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Ovarian morphology, oogenesis, and changes through the annual reproductive cycle of the female blue crab, Callinectis sa...

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Ovarian morphology, oogenesis, and changes through the annual reproductive cycle of the female blue crab, Callinectis sapidus Rathbun, in Tampa Bay
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English
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Brown, Catalina E
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University of South Florida
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Subjects / Keywords:
Germinal zone
Primary growth
Secondary growth
Atresia
Follicle cells
Dissertations, Academic -- Marine Science -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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ABSTRACT: The blue crab, Callinectes sapidus Rathbun, 1896, was studied because of its high dollar value to Florida's commercial and recreational fisheries. The purpose of this study was to describe the structure of the ovary and oogenesis in the blue crab and the morphological changes in the female reproductive developmental stages over time. Histological techniques for high-resolution light microscopy were used to determine sexual maturity of female blue crabs. The ovarian morphology, oogenesis, and changes through the annual reproductive cycle of blue crabs in Tampa Bay were investigated for a period of two years, from January 2005 to January 2007. Ovarian structure was assessed by analyzing histological sections embedded in plastic epoxy resin, which provided a higher resolution than any other embedding material previously used in research on blue crab reproduction.Qualitative analyses of female gonads were made by describing the structure of the oocytes and determining the developmental stage of the oocytes from oogonia to full-grown oocytes. This study developed and introduced a new reproductive staging criteria for the species. Morphological characteristics of ovarian tissues and oocytes were determined to develop a classification for oocyte maturation stages. Morphological changes in the oocytes are well defined, and these were used to develop the staging schema. In this study, it was found that carapace width is not a good indicator of maturity or developmental stage. Examination of the annual reproductive cycle indicates that late secondary growth occurs from July to March, and gravid crabs were found during November and December. Histological examination of ovarian tissue is essential for determining maturity in female blue crabs.By observing ovarian characteristics and by establishing the length of secondary growth during oogenesis in blue crabs of Tampa Bay, a more thorough understanding of the cyclic reproductive aspects of this species was obtained and specifically that animals at a carapace width between 100 mm and 125 mm may have mature oocytes, yet external features may not indicate that they are mature.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
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Includes bibliographical references.
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by Catalina E. Brown.
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Title from PDF of title page.
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Document formatted into pages; contains 48 pages.

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oclc - 567802468
usfldc doi - E14-SFE0003099
usfldc handle - e14.3099
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Ovarian Morphology,Oogenesis,and Changes through the Annual Reproductive Cycle of the Female Blue Crab, Callinectes sapidus Rathbun,in Tampa Bay by Catalina E. Brown A thesis submitted in partial fulllment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor:Norman Blake,Ph.D. David Mann,Ph.D. Maria del Carmen Uribe-Aranzabal,Ph.D. Harry Grier,Ph.D. Date of Approval: April 10,2009 Ke ywords:Germinal zone,Primary Growth,Secondary Growth,Atresia,Follicle Cells, Ovarian Lobe Copyright 2009,Catalina E. Brown

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DEDICATION To Alex F or your strength,dedication,support and love. For all the times you knew what to say, and everything you say is just right. Mostly for all the smiles and laughter. To Ariana F or your compassion,understanding,sweetness,and caring,loving words. You are a light of joy and a source of love and happiness always. To Erika F or your sense of humor and continual running commentary and curiosity on everything. I hope you learned something. I love you all,and I am so glad that we made it through this. We move on forward to the next adventure.

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A CKNOWLEDGMENTS I thank my committee members,Dr. Blake,Dr. Uribe-Aranzabal,Dr. Grier,and Dr. Mann,for their helpful suggestions and advice for improving the manuscript. I greatly appreciate the long hours and great efforts of Llyn French in her assistance and technical support for formatting and graphics. I thank the FWCFWRI Crustacean Fisheries group,especially Charles Crawford and Angie Machniak,for their assistance in collecting samples and Joe O'Hop for helpful statistical advice. I extend a special thanks to Noretta Perry and Yvonne Waters for all their histology knowledge.

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T ABLE OF CONTENTS List ofTables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ii List ofFigures . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 General Biology of the Female Blue Crab . . . . . . . . . . . . . . . .6 Exterior Structure of the Ovary . . . . . . . . . . . . . . . . . . . .6 Sexual Maturity . . . . . . . . . . . . . . . . . . . . . . . . . .7 Annual Reproductive Cycle . . . . . . . . . . . . . . . . . . . . .8 Ovary and Oogenesis . . . . . . . . . . . . . . . . . . . . . . . .9 Perinuclear Yolk Complex . . . . . . . . . . . . . . . . . . . . .11 Reproductive Characteristics of the Blue Crab . . . . . . . . . . . . .11 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . .12 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 General Morphometric Analyses . . . . . . . . . . . . . . . . . . .15 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Oocyte Descriptive Statistics . . . . . . . . . . . . . . . . . . . .16 Histological Structure of the Ovary . . . . . . . . . . . . . . . . . .16 Oocyte Staging Schema . . . . . . . . . . . . . . . . . . . . . .19 Oogonia . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Early Primary Growth . . . . . . . . . . . . . . . . . . . . . . .21 Late Primary Growth . . . . . . . . . . . . . . . . . . . . . . .21 Secondary Growth . . . . . . . . . . . . . . . . . . . . . . . .24 Full-Grown Oocytes . . . . . . . . . . . . . . . . . . . . . . . .26 Ovigerous Females . . . . . . . . . . . . . . . . . . . . . . . .27 Atresia . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 Annual Reproductive Cycle . . . . . . . . . . . . . . . . . . . . .29 Correlation of Carapace Width and Oocyte Diameter with Developmental Stages . . . . . . . . . . . . . . . . . . . . . . .30 Percentage of Females in Different Developmental Stages . . . . . . . . .32 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45 i

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LIST OF TABLES Ta ble 1.Blue crab landings in Florida for the years 2006 to 2008,with only preliminary data for 2008. . . . . . . . . . . . . . . . . .2 Ta ble 2.Blue crab taxonomy. . . . . . . . . . . . . . . . . . . . . .2 Ta ble 3.Macroscopic changes and staging of the ovaries of blue crab, Callinectes sapidus, during the reproductive cycle. . . . . . . . . .8 Ta ble 4.Morphological changes of the oocytes during oogenesis in the red frog crab, Ranina ranina. . . . . . . . . . . . . . . . . .10 Ta ble 5.Crab carapace texture scale according to the Crustacean Fisheries group,FWCFWRI. . . . . . . . . . . . . . . . .12 Ta ble 6.Conversion table showing objective size and conversion factor to change diameter into microns conversion factor. . . . . . . . .15 Ta ble 7.Descriptive statistics for oocyte diameter ranges. . . . . . . . . .16 Ta ble 8.Oocyte staging schema with developmental stages,cellular steps,and morphological characteristics. . . . . . . . . . . . .19 Ta ble 9.Oocyte developmental stages,showing oocyte diameter ranges in microns and carapace-width ranges in mm. . . . . . . . . . .20 Ta ble 10.Descriptive statistics for the average diameters of oogonia and oocytes by developmental stage. . . . . . . . . . . . . . . . .30 Ta ble 11.Comparison of oocyte diameter ranges between this study and the study by Lee et al. (1996). . . . . . . . . . . . . . . . . .38 ii

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LIST OF FIGURES Figure 1.Mature and immature female aprons. . . . . . . . . . . . . .3 Figure 2a.Ventral view of mature female blue crab. . . . . . . . . . . . .6 Figure 2b.Fertilized oocytes attach to the setae of the pleopods to form a large mass referred to as a spongein a female blue crab. . . . .6 Figure 3a.Dorsal ovary covering cardiac stomach. . . . . . . . . . . . .7 Figure 3b.Immature ovaries and immature seminal receptacles. . . . . . . .7 Figure 3c.Mature seminal receptacles. . . . . . . . . . . . . . . . . .7 Figure 3d.Mature ovary and medial connection. . . . . . . . . . . . . .7 Figure 4.Map of the Tampa Bay area showing sites where blue crabs were collected for this study. . . . . . . . . . . . . . . . .12 Figure 5.Carapace width (CW) and carapace length (CL). . . . . . . . .13 Figure 6a.Necropsy of the blue crab carapace to dissect ovaries. . . . . . .13 Figure 6b.Internal organs of blue crab,including mature ovaries. . . . . . .13 Figure 6c.Ovarian structure showing places where sections were obtained for histology. . . . . . . . . . . . . . . . . . . .13 Figure 7.Oocytes were measured only when the whole nucleus was visible. . . . . . . . . . . . . . . . . . . . . . . . . .15 Figure 8a.Ovarian lobe containing secondary-growth oocytes. . . . . . . .17 Figure 8b.Several ovarian lobes (OL) containing secondary-growth oocytes and germinal zones (GZ). . . . . . . . . . . . . . .17 Figure 9.Percentage of oocytes at a specic developmental stage inside ovarian lobes of four randomly chosen female blue crabs. . . . . . . . . . . . . . . . . . . . . . . . . .17 Figure 10a.Germinal zone (GZ) or germaria (PAS/MY). . . . . . . . . . .18 Fi gur e 10b.Germinal zone (GZ) or germaria in a mid-secondary growth oocyte (H&E). . . . . . . . . . . . . . . . . . . . . . .18 Figure 10c.Mid-secondary-growth oocytes in periphery (H&E). . . . . . . .18 Figure 10d.Late secondary-growth oocytes on periphery of ovarian lobe (PAS/MY). . . . . . . . . . . . . . . . . . . . . . . .18 Figure 11a.Germinal zone is not evident in ovarian lobe in primary growth (PAS/MY). . . . . . . . . . . . . . . . . . . . .18 Figure 11b.Dened germinal zone in late secondary growth ovarian lobe (PAS/MY). . . . . . . . . . . . . . . . . . . . . . . .18 iii

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Figure 12.Oogonia (OOG) with characteristic clear ooplasm,pale nucleus,and interphase chromosomes (IC) on periphery of ooplasm. . . . . . . . . . . . . . . . . . . . . . . . .20 Figure 13a.Early primary growth (ePG) cell (PAS/MY). . . . . . . . . . .21 Figure 13b.Early primary growth (ePG) cell (H&E). . . . . . . . . . . .21 Figure 14.Primary growth oocyte showing hemolymph (HL) and germinal zone (GZ),although not as evident. . . . . . . . . . .22 Fi gur e 15a.Perinuclear yolk complex (PNYC) (P AS/MY). . . . . . . . . .22 Figure 15b.Perinuclear yolk complex (PNYC) as seen in primary growth (H&E). . . . . . . . . . . . . . . . . . . . . . . . . .22 Figure 16a.Lampbrush chromosomes (LBC) and dispersing perinuclear yolk complex (DPNYC). . . . . . . . . . . . . . . . . . .23 Figure 16b.Lampbrush chromosomes (LBC),nucleus (n),and nucleolus (nu). . . . . . . . . . . . . . . . . . . . . . . . . . .23 Figure 17.Follicle cells nuclei (FCN) surrounding the follicles. . . . . . . .23 Figure 18.Somatic cells (SC),follicle cells nuclei (FCN),and basal lamina (BL). . . . . . . . . . . . . . . . . . . . . . .23 Figure 19a.Cortical alveoli (CA) in the periphery of the oocytes. . . . . . .24 Figure 19b.Cortical alveoli (CA),basal lamina (BL),and follicle cell nucleus (FCN). . . . . . . . . . . . . . . . . . . . . .24 Figure 20.Oil droplets (OD) appear as one of the last stages of late primary growth. . . . . . . . . . . . . . . . . . . . . .25 Figure 21.Early secondary growth stage. . . . . . . . . . . . . . . . .25 Figure 22a.Mid-secondary growth showing better-dened germinal zone and rapid formation of yolk globules. . . . . . . . . . . . . .26 Figure 22b.During mid-secondary growth (mSG),yolk globules (YG) begin to ll the ooplasm as development progresses. . . . . . . .26 Figure 23.Late secondary growth oocytes are bright pink,and yolk globules have reached maximum diameter. . . . . . . . . . . .26 Figure 24.Late secondary growth:large yolk globules (YG),somatic cells (SC),and basal lamina (BL). . . . . . . . . . . . . . .26 Figure 25a.Full-grown oocytes showing fused yolk globules (arrows) in oocytes at the periphery of the ovarian lobe. . . . . . . . . . .27 Figure 25b.Follicle cells (arrows) in full-grown oocytes. . . . . . . . . . .27 Figure 26.Mature fertilized egg in ovigerous female,showing yolk globules similar to those in full-grown oocytes. . . . . . . . . .27 Figure 27.Ovaries of ovigerous females in early and late primary growth. . . . . . . . . . . . . . . . . . . . . . . . . .27 Figure 28.Progress of fertilized egg from early division to almost fully developed (arrow). . . . . . . . . . . . . . . . . . . . .28 iv

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Figure 29a.Collapsing membranes among oocytes (arrows),a condition referred to as atresia or atypical oocytes. . . . . . . . . . . .28 Figure 29b.Atresia in oocytes,showing fusion and collapsing of ooplasm among oocytes (arrows). . . . . . . . . . . . . . . . . . .28 Figure 29c.Atresia in oocytes. . . . . . . . . . . . . . . . . . . . .28 Figure 30.Annual reproductive cycle of blue crabs in Tampa Bay. . . . . . .29 Figure 31.Field data showing how Tampa Bay blue crab females are scored for maturity and immaturity. . . . . . . . . . . . . .31 Figure 32.Histogram of the percentage of females in different developmental stages vs. carapace width. . . . . . . . . . . .32 v

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OV ARIAN MORPHOLOGY,OOGENESIS,AND CHANGESTHROUGHTHE ANNUAL REPRODUCTIVECYCLE OFTHEFEMALEBLUECRAB, CALLINECTES SAPIDUS RATHBUN,INTAMPABAY Catalina E. Brown ABSTRACT The blue crab, Callinectes sapidus Rathbun,1896,was studied because of its high dollar v alue to Floridas commercial and recreational sheries. The purpose of this study was to describe the structure of the ovary and oogenesis in the blue crab and the morphological changes in the female reproductive developmental stages over time. Histological techniques for high-resolution light microscopy were used to determine sexual maturity of female blue crabs. The ovarian morphology,oogenesis,and changes through the annual reproductive cycle of blue crabs in Tampa Bay were investigated for a period of two years,from January 2005 to January 2007. Ovarian structure was assessed by analyzing histological sections embedded in plastic epoxy resin,which provided a higher resolution than any other embedding material previously used in research on blue crab reproduction. Qualitative analyses of female gonads were made by describing the structure of the oocytes and determining the developmental stage of the oocytes from oogonia to full-grown oocytes. This study developed and introduced a new reproductive staging criteria for the species. Morphological characteristics of ovarian tissues and oocytes were determined to develop a classication for oocyte maturation stages. Morphological changes in the oocytes are well dened,and these were used to develop the staging schema. In this study,it was found that carapace width is not a good indicator of maturity or developmental stage. Examination of the annual reproductive cycle indicates that late secondary growth occurs from July to March,and gravid crabs were found during November and December. Histological examination of ovarian tissue is essential for determining maturity in female blue crabs. By observing ovarian characteristics and by establishing the length of secondary growth during oogenesis in blue crabs of Tampa Bay,a more thorough understanding of the cyclic reproductive aspects of this species w as obtained and specically that animals at a carapace width between 100 mm and 125 mm may have mature oocytes,yet external features may not indicate that they are mature. vi

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INTRODUCTION The study of the morphology and physiology of the reproductive systems is essential to dene the reproductive cycles of an animal species. It is established that reproduction is adapted to environmental conditions,in particular,temperature,photoperiod,food supply,and predators. Consequently,reproduction is cyclical and based on the season of the year in which conditions are adequate for the survival of the offspring. The foundation of reproduction for any species originates from the development of germinal cells during the process of gametogenesis,which is oogenesis in the females and spermatogenesis in the males. Therefore,the development of oocytes in the females and of spermatozoa in the males is a fundamental function of reproduction. Oogenesis is a complex process of cellular and molecular changes that occur during the formation, growth,and maturation of the female germinal cells. The development of the oocyte is remarkable,and the study of this process is essential to the understanding of reproduction. The determination of the annual activity of the ovaries of blue crabs, Callinectes sapidus Rathbun (Portunidae),and the characterization of the developmental stages of the oocyte,are fundamental for an efcient management of the blue crab shery. Blue crab sheries are signicant to the state of Florida and are an integral part of the state's economic and cultural livelihood. In 2007,commercial landings of blue crabs in Florida were estimated to be over 10,300,000 pounds (Florida Fish and Wildlife Conservation Commission,Fish and Wildlife Research Institute [FWCFWRI] Fisheries-Dependent Monitoring [FDM] data,20062008). Even though recreational sheries of blue crabs are not monitored,blue crabs also support a large recreational shery in the state. Statewide,Florida commercial blue crab landings in 2006 were 11,919,742 pounds, having a total value of $10,936,905. In 2007,commercial landings were 10,371,523 pounds,having an estimated value of $10,302,312. Unofcial landings data for 2008 show the total pounds of blue crabs in the Tampa Bay area to be about six million pounds (Table 1; FWCFWRI,FDM data,20062008). Blue crabs occupy a large geographical distribution and a diversity of habitats, ranging along the eastern seaboard of North America,throughout the Gulf of Mexico, into the Caribbean,and down to northern Argentina (Steele and Bert,1994) The blue crab life history entails a comprehensive cycle of planktonic,nektonic, and benthic stages that occur throughout the estuarine and nearshore marine environments,depending upon the specic physiological requirements of each lifehistory stage. Callinectes sapidus are classied as true crabs (Table 2). 1

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The blue crab produces large numbers of young,grows rapidly,and attains early sexual maturity. Offsetting their fecundity are high mortality rates and a short life span (Steele,1982). Three types of migration have been described:alimental,climatic,and gametic. Gametic migration involves reproduction and spawning (Steele,1982). Blue crabs have a divided life cycle dened by migration,in which the female blue crab are catadromous and migrate from hyposaline waters (<35) to higher salinity waters to spawn and hatch their eggs. The high salinity of oceanic waters not only serves as habitat for the spawning female but also aids in the development of the larvae,improves dispersal capabilities, reduces osmoregulatory stress,and decreases predation (Steele,1982). The distribution of the species varies depending mostly on season,sex,and age. Adult blue crabs inhabit a range of bottom types including fresh,estuarine,and shallow oceanic waters. Large blue crabs are prevalent in larger bays and bayous (Oesterling, 1976). Differences in salinity,temperature,and habitat are among the factors that affect blue crab distribution and abundance (Steele and Bert,1994). Although adult blue crabs are ubiquitous throughout an estuarine system,they are distributed seasonally with 2 Ta ble 1. Blue crab landings in Florida for the years 2006 to 2008,with only preliminary data for 2008. (FWCFWRI,2008) Ta ble 2. Blue crab taxonomy. (Zinski,2006)

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respect to salinity and sex. In general,males are more prevalent in low-salinity areas, whereas females predominate in high-salinity areas (Oesterling,1976). Some studies have shown that blue crab populations are cyclic,and their abundance can uctuate dramatically from year to year. These inter-annual uctuations in abundance are thought to be due to physical,chemical,and biological factors that strongly inuence the characteristics of the populations (Steele and Bert,1994). Blue crabs are heterosexual and exhibit distinct sexual dimorphism. Female blue crabs look distinctly different from male blue crabs. Macroscopic examination of the animals shows that females have red pinchers,and males have blue pinchers. Male and females are also easily distinguishable by the shape of their aprons. The male blue crab has the same shape apron throughout its life. The female's apron changes from a narrow shape to a broader,wider apron as she become sexually mature (Fig. 1). Hermaphroditic blue crabs have been observed in Chesapeake Bay,Virginia (Johnson,1980). 3 Figure 1. Mature and immature female aprons. Mature females have a broader apron than immature females. Females attain sexual maturity in 12 to 14 months,reaching adult size at approximately 130139 mm or 5.255.50 in. after 1820 molts. When they molt at maturity,growth ends; consequently,it is known as the "terminal molt"(FWCFWRI, 2006). Females mate only once,but males may mate numerous times. Mating takes place in brackish water after the female's terminal molt. Unlike most marine organisms,blue crab mating and spawning (shedding of eggs) occur at different times (Steele,1982). The sperm is stored in the female's body for up to a year and so will be accessible for successive spawnings,even though mating for the female is a one-time occurrence. She may spawn one to nine months after mating,depending on water temperatures. After mating,females migrate to high-salinity waters in lower estuaries,sounds,and nearshore spawning areas (FWCFWRI,2006). In brachyurans,the spermatophores are deposited directly into the seminal receptacle of the female. After entering her pubertal molt,the female blue crab is carried by the male. Directly following the molting of the cuticula,known as ecdysis,copulation occurs. Unlike blue crabs,females of other brachyurans undergo more than one molt and thus successive mating (Johnson,1980).

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4 F ollowing the pubertal-molt mating,the female enters a prolonged period without ecdysis known as anecdysis and normally will not molt or mate again. Sperm are stored in the seminal receptacles and used during both ovulations that normally occur in this species (Steele and Bert,1994). Eggs are laid and held together by adhesion to the setae of the endopodites of the abdominal segments and the maturing egg mass (Johnson,1980). A precise analysis of the microscopic structure of the ovaries provides a better comprehension of the development of the female germ cell,the oocyte during the process of oogenesis,and its cyclical changes that dene the reproduction of this species. Oogenesis is the sequence of stages that oocytes undergo,from the oogonium to oocyte maturation. To properly describe the process of oogenesis,several essential aspects need to be studied:the examination of the germinal zone of the ovary,in order to distinguish where the oogonia are located; and the identication of the process through which oogonia give rise to the oocytes,including the cellular morphological characteristics during the different stages of development,from early primary growth to late secondary growth. The development of the oocyte involves active and complex increases of ooplasm,and the deposit of abundant nutrients enclosed in structures such as yolk platelets and lipid droplets in the ooplasm during a precise sequence of changes. Morphological changes in the ooplasm that occur during this process aid in identifying the stages of the oocytes throughout oogenesis and thus the phase of the reproductive cy cle of the specimen. This maturation sequence is the basis of the analyses of reproduction in the species and can be identied and quantied through histological e xamination.

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5 OBJECTIVES Characterize the structure of the ovary and its morphological changes in blue crab populations of Tampa Bay. Identify and describe the morphological characteristics of the germ cells during the process of oogenesis and particularly the germinal zone of the ovary,providing an accurate description of the oocytes by using histological techniques. Dene the annual development and seasonal changes of the oocytes in blue crab populations of the Tampa Bay area. Introduce a histology-based staging schema for oocyte developmental stages in blue crabs. Correlate oocyte developmental stages to carapace width.

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Exterior Structure of the Ovary The ovaries lie dorsal to the hepatopancreas and extend on both sides of the anterior margin of the body cavity to the cephalothorax. The hepatopancreas and the ovaries are intermingled along the anterior marginal dorsal portion of the carapace (Fig. 3a). The ov aries continue down towards the cardiac stomach in a posteromedial course. Then they 6 LITERATUREREVIEW Several authors have studied the reproductive characteristics of females of several species of decapoda and have described with macroscopical and microscopical observations the condition of the ovaries at sexual maturity,the annual reproductive cycles,and the morphology of the ovary and the process of oogenesis. General Biology of the Female Blue Crab Female blue crabs have four pairs of pleopods on abdominal segments 2 through 5. The rst coxa of a pleopod is connected to the body by a soft and exible articulating membrane. The coxa is undersized and barely calcied; but the next article,the basis,is large and harder. Two rami,the exopod and endopod,arise from the basis. (Fig. 2a). The oocytes pass through the seminal receptacles,where they are fertilized as they move out from the ovaries. The fertilized oocytes are then expelled from the gonopores and attach to the setae of the pleopods to form a large,cohesive mass or spongethat remains attached to the female until they hatch. This constitutes an ovigerous female (Fig. 2b). Figure 2a. Ventral view of mature female blue crab. Figure 2b. Fertilized oocytes attach to the setae of the pleopods to form a large mass referred to as a spongein a female blue crab.

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are attached by a medial connection that joins the left and right ovaries at the level of the medial stomach. The ovary continues downward toward the posterior stomach and cover it. The ovaries show several gross stages as they go from immature to mature stages. The immature ovary resembles a small pink lament. At both distal regions of the oviduct are the seminal receptacles. These are contained in a hard,chitinous wall. The seminal receptacles are positioned laterally to the midline between the stomach and heart along the posterolateral border of the stomach. The size and color of the seminal receptacles va ry depending on the maturity level of the ovary. Seminal receptacles change from small,thin,white,and membranous disks (Fig. 3b) to large,hard,and pink rounded spheres (Fig. 3c). As the ovary matures,the seminal receptacles shrink and the ovaries become thicker. The ovaries change from the light,soft pink of the immature ovary to the bright orange of the mature ovary (Fig. 3d). Sexual Maturity In Chesapeake Bay,female blue crabs reach sexual maturity after 1820 post-larval molts,at the age of 1 to 1.5 years (Williams,1965). Mature condition of the female blue crabs that have completed the pubertal molt is determined by examining the semicircular abdomen (Mense and Wenner,1989; Steele and Bert,1994). In stone crabs, Menippe mercenaria, mature condition of the ovaries was determined by gross analysis of the 7 Figure 3a. Dorsal ovary covering cardiac stomach. Figure 3b. Immature ov aries and immature seminal receptacles. Figure 3c. Mature seminal receptacles. Figure 3d. Mature ovary and medial connection.

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organ and by establishing a color-coded staging system based upon the color of the ov aries (Gerhart and Bert,2008). This color-coded system includes physiologically mature females as having orange gonads,which determined that the female was gravid. In blue crabs,it has been observed that size at maturity is inconsistent. Tagatz (1968) noted that maturity of blue crabs could be attained at various widths. Tagatz (1968) recorded a mature female with a carapace width of 99 mm and an immature female with a carapace width of 177 mm. It was noted that overlap in size ranges of immature and mature female blue crabs was considerable. Fischler (1959) recorded the smallest adult female blue crab in an ovigerous condition with a carapace width of 55 mm,off the North Carolina coast. It was not established whether these dwarf forms of C. sapidus normally occurred in the areas in which they were collected,or whether they were the result of genetic or environmental f actors inuencing growth and thereby causing dwarsm (Fischler,1959). In contrast, Steele and Bert (1994) found that the size at sexual maturity shows no obvious latitudinal va riation; most female blue crabs attain sexual maturity within the carapace width range of 130139 mm. In addition,it has been suggested that variations in temperature may affect the development of mature oocytes. Steele and Bert (1994) found that there is a virtual cessation of spawning by females in July. This observation suggests that midsummer water temperatures produce physiological stress that inhibits reproduction. Annual Reproductive Cycle Hard (1942) described the macroscopic aspects of the ovaries in the blue crab during the annual reproductive cycle,dividing the process into ve stages (Table 3). 8 Ta ble 3. Macroscopic changes and staging of the ovaries of blue crab, Callinectes sapidus, during the reproductive cycle. (Hard,1942)

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Perry (1975) described characteristics of the reproductive cycle in female blue crabs with developing ovaries and recently mated females during the spring,summer,and f all. Females with mature ovaries occurred throughout the year,and berried"females were present in March and April. It was noted that females in the area spawned when w ater temperatures began to rise in the spring. Females of Po rtunus trituberculatus (Hamasaki et al.,2006) were shown to have vitellogenic oocytes from October to December,and their developmental stage did not change over the winter from December to March. Blue crab females from Chesapeake Bay showed arrested vitellogenesis during the winter months. Crabs collected in the same locality for histological examination during the winter months showed that no progression of oocyte development occurred during those months (Johnson,1980). In the paddle crab, Ovalipes catharus (Armstrong,1988),it was observed that the percentage of females with moderately developed to well-developed ovaries increased steadily from March to October. Females with well-developed ovaries appeared in September. After the females with well-developed ovaries were collected in October, there was an increase of females in early stages of oocyte development. This would indicate that females of the paddle crab undergo a period of oogenesis over the winter months followed by spawning in the spring (Armstrong,1988). Minagawa et al. (1993) found that in the Izu islands of Japan,spawning females of the red frog crab, Ranina ranina, occurred only during the months of May to September. No oocytes were observed to develop beyond the primary yolk platelet stage during December and January. From August to November,most oocytes remained in stages younger than the secondary growth stage. Minagawa et al. (1993) divided oocyte development into stages based on the morphological characteristics of each stage. Sastry (1983) indicated that the duration of reproductive cycles may differ in populations of the same species of crabs according to environmental conditions. Swiney and Shirley (2001) found that females of the dungeness crab, Cancer magister, spawned ev ery two years in high-altitude populations in Alaska. Swiney and Shirley (2001) observed that the crab females do not extrude eggs annually,but could extrude eggs biannually. Other authors propose that adding specic hormones brings about changes in the process of oocyte development. In the freshwater rice eld crab, Oziotelphusa senex senex, Nagaraju et al. (2006) induced ovarian growth and vitellogenesis with the introduction of hormones,suggesting that vitellogenesis is a process in which female crabs store nutrients for subsequent use by developing oocytes. Ovary and Oogenesis Minagawa et al. (1993) divided the process of oogenesis in Ranina ranina into ve main stages and ten substages based on the morphology of the oocytes. They described the process of proliferation as occurring in the oogonia and all of the following stages occurring in the oocytes (Table 4). 9

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Lee et al. (1996) compared the levels of vitellin-immunoreactive proteins with the developmental changes in relation to vitellogenesis of the blue crab and established ve staging levels. These are as follows: Stage 1:Immature females. In this stage,the ovary was composed of previtellogenic oocytes with scant basophilic ooplasm,with a mean oocyte diameter of 1624 m. A layer of prefollicle cells enclosed the oocytes. Stage 2:Females recently completing pubertal molt. This stage is characterized by the presence of swollen seminal receptacles in the oviduct of the females. The previtellogenic oocyte diameter ranged 3060 m. The ooplasm was intensely basophilic and contained a distinctive perinuclear yolk complex. Prefollicle cells migrating to the lobule periphery appeared to be in the process of surrounding the oocytes. Stage 3:Early vitellogenesis. During this stage,the mean oocyte diameter ranged 66100 m. The perinuclear yolk complex had disappeared from the ooplasm, which now contained eosinophilic yolk bodies. The previtellogenic oocytes in stage 2 were found in the central regions of the ovarian lobes. Stage 4:Mid-vitellogenesis. During this stage,the mean diameter of the oocytes was 103160 m. The ooplasm was full of eosinophilic yolk bodies. Stage 5:Late vitellogenesis. During this stage,the mean oocyte diameter was 168288 m. Similar to stage 4; a layer of attened follicle cells enclosed the oocytes. The ooplasm was full of eosinophilic yolk bodies. 10 Ta ble 4. Morphological changes of the oocytes during oogenesis in the red frog crab, Ranina ranina. (Minagawa et al.,1993)

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Perinuclear Yolk Complex During primary growth,a body known as the perinuclear yolk complex becomes apparent proximal to the nucleus. Several authors have described the perinuclear yolk complex in different species,including sh (Glsoy,2007) and hermit crabs (Komm and Hinsch,2005). The perinuclear yolk complex of blue crabs has not been described in detail. Johnsons (1980) description of this structure in blue crabs is limited; she only refers to it as a single large spherical inclusion that is PAS positive. In the trout Oncorhynchus mykiss, Glsoy (2007) describes an inclusion that appears during primary growth in the basophilic cytoplasm of the trout. This inclusion is further portrayed as an area with the appearance of loosely aggregated particles that begins to emerge around the nucleus during primary growth. Glsoy (2007) explains that this structure may be called perinuclear yolk body in different articles,but that the structure is not yolk; yet this term has widely been used for a long time in sh literature. Similarly,Nayyar (1964) agrees that authors throughout the literature have diverse views regarding the origin and the morphology of the perinuclear yolk complex,but that the majority of authors believe it to be related in some way with yolk formation; hence the term yolk nucleushas been universally accepted. Azevedo (1984) explains that this dense material consists mainly of ne granulo-brillar components and seems to be a common structure in germinal cells within different animal groups. Nayyar (1964) similarly illustrates the perinuclear yolk complex. In his study,the author describes the process of the yolk nucleus appearing as a mass of lipid granules and mitochondria located at rst beside the nucleus. As the perinuclear yolk complex becomes spherical,it migrates to the peripheral region of the c ytoplasm to lie just below the cell membrane (Nayyar,1964). Nayyar (1964) compared this structure in eight species of sh,and no differences were found in the morphology of the yolk nucleus among these species. Reproductive Characteristics of the Blue Crab Florida blue crabs mate from March to December when water temperatures exceed 22C. Female blue crabs mate once in their lifetime following the last,or terminal,molt (Steele, 1982). In Chesapeake Bay,blue crabs mate from May to October (Millikin and Williams, 1984),occurring regularly in areas with low salinity such as upper estuaries or lower portions of rivers (Tagatz,1968). According to Hard (1942),female blue crabs in Chesapeake Bay ovulate twice after maturation and attain sexual maturity in 12 months at a size of 5 inches. During years with longer and colder winters,the spawning season was signicantly shorter (Daugherty,1952). In the Mississippi region,a project carried out by the Gulf Coast Research Laboratory studied the distribution and abundance of blue crabs in 1975. During this project,they collected and examined mature blue crab females. They found that spawning of blue crabs in northern gulf waters is protracted and that egg-bearing females occur in coastal and estuarine waters in the spring,summer,and fall (Perry,1975). 11

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12 Ta ble 5. Crab carapace texture scale according to the Crustacean Fisheries group,FWCFWRI. (C. Crawford,personal communication,2007) MATERIALS AND METHODS A total of 74 females were collected monthly from January 2005 to December 2006 from the Tampa Bay area (Fig. 4). Blue crabs were harvested following collection protocols described in Steele and Bert (1994). For the duration of the study,temperatures were recorded at collection times. Mean temperatures (four measurements per month) in the Tampa bay area during these months ranged 20¡28¡C. Temperature ranges showed little variation. Blue crab carapace texture was rated according to the subjective scale described in the protocol that the FWCFWRI Crustacean Fisheries group uses for their data (Table 5). Figure 4. Map of the Tampa Bay area showing sites where blue crabs were collected for this study.

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13 Live crabs were transported to the FWCFWRI histology lab where they were placed on ice for 20 minutes for anesthetizing prior to dissection. All crabs were weighed on a Denver Instruments¨scientic balance,model 4100,to the nearest 0.1 g. Carapace width (CW) was dened as the distance between the anterior lateral spine and the mostposterior lateral spine (Fig. 5). Carapace length (CL) was dened as the distance between the centers of the frontal interorbital carapace margin and the posterior margin. Carapace width and length were measured to the nearest millimeter (mm). Crabs with missing limbs,broken carapaces,or any signs of disease were not used. Histology Dissection followed methods described in Johnson (1980). The rst carapace cut was made from the dorsal articulation above the right posterior leg,anterior to the frontal margin at the head. A second cut was made parallel to the rst cut,on the left side. The lateral cuts were then joined by a transverse cut at the posterior margin of the carapace (Fig. 6a). The blue crab ovaries are located covering the cardiac stomach and under the spines,intermingled with the hepatopancreas. Gills are located to the side of the ovaries Figure 5. Carapace width (CW) and carapace length (CL). Figure 6a. Necropsy of the blue crab carapace to dissect ovaries. CW CL

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14 (Fig. 6b). Ovaries were removed from the abdominal cavity and sectioned for histology as anterior,mid,and posterior regions. Each section was processed in order to identify the reproductive stage of the ovary (Fig. 6c). Ovarian samples were placed in a xative solution of 5% paraformaldehyde (PFMA) 0.1 molar phosphate buffer (Humason,1972) for 24 hours (Appendix,Table A-1). F ollowing xation,the ovaries were thoroughly rinsed in tap water for an hour and placed in 70% ethanol inside scintillation vials. The dehydration process continued with 95% ethanol and an inltration progression to 100% fresh glycol methacrylate resin, JB-4¨resin (Appendix,Table A-2). Ovaries were then embedded in JB-4¨, an epoxy resin distributed by Electron Microscopy Sciences. Embedded tissue was sectioned at 4-m thickness on a Leica¨RM 2165 microtome using a 9-mm glass knife. A minimum separation of 60 m (the approximate maximum diameter of an oocyte) was made between sections. The sections were mounted on Fisher¨slides that are pretreated with an acid-cleaned 0.1% HCl solution. (Appendix,Table A-3). Three slides per tissue sample were made. One was stained with hematoxylin and eosin (H&E) (Appendix,Table A-4). A second slide was stained with periodic acid Schiff's/metanil yellow (PAS/MY) (Quintero-Hunter et al., 1991; Appendix,Table A-5). A third slide was kept unstained. Stained sections were e xamined at a total magnication of 1001000 on an Olympus¨compound microscope. Each ovary was assigned to a reproductive stage following a classication scheme based on oocyte diameter ranges and oocyte morphological characteristics. Power of the test for a two-sample t-test comparison was used to determine how many oocytes needed to be measured per ovarian lobe to get an accurate mean and va riance. Only oocytes sectioned through the nucleus were measured (Armstrong,1988) (Fig. 7). Some samples had fewer cells inside the ovarian lobe; in that case,all the cells that had a fully viewable nucleus were counted. Figure 6b. Internal organs of blue crab,including mature ovaries. Figure 6c. Ovarian structure showing places where sections were obtained for histology. Anterior (a), mid (m),and posterior (p) sections were dissected in order to quantify that the ovary was all in the same developmental stage.

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15 Using an Olympus¨BH2 model teaching microscope,oocyte diameters were measured to the nearest m with an ocular micrometer. Then with the calibration of 2mm divisions into units of 100,the conversion per magnication was obtained (Table 6). Photomicrographs of sections illustrating the classication criteria were made using the Olympus¨V anox-T AH-2 camera. General Morphometric Analyses Blue crabs were weighed and measured in the eld,and the results were analyzed by linear regression methods after log transformation of body weight and carapace width. Linear regressions were used to derive the parameters of the power curve. Because there w as a signicant relationship between body weight and carapace width,analysis of covariance (ANCOVA; Zar,1996) was used to examine the relationship of body weight and reproductive stage (determined from histological samples from female blue crab gonads) using carapace width (log-transformed) as the covariate. The covariate,carapace width,adjusts the observations of body weight (log-transformed) for the average body size of crabs in the samples and allows the comparison of body weight versus the oocyte developmental stages dened in this study. Ta ble 6. Conversion table showing objective size and conversion factor to change diameter into microns conversion factor. Figure 7. Oocytes were measured only when the whole nucleus was visible.

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16 Histological Structure of the Ovary The ovary is contained inside units called ovarian lobes or ovarian pouches (Fig. 8a,b). Ando and Makioka (1999) used the later nomenclature. The ovarian lobes encompass the oocytes. Most oocytes inside the ovarian lobes are at about the same developmental stage. Oogenesis is a continuous process in which oocytes undergo development from primary growth to secondary growth in a somewhat rapid fashion. Therefore,the percentage of oocytes inside an ovarian lobe at a specic developmental stage can correspond with the occurrence of another developmental stage and overlap at roughly 75% to 25% of each stage. Some lobes are congured with two main stages,but they may also have a small percentage of a third stage in them. Therefore,an ovarian lobe that is mainly in mid-secondary growth (75%) can have some oocytes in primary growth (23%) and a few oocytes representing the early primary growth stage (2%). Development RESULTS Oocyte Descriptive Statistics Descriptive statistics for the average diameters of oogonia and oocytes by developmental stage are shown in Table 7. Fifty-ve cells were measured to obtain a meaningful average of oocyte size range. Condence intervals are tight,and a reasonable difference in means w as detected. Ta ble 7. Descriptive statistics for oocyte diameter ranges.

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17 is a uid process and oogonia are always present,but they may not always be evident. Early primary growth oocytes are also always present because along with oogonia,they compose the germinal zone,and all ovarian lobes have a germinal zone. Therefore,there can be more than one developmental stage inside one ovarian lobe,but mostly the ov arian lobes are in the same developmental stage per crab (Fig. 9). The structure of the blue crab ovary consists of oocytes that develop from the center of the ovarian lobe to the periphery as oogenesis advances. This distribution of germinal cells in the ovarian lobes is characterized by the developmental progress of cells Figure 8a. Ovarian lobe containing secondary-growth oocytes. GZ = germinal zone. Figure 8b. Several ovarian lobes (OL) containing secondary-growth oocytes and germinal zones (GZ). Figure 9. Percentage of oocytes at a specic developmental stage inside ovarian lobes of four randomly chosen female blue crabs. This gure illustrates that oocytes at different stages of development can be found inside a specimens ov arian lobe. OOG = oogonia,lSG = late secondary growth, emSG = early mid-secondary growth,lPG = late primary growth,ePG = early primary growth. GZ } GZ } oocyte OL

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18 from oogonia,which are found in a central germinal zone also known as a germaria (Fig. 10a,b),to late secondary growth stage,which are found in the periphery of the ovarian lobes (Fig. 10c,d). The germinal zone is a row of germinal cells at the center of the ovary. During primary growth,there are no well-dened germinal zones and oogonia are difcult to recognize (Fig. 11a). In secondary growth,the germinal zones are well-dened and consist mostly of primary growth oocytes. Germinal zones provide new cells for continual development of oocytes as cells mature when they reach the periphery (Fig. 11b). Figure 10a. Germinal zone (GZ) or germaria (PAS/MY). Figure 10b. Germinal zone (GZ) or germaria in a mid-secondary growth oocyte (H&E). Figure 10c. Mid-secondary-growth oocytes in periphery (H&E). Figure 10d. Late secondary-growth oocytes on periphery of ovarian lobe (PAS/MY). Figure 11a. Germinal zone is not evident in ov arian lobe in primary growth (PAS/MY). Figure 11b. Dened germinal zone in late secondary growth ovarian lobe (PAS/MY). GZ GZ GZ

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19 Ta ble 8. Oocyte staging schema with developmental stages,cellular steps,and morphological characteristics. (n = 59) Oocyte Staging Schema A new staging schema is introduced in this study. This schema is based on morphological changes that occur through development. The oocyte staging schema is detailed in Table 8. Oocyte developmental stages and carapace-width diameters are compared and summarized in Table 9.

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20 Ta ble 9. Oocyte developmental stages,showing oocyte diameter ranges in microns and carapace-width ranges in mm. (n = 36) Oogonia Oogonia are the female germinal cells,small oval cells found in the germinal zone. These cells cannot be classied as oocytes because oogonia are capable of dividing via mitosis to form other oogonia or via meiosis to form an oocyte. They are diploid cells in which meiosis has not started. Oogonia have a characteristic scant,very clear ooplasm. The nucleus is pale,and a single nucleolus is prominent (Fig. 12). Oogonia appear to have interphase chromosomes prior to commencement of meiosis. Interphase chromosomes are found on the periphery of the cell. Basal lamina is evident in the germinal zone where oogonia are found. Oogonia range in diameter 912 m. Figure 12. Oogonia (OOG) with characteristic clear ooplasm,pale nucleus,and interphase chromosomes (IC) on periphery of ooplasm. GZ = germinal zone,BL = basal lamina. BL }OOG IC GZ

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21 Figure 13a. Early primary growth (ePG) cell (PAS/MY). Figure 13b. Early primary growth (ePG) cell (H&E). Early Primary Growth Early primary growth oocytes have begun meiosis,and therefore the cell has become a primary oocyte. The appearance of the early primary growth oocytes is similar to oogonia; however,the ooplasm is characterized by ooplasmic basophilia (Fig. 13a). When the ooplasm changes from the clear,scant appearance that it had during oogonia to a blue hue,that is the indication that the oogonia is now in early primary growth (Fig. 13b). These cells are larger than oogonia,ranging 1550 m,and can no longer divide via mitosis again. Therefore,the early primary growth oocyte is an oocyte and remains as such throughout the rest of its development,until full-grown. Oocytes,unlike oogonia, are diploid cells in meiosis that have duplicated chromosomes. Late Primary Growth The beginning of late primary growth is characterized by gradual basophilia of the ooplasm and by the absence of yolk. The basophilic ooplasm indicates that the cell is active with production of organelles,which increase the volume of the ooplasm,another marker for this stage. Oocyte diameter range for this stage is 51114 m. Throughout late primary growth,the formation of organelles such as mitochondria,Golgi complexes,and abundant quantities of endoplasmic reticulum, ribosome,and fragmented glycogen may contribute to the blue staining of the ooplasm. F or example,ribosomes are basophilic organelles that have a chemical attraction for basic stains,like hematoxylin stains,which give the cell its unique blue hue, characteristic of late primary growth. During this stage,the germinal zone is not as e vident as it will be in more developed oocytes,yet the germinal zone with the oogonia ePG ePG ePG

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22 Figure 15a. Perinuclear yolk complex (PNYC) (P AS/MY). Figure 14. Primary growth oocyte showing hemolymph (HL) and germinal zone (GZ),although not as e vident. Figure 15b. Perinuclear yolk complex (PNYC) as seen in primary growth (H&E). and the early primary growth oocytes is always present. Hemolymph is evident,and follicular cells and somatic cells are also present but not as obvious (Fig. 14). In the course of the beginning stages of late primary growth stage,a distinct PAS positive body is present in the ooplasm,usually near the nucleus (Fig. 15a,b). The PAS positive body,also known as the perinuclear yolk complex,is only evident during the early stages of late primary growth,and it disperses before the cortical alveoli can be observed. Although the biochemical properties of this body were not analyzed in this study, its description is similar to the description given for the inclusion called the perinuclear yolk complex by other authors (Nayyar,1964; Komm and Hinsch,2005; GŸlsoy,2007). The function of the perinuclear yolk complex may be to assist in assembling the proteins that later form the yolk globules. The PAS positive body is a cellular structure that will organize the yolk. The perinuclear yolk complex disappears as the oocyte develops and growth continues into the next stage. The PAS positive body disperses its contents into the ooplasm as late primary growth continues. Once the perinuclear yolk complex has dispersed,laments similar in appearance to lampbrush chromosomes are observed in the nucleus of blue crab oocytes. These structures are an indication that the oocyte is in arrested meiosis (Fig. 16a,b). HL GZ PNYC PNYC

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23 Figure 16a. Lampbrush chromosomes (LBC) and dispersing perinuclear yolk complex (DPNYC). Figure 16b. Lampbrush chromosomes (LBC), nucleus (n),and nucleolus (nu). Figure 17. Follicle cells nuclei (FCN) surrounding the follicles. Figure 18. Somatic cells (SC), follicle cells nuclei (FCN),and basal lamina (BL). In late primary growth,somatic cells and follicular cells are evident. These cells do not disappear during secondary growth and have always been present,but it is at the end of late primary growth and at the beginning of early secondary growth that follicular cells,basal lamina,and somatic cells become more evident. Follicle cells are distinguishable because of their elongated nucleus,and they can be found surrounding the oocytes. Follicle cells surround the oocyte,the follicle,and are found in the germinal compartment. The elongated nucleus of the follicle cells is more apparent in the later stages of development (Fig. 17). Somatic cells are found (Fig. 18) outside the basal lamina and within the germinal compartment. Somatic cells can become follicle cells,but until they are not found in the LBC nu n LBC DPNYC FCN SC SC FCN BL

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24 Figure 19a. Cortical alveoli (CA) in the periphery of the oocytes. Figure 19b. Cortical alveoli (CA),basal lamina (BL),and follicle cell nucleus (FCN). germinal compartment and surround the follicle,they are not described as follicle cells b ut only as somatic cells. Basal lamina is the structure located at the base of all epithelium and acts as a barrier between connective tissue and epithelium. Basal lamina had not been described in past studies of blue crabs. Cortical alveoli are another morphological characteristic that emerge during late primary growth,and they occur immediately prior to the onset of secondary growth. At times,formation of cortical alveoli and the formation of yolk may proceed simultaneously in the latest stages of late primary growth. The cortical alveoli have never been described in studies of blue crabs. Although this study did not include a biochemical analysis,the staining procedures indicate that these may be cortical alveoli because they are PAS positive,and cortical alveoli contain polysaccharide components and protein components that would give them a strong purple hue (H. Grier,personal communication). The cortical alveoli are synthesized by the oocyte and become visible in the perimeter of the ooplasm. When cortical alveoli appear,they remain for the duration of development. This is the only morphological feature that was found in late primary growth and throughout secondary growth (Fig. 19a,b). During the nal stages of late primary growth,there are increasing numbers of lipid droplets in the ooplasm. The oil droplets are identied in tissue that has been inltrated as vacuoles in the tissue. The lipids that compose these oil droplets dissipate during the inltration process with ethanol. Oil droplets are also evident during early secondary growth but not midor late secondary growth (Fig. 20). Secondary Growth Secondary growth,or vitellogenesis,begins with the inclusion of yolk globules in the ooplasm. This initialization of yolk proteins in the ooplasm indicates that the oocyte has moved into secondary growth. Secondary growth was divided into 3 steps:early,mid-, CA CA BL FCN

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25 Figure 21. Early secondary growth stage. Ooplasm becomes less basophilic,and yolk begins to form. Figure 20. Oil droplets (OD) appear as one of the last stages of late primary growth. Oocytes moving uidly from one developmental stage to the next can show several morphological characteristics,including perinuclear yolk complex (PNYC),oil droplets,and yolk (Y) formation. FCN = follicle cell nucleus; EV = early vitellogenic oocyte. and late secondary growth. Each stage was based upon the size of oocyte diameter,yolk globules,and variableness of the ooplasm,such as the polymorphism of yolk globules. When secondary growth begins,the ooplasm loses the basophilic characteristics, becoming clearer in appearance and staining less blue/purple and more a lighter pink (Fig. 21). In early to mid-secondary growth,oocyte diameter range was 115150 m and the yolk globules size range was 3365 m. During early secondary growth,the PAS positive body or perinuclear yolk complex that was apparent in primary growth disappears; however,the follicle cells still surround the oocyte During mid-secondary growth,the yolk globules are more evident in the ooplasm as they grow larger. There is also a well-differentiated germinal zone that contains only primary growth oocytes (Fig. 22a,b). During late secondary growth,the ooplasm stains a bright pink,and the germinal zone is evident. When the yolk globules reach a maximum diameter,the oocyte is in late secondary growth. Vitelline becomes more uid and lighter in color. Large yolk globules coalesce,and the ooplasm has a more homogenous consistency (Fig. 23). Near the germinal zone,several other cellular structures can be observed. Somatic cells are found outside the basal lamina and the germinal compartment,and follicle cells are found within the germinal compartment. Follicle cells surround the oocytes,and their nuclei are clearly seen around oocytes. Somatic cells can become follicle cells; but while they are found outside the germinal compartment,they are described as somatic cells. A basal lamina is also present. This structure is located at the base of the epithelium, separating the connective tissue from the epithelium and acting as a barrier (Fig. 24). FCN Y OD PNYC EV

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Figure 23. Late secondary growth oocytes are bright pink,and yolk globules have reached maximum diameter. Figure 24. Late secondary growth:large yolk globules (YG),somatic cells (SC),and basal lamina (BL). 26 Figure 22a. Mid-secondary growth showing a better-dened germinal zone GZ) and rapid formation of yolk globules. Figure 22b. During mid-secondary growth (mSG),yolk globules (YG) begin to ll the ooplasm as development progresses. Full-Grown Oocytes Full-grown oocytes have reached their maximum size. Most of the ooplasm retains a bright-pink stain. With the absence of large accumulation of cell organelles,the tissue now has a greater afnity for acid stains,and as a result,eosin stains them pink. Yolk becomes fused in full-grown oocytes (Fig. 25a). These oocytes are found mostly on the perimeter of the ovarian lobe at rst; then when development continues,the whole ov arian lobe will appear with fused yolk oocytes. When full-grown,oocytes retain follicle cells,and yolk platelets are at their maximum size of 95 m (Fig. 25b). YG mSG GZ YG BL SC

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27 Figure 25a. Full-grown oocytes showing fused yolk globules (arrows) in oocytes at the periphery of the ovarian lobe. Figure 25b. Follicle cells (arrows) in fullgrown oocytes. Figure 26. Mature fertilized egg in o vigerous female,showing yolk globules similar to those in full-grown oocytes. Figure 27. Ovaries of ovigerous females in early and late primary growth. GZ = germinal zone. Ovigerous Females After the oocytes are full-grown,they mature and are fertilized. This signies the end of oogenesis. The oocyte reactivates meiosis. The appearance of yolk in the mature fertilized egg is very similar to the appearance of the full-grown oocyte (Fig. 26). Fertilized eggs make up the sponge. The ovaries of ovigerous females were found to always be in primary growth (Fig. 27). The fertilized egg mass itself or the sponge area can be described as having fertilized eggs in various stages of development that may range from an early division stage to almost fully developed (Fig. 28). Atresia Some of the oocytes that were studied presented a condition similar to what has been described as atypical oocytes or atresia (Fig. 29a). The atresia stage is characterized by GZ

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28 Figure 28. Progress of fertilized egg from early division to almost fully developed (arrow). Figure 29a. Collapsing membranes among oocytes (arrows),a condition referred to as atresia or atypical oocytes. Figure 29b. Atresia in oocytes,showing fusion and collapsing of ooplasm among oocytes (arrows). e vidence of oocyte disintegration. Atresia was observed in small females with a carapace width usually less than 100 mm. During atresia,oocytes showed a lack of structural integrity,with a tendency for oocyte membranes to fall apart and appear to fuse between oocytes. Oocytes undergoing atresia exhibit shrinking of the ooplasm,folding or collapsing of the oocyte membranes (Fig. 29b),and fusion of nucleus (Fig. 29c). The oocyte structure degenerates to form one continuous mass. This step was found only in samples from crabs measuring less than 100 mm CW and only during early primary growth. Figure 29c. Atresia in oocytes.

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29 Figure 30. Annual reproductive cycle of blue crabs in Tampa Bay. SPG = sponged or ovigerous female,lSG = late secondary growth,emSG = early mid-secondary growth,lPG = late primary growth,ePG = early primary growth,OOG = oogonia, JUV = Juvenile. Annual Reproductive Cycle T ampa Bay blue crabs with oocytes in the late secondary growth stage occurred more frequently from December to March than at any other time of the year. Of nine samples collected in December,seven had late vitellogenic oocytes,one was in midvitellogenesis,and one was a sponged female. Oocytes in the late vitellogenesis step were also observed in specimens collected during July,August,and September. From April to June,oocytes in most females were undergoing primary growth or early vitellogenesis,and no oocytes in the late vitellogenesis stage were observed in any of the specimens. The only sponged females collected were found in November and December. Blue crabs with oocytes in early primary growth and juvenile crabs were found mostly during the months of March,May,and June (Fig. 30); however,sample size restricts this conclusion. While sponged crabs were found only in November and December,it is possible that sponged crabs may be found at other times of the year. A larger sample size w ould be needed to determine whether ovigerous females are present in other months of the year. During this study,no evidence was found of blue crabs in a state of arrested development (Johnson,1980) during the winter months. Temperature ranges in the sampling locations were not extreme,and subtropical regions such as Tampa Bay may provide favorable environmental conditions that support year-round production of eggs. Therefore,the annual reproductive cycle of the blue crab in the Tampa Bay area may differ from the annual cycle of the blue crabs in locations where water temperature ranges are cooler during winter months. Tampa Bay area blue crabs appeared to have a reproductive cycle that is continuous throughout the year.

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30 Ta ble 10. Descriptive statistics for the average diameters of oogonia and oocytes by developmental stage. Correlation of Carapace Width and Oocyte Diameter with Developmental Stages The average oocyte diameter per month for female blue crabs in the Tampa Bay area had a large variance,as would be expected (Table 10). During this study,crabs in all developmental stages were found during most of the year. Therefore,there is a large standard deviation each month,which signies that the center of mass is spread wider about the mean. The relationships between carapace width (CW) and oocyte diameters with gonad developmental stages were examined. Carapace width varied for blue crabs in different gonadal stages and was not clearly correlated with the progression in gonadal development. Although size (e.g.,CW) is likely to be related to reproductive output (e.g., number of eggs produced),size alone may not be a good indicator of sexual maturity or developmental stage in blue crabs. The average diameters of oocytes appeared to be more strongly correlated to gonad developmental stage from oocyte primary growth through nal oocyte maturation (OM). A slight trend of increasing carapace width from primary growth to early and mid-secondary growth was observed,but the sample sizes were too small to make any denite statements on this relationship if it exists. Blue crabs in later stages of gonad development were sometimes smaller than those in earlier stages of development,and it is probable that carapace width alone is not the best indicator of maturity or developmental stage in blue crabs.

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31 Figure 31. Field data showing how Tampa Bay blue crab females are scored for maturity and immaturity. Field data shows that 88% of females at 130 mm CW are externally mature,and most females do not show external sexual maturity characteristics until they reach 125 mm CW (Fig. 31). There is a range from 80 mm CW to 120 mm CW in which females are scored as being immature but may be internally mature. Twelve percent of females under 130 mm CW were scored as mature,and one percent of females over 130 mm CW were scored as immature based on external characteristics,but they are most likely mature internally.

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Percentage of Females in Different Developmental Stages During this study,specimens at any size were found in different developmental stages (Fig. 32). Females of 90124 mm CW included immature and mature individuals. Immature individuals were characterized by having no obvious oogonia,oogonia only,or oocytes in the preprimary growth stage. Individuals that were scored as mature (even if they had not yet undergone the molt to the adult instar) showed oocytes undergoing primary growth or secondary growth (early to late). Animals with the larger carapace widths had ovaries in any of the developmental stages from primary growth to late secondary growth. 32 Figure 32. Histogram of the percentage of females in different developmental stages vs. carapace width. lSG = late secondary growth,emSG = early mid-secondary growth,lPG = late primary growth,ePG = early primary growth,OOG = oogonia.

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33 DISCUSSION One of the most signicant processes in the reproductive biology of any animal species is the development of oogonia into mature oocytes. Current investigations into oocyte maturation have classied the process into discrete stages based upon microscopic characteristics,from oogonia to mature oocytes. The characteristics of the ovary of blue crabs have not been extensively analyzed using modern histological methods (Johnson, 1980). No previous assessment of blue crab oocyte development using glycol methacrylate embedding resin JB-4¨, the embedding medium used during this study,has been published. This embedding technique allows the tissue to be sectioned at 4 m, providing much better resolution of the cells with minimal artifact or distortion. Furthermore,the inltration process itself is less damaging to the tissue than the traditional parafn inltration process. Cells do not collapse,shrink,or swell during processing as they do when other embedding techniques are used. Overall,using the glycol methacrylate resin JB-4¨embedding technique increases clarity and resolution,reduces distortions,and provides a higher-quality histological section that presents much more cellular detail than tissue processed by traditional parafn methods. Using this embedding technique,the reproductive cycle and the ov arian morphology of the blue crab in the Tampa bay area can be better depicted. F or this research,the process of oocyte maturation in blue crabs is described and a classication system for stages is developed to best categorize the morphological changes occurring throughout the maturation process of the oocytes. The morphological characteristics of blue crab oogenesis have never been depicted as well and with such high denition as they have for this study. Because of these advanced techniques,an updated oocyte development schema for blue crabs is introduced. In blue crabs,oocytes are contained within an ovarian lobe. The connective tissue that surrounds the ovarian lobe and retains all the oocytes inside is visible. As oocytes mature,they move to the periphery of the ovarian lobe,and only oogonia and early primary growth oocytes are found closer to the center of the ovarian lobe. This area, where the younger stages originate,is known as the germinal zone or germaria,which is represented as specialized germ cell areas. In a study by Hinsch (1972),it was observed that in the spider crab, Libinia emarginata, the oogonia and primary growth oocytes were found in central regions of the ovary. In the freshwater crab, Po tamon dehaani, when the oogonia reach about 20 m in diameter,they move out of the germinal zone. Ando and Makioka (1999) noted that in P. dehaani, the oogonia are not enclosed in the same regions where larger oocytes are found. Ando and Makioka (1999) observed that oocytes larger than 100 m in diameter are enclosed within their own regions. The authors call these regions the oogenetic lobes.

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Hence,in P. dehaani, there are large oogenetic lobes that contain only mature eggs (Ando and Makioka,1999). In contrast,during this study it was noted that blue crab oocyte stages are only partially synchronous because cells are in different developmental stages,and all can be found in the same histological section. Different developmental stages were found within one ovarian lobe. Contained by the ovarian lobes,a large percent of the oocytes are all in a comparable developmental stage (Fig. 8a). Ovarian lobes were noted to have two main oocyte developmental stages strongly represented; for e xample,mid-vitellogenic and primary growth,or late vitellogenic and mid-vitellogenic. W ith the more mature stage germinal cells encompassing the majority of the oocytes present,the younger developmental stage oocytes are represented in smaller percentages of the total oocyte count. Oogenesis is a uid process,and the different developmental stages of oocytes inside the ovarian lobes correspond to the different times in the annual reproductive cycle in which the crab will spawn. The younger oocytes,occupying less of the total percentage of the ovarian lobe,will eventually develop to occupy most of the ov arian lobe as they become mature. Similarly,Lee et al. (1996) found that germ cells of va rious developmental stages were present simultaneously within the ovaries of blue crabs. However,for this reason,when Lee et al. (1996) measured the oocytes,they measured only the most developmentally advanced oocytes within that ovary. For the study of blue crabs in Tampa Bay,in order to decide the number of oocytes to be measured,power of the test calculations for two-sample t-test was used. It was determined that 55 oocytes within the ovarian lobe needed to be measured to obtain a meaningful average of oocyte size range. In blue crabs,oogonia and early primary growth oocytes are found in a germinal zone located in the center of the ovary. Ovarian lobes that contained mostly late vitellogenic oocytes had a distinct germinal zone assembled by primary growth oocytes. Therefore,ovarian lobes with secondary growth oocytes were noted to have an evident germinal zone. This germinal zone in the center of the ovarian lobe contains the late vitellogenic oocytes,and it consists of oocytes in an earlier stage of maturation. The germinal zone characteristic of ovarian lobes in late secondary growth,made up mainly of primary growth oocytes,ranged 4050 m in oocyte diameter. The germinal zone is made up of oogonia and oocytes in early primary growth (Fig. 10a). In the freshwater crab, P. dehaani, Ando and Makioka (1999) recognized a similar germinal zone,which they called germaria."In P. dehaani, Ando and Makioka (1999) noticed that germaria are located in an ovarian epithelium. In contrast,blue crabs did not appear to have a separate epithelium,but rather the germinal zone is found in the center of the ovarian lobe,and the germinal zone contained the oogonia and early preprimary growth. In the freshwater crab, P. dehaani, oogonia were dened as being basophilic and smaller than 20 m in diameter. In blue crabs,oogonia have a scant ooplasm that is clear and range 912 m in diameter. The oogonia of blue crabs have interphase chromosomes in the periphery. Basal lamina can also be observed near the oogonia (Fig. 12). Ando and Makioka (1999) observed that in P. dehaani, the oogonia are found only in the germaria,which in 34

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turn is found inside an ovarian epithelium. This similarity is shared by the freshwater crab, P. dehaani, and blue crabs. In both species,the oogonia are noted to be found only inside the germinal zone. However,in blue crabs,early primary growth oocytes were also found in the germinal zone,not just oogonia. This is why the germinal zone is less e vident during the early primary growth stage (Fig. 11a). Unlike the freshwater crab, P. dehaani, no ovarian epithelium was evident in the blue crab. Basal lamina was evident in blue crabs (Fig. 24). The advanced histological techniques used during this project provided outstanding resolution. The tissues obtained corroborated the results described by Johnson (1980),that oogonia in blue crabs are found in the germinal zone. Furthermore,this study advances to show that not only oogonia but also primary growth oocytes are in the germinal zone. The improved resolution that comes from using glycol methacrylate resin to embed the tissue provides e vidence that oogonia are even more scant and smaller than described by Johnson (1980). The interphase chromosomes of oogonia were also observed. Early and late primary growth oocytes in blue crabs are characterized by c ytological changes in the ooplasm. Primary growth oocytes have a basophilic ooplasm. As oogonia develop into primary growth oocytes,there is an increase in basophilia (see staging schema,Table 8). Throughout development,one nucleus and one nucleolus are present (Fig. 16b). These are particularly evident during primary growth. A perinuclear yolk complex,also known as the PAS positive body,appears during primary growth (Fig. 15a,b). This structure is observed as one of the early stages of late primary growth. This perinuclear yolk complex,or PAS positive body,aids in the future production of yolk. When it is present in the primary growth oocyte,it is assembled of organelles,not vitelline. The PAS positive bodies are noted rst near the nucleus and later disappear by dispersing in the ooplasm as the oocyte develops further (Fig. 15a). With high-resolution light microscopy,the elements in this structure can be described as containing polysaccharides because it is PAS positive. In the course of primary growth,the nucleus appears to have a lamentous characteristic. These laments suggest that there may be lampbrush chromosomes in the nucleus (Fig. 16a),which would indicate that an oocyte with this structure might be in arrested meiosis I. This aspect of development needs to be studied further by using EM and comparing xatives. F ollicle cells and somatic cells are always present in the oocyte; however,they are more apparent during the later stages of development. Nuclei of follicle cells can be observed surrounding the oocytes and are found inside the germinal compartment (Fig. 17). This relationship between the follicle cells and the oocyte is evident throughout the developmental process. Follicle cells do not disappear during secondary growth. After o vulation,the follicle is broken and the oocyte comes out of the follicle cells that surround it. The follicle cells remain in the ovarian lobe,and the oocyte comes out of the ov arian lobe,where it is fertilized and embryonic development begins. Therefore,the embryo is not surrounded by the follicle cells that are seen enclosing the oocytes prior to fertilization. Ando and Makioka (1999) did not observe true follicle cells surrounding the 35

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oocytes and eggs throughout oogenesis in the freshwater crab, P. dehaani. In the spider crab, L. emarginata, Hinsch (1972) identies follicular cells surrounding the oocytes as they differentiated. Throughout development,the encompassing follicular cells become located on the external boundaries in the spider crab (Hinsch,1972). Lee et al. (1996),using parafn preparations,also observed that the blue crab ov arian lobes contained oocytes and that a layer of follicle cells enclosed these lobes,but that follicle cells were rarely found surrounding individual oocytes. It is possible that because this study used JB-4¨epoxy resin and therefore obtained better resolution,the follicle cells surrounding the oocytes became apparent; and that Lee et al. (1996) were not able to distinguish the follicle cells because of the lower resolution achieved with methods using parafn. Somatic cells are located outside the germinal compartment and do not surround the follicle. Basal lamina acts as a barrier from the connective tissue. (Fig. 18). Cortical alveoli appear in the periphery of the oocyte in late primary growth and remain through development (Fig. 19a,b). Cortical alveoli have not been described in studies of blue crabs. The empty vesicles that had contained lipids become evident during the last phases of late primary growth. Lipids are lost during the inltration processes used for histological tissue preparation because the 70% and 95% ethanol dissolve the lipids. Therefore,the vesicles that had contained the lipids appear microscopically as empty vesicles (Fig. 20). In blue crabs,the lipid droplets can still be observed during the period between early secondary growth and late primary growth; they are a uid feature that progresses from one stage to the next and then disperses. These empty vesicles are conrmation that lipids were formed. This observation is concurrent with Ando and Makioka's (1999) description of small oil droplets occurring in the ooplasm around the germinal vesicles of the largest previtellogenic oocytes seen during their study. Secondary growth is a clear and evident process involving morphological changes in the oocyte. During secondary growth in blue crabs,the ooplasm shows a signicant decrease in basophilia. Oocytes increase in size because of a considerable increase in the v olume of yolk globules. Yolk globules begin to appear in one hemisphere of the oocyte (see Fig. 21),and as the yolk continues to grow,the oocyte becomes more acidophilic. As development continues,the yolk globules increase in volume (see Fig. 22a). Yolk globule size is the main distinguishing difference between early to mid-vitellogenic and late vitellogenic oocytes. This process coincides with the observations made by Ando and Makioka (1999) of the freshwater crab, P. dehaani, where it was noted that ne yolk granules appear rst at the periphery of the ooplasm of the earliest vitellogenic oocytes. Then yolk granules increase in size and coalesce to become larger and ll the ooplasm. Hinsch (1972) noted that as vitellogenesis progresses in the spider crab, L. emarginata, there is a large amount of endoplasmic reticulum in the vitellogenic oocyte (Hinsch, 1972). In blue crabs during late secondary growth,the ooplasm is acidophilic. During secondary growth,the germinal zone becomes more evident. Ovarian lobes with oocytes in the late secondary growth stage have a signicant germinal zone that comprises primary growth oocytes and oogonia (Fig. 8b). 36

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As the oocyte develops into full growth,the ooplasm is less basophilic and becomes more acidophilic and more uid. The appearance of a full-growth oocyte is then similar to the appearance of the ovulated eggs (Fig. 26). In this study,it was observed that some oocytes had ooplasms and cellular membranes that fused. This fusion of ooplasm and cellular membranes most likely represents atresia,a state in which the cellular material of the oocyte is being resorbed by the ovary (Fig. 29c). Atresia or atypical oocytes were observed in small females of less than 100 mm CW,and some atresia was observed in the ovarian lobes of sponged crabs (post-spawning) During atresia,oocytes are characterized by a lack of structural integrity,with a tendency for oocyte membranes to fall apart and appear to fuse between oocytes. Fusion of ooplasm and folding of membranes was typical of this condition (Fig. 29b). Lee et al. (1996) also found atypical oocytes in the blue crabs. In their study,Lee et al. (1996) referred to oocytes experiencing atresia as atypical oocytes and described them as lacking distinct structures such as nuclei and yolk bodies. The atypical oocytes or atretic oocytes seen during this study were mostly in primary growth; therefore,no disintegration of yolk globules was observed. Lee et al. (1996) assigned each oocyte to a level from one to six based on its maturation. This classication system was valuable to this analysis to establish a base line. However,there are considerable size differences that arise from using different xatives and from using parafn instead of JB-4¨. The diameter ranges found in this study corroborate that because of the better resolution,more detail can be distinguished and smaller cells can be measured and described. Maturation stages for this study were based on the diameter ranges of the 55 oocytes per ovarian lobe. This sample size was based on the power of the test calculations,which concluded that measuring 55 oocytes w ould provide an accurate mean and an accurate variance. Lee et al. (1996) established that in the blue crabs they analyzed,ovaries in immature females contained oocytes 1624 m wide. In this study,the comparable stage is oogonia,which ranged 912 m in diameter. For stage 2,Lee et al. (1996) had blue crab oocyte diameter ranges of 3060 m. In this study,the early primary growth oocytes had a diameter range of 1550 m. In primary growth,the diameter ranges in this study were 51144 m,which is comparable to Lee et al. (1996) stage-three diameters ranging 66100 m. Early to mid-secondary growth oocytes in this study ranged 115150 m wide. There was no stage in Lee et al.'s (1996) study that was comparable to the early to mid-secondary growth stage found in the Tampa Bay blue crabs. It is possible that because of the higher resolution and the different embedding procedures used in this study,the early to mid-vitellogenic stage was more apparent than it would have been using the parafn histology used by Lee et al. (1996). This study has secondary growth oocytes measuring 151357 m,and Lee et al. (1996) observed oocyte ranges in this stage of 168288 m. Lee et al. (1996) noted that the appearance of the most advanced oocytes of the post-spawning females was similar to the oocytes in early developmental stages (Table 11). During this study,it was also noted that females that carried fertilized e ggs in a sponge had primary growth oocytes in the ovarian lobes. This is an important 37

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feature because when determining sexual maturity based on carapace width,females that have had a sponge will have ovarian lobes in the primary growth developmental stage and yet they are sexually mature. In this study,a new staging schema is introduced (Table 8). This is a very important contribution because the staging schemas used for blue crabs either are not based on histology or lack morphological detail. In recent publications,less than one paragraph is dedicated to the internal structure of the female blue crab,and these are based on descriptions from staging schemas