a tropical oyster, C. corteziensis sampled in a coastal lagoon in Northwest, Mexico. Males were larger than immature oysters, with females presenting intermediate values. In this species, mature organisms were found most of the year, and there were at least two strong spawning periods, one in summer and the second in autumn. The presence of mature oocytes most of the year did not allow for differentiation of an annual reproductive pattern using average oocyte diameter, as has been used in other species. There was a very short resting period in winter, particularly in December 2005, and by January to February 2006 postvitellogenic oocytes can already be found. Both sexes tended to have more lipids in the gonad tissue as maturation advanced, with an inverse correlation to carbohydrate in gonad and in vesicular tissue in females. No differences in lipids or carbohydrates content were found in digestive gland. A negative correlation was found between chlorophyll a content and gonad coverage area in males and females. Maturation occur at sea surface temperatures higher than 20°C, and spawning when temperature increases above 27°C.

Comparison of ovary histology between different-sized wild and pond-reared shrimp Litopenaeus vannamei (=Penaeus vannamei)

Summary Ovarian maturation of pond-reared and wild Litopenaeus vannamei (= Penaeus vannamei) broodstock was compared by analyzing morpho-histological characteristics. The number of spawns by each female was also considered to determine if successive rematurations can affect ovarian development. Individually tagged 42 wild and 47 pond-reared females were sampled at the end of a production period in a commercial hatchery. Morphological variables were recorded and ovaries were histologically evaluated for oocyte type and diameter. Although wild shrimp were bigger than pond-reared, no differences were seen in gonadosomatic indexes, either considered as a group or within stages of maturation. Differences between pond-reared and wild shrimp were observed in previtellogenic and early-vitellogenic oocyte distribution in vitellogenic and cortical stages. However, no differences were obtained in relation to the proportion of late vitellogenic oocytes and mature oocytes with cortical rods, or oocyte diameter in each...

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.

Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus

Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-D-glucosamine, α-D-glucose, α-D-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops.

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.

Reproductive indicators and gonad development of the Panama brief squid Lolliguncula panamensis (Berry 1911) in the Gulf of California, Mexico

ABSTRACT The reproductive biology of the Panama brief squid was evaluated using reproductive indicators, and histological and histochemical analyses. A total of 2,460 squid were analyzed, which were captured during 15 exploratory fishing surveys in the Gulf of California during 2003 to 2006 and 2008. Of the total sample, 61% were female, 15% were male, and the rest were undifferentiated. Based on the frequency of the gonad developmental stages, the largest number of mature females was identified in February and October, whereas mature males were found in April and August. This result coincided with the gonadosomatic index. According to the histological analyses, we characterized 4 stages of oogenesis: previtellogenesis, vitellogenesis, postvitellogenesis with mature oocytes, and postspawning. We also characterized 6 substages of oocyte and 2 structural indicators of spawning: oogonia (Po0), previtellogenic oocyte primary (Pol), previtellogenic oocyte secondary (Po2), vitellogenic primary oocyte (Vol), secondary vitellogenic oocyte (Vo2), postvitellogenic oocyte (Pvo), postovulatory follicles, and atresia. Oocyte size among types showed significant differences (P < 0.05). The presence of postovulatory follicles, and oocytes of different sizes and various developmental stages throughout the study period indicates that the Panama brief squid has a synchronous ovarian development with multiple spawning. The size at first maturity (L50) indicates that males (mantle length, 51 mm) mature at lengths shorter than females (mantle length, 85 mm).

Histological characterization of the spawning process in the catarina scallop, Argopecten ventricosus (Sowerby II, 1842) induced by thermal shock and serotonin injection

Summary The histology of the spawning catarina scallop Argopecten ventricosus was studied to characterize cellular and anatomical changes occurring along the gonad-kidney pathway. Broodstock were conditioned for 25–30 days and induced to spawn with two methods: thermal shock and serotonin by intracardiac injection. Samples of the kidney-gonad-digestive gland complex were fixed at the sperm and oocyte stages of gamete emission and at the end of spawning. Both induction methods elicited gametes, and no particular difference in the microscopic anatomy of the acini-kidney pathway was detected. However, serotonin injection, apart from producing a rough valve opening, sporadic movement (opening and closure of valve), and pronounced foot extension, induced gamete emission in less time than thermal shock. Primary gonad ducts opened to acini to permit sperm emission; at the same time, germinal vesicles broke down in female acini and meiosis restarted. Shortly afterward, oocytes gradually changed form from typically poly-hedral to a more malleable form. This probably contributed to oocyte transport through ciliary activity along the gonad and kidney ducts. Nucleoli disintegration, chromosome condensation, and appearance of meiotic figures were observed along the acini-kidney pathway. Although sperm and oocytes were sometimes observed in gonad or kidney ducts, self-fertilization was rarely observed.

BIOCHEMICAL AND HISTOCHEMICAL CHANGES ASSOCIATED WITH GONAD DEVELOPMENT OF THE CORTEZ GEODUCK, PANOPEA GLOBOSA (DALL 1898), FROM THE GULF OF CALIFORNIA, MEXICO

ABSTRACT Thirty adult organisms of Panopea globosa, an endemic geoduck species from Baja California, Mexico, were collected monthly during a maturation cycle (October through January). Histological and semiquantitative histochemical changes (total lipids and carbohydrates) were assessed in gonad tissue during the maturation process. Biochemical quantification of proteins, lipids, carbohydrates, vitellogenin, and vitellin was performed on hemolymph and gonad tissues of all specimens. The theoretical diameter of oocytes was also estimated from their surface area, measured in histological sections of gonads. Five distinct gonadal stages were identified in both male and female geoducks, with previtellogenic (PVt)/early spermatogenic (ES) gonads being observed in October. Spawning took place in December/January when the temperature reached a plateau of ∼18.5°C. Oocyte diameter varied between 25.5 µm(PVt stage) and 74.8 µm during the late vitellogenic stage (LVt). The percent of lipids and carbohydrates evaluated histochemically in females closely mirrored the gonadal stages, as well as the diameter. Quantitative changes in proteins, lipids, and carbohydrates in the hemolymph did not show a clear temporal pattern, yet the level of vitellogenin increased from 16.4 (PVt stage)—47.8 µg/mL (LVt stage) and correlated with the oocyte diameter. The weightspecific content of proteins, lipids, and carbohydrates in the gonads was significantly greater during PVt and ES than the other gonadal stages, and remained relatively constant thereafter. Vitellin content mirrored the evolution of each maturation stage and correlated significantly with oocyte diameter. Vitellogenin or vitellin can be used as a reliable quantitative index of gender and degree of gonadic maturity in females. Hemolymph can be obtained readily from the pericardial cavity without affecting the performance of the organism significantly, which further makes vitellogenin a valuable, nonlethal marker for this genus.

Seasonal reproduction of sailfish Istiophorus platypterus from the southeast Mexican Pacific

Abstract Quantitative histology was used to analyse the seasonal reproductive characteristics of the Istiophorus platypterus sailfish obtained from bycatch and recreational catches in the southeast Mexican Pacific. The analysis revealed five stages of ovarian development, eight kinds of oocytes, and one postovulation structure. The species shows an asynchronous ovary development and indeterminate fecundity. Gonadosomatic indices (GSI) indicated that sailfish have a reproductive period with peaks in September and October (mean monthly GSI=1.9 and 1.8). Females were more abundant during the second quarter, whereas males were more abundant in the third and fourth quarters. We found that the estimates of the frequency distribution of whole oocytes showed that this analysis may be useful as a tool to assess maturity. The estimated size at sexual maturity for this species in the Gulf of Tehuantepec was 150.2 cm eye–fork length.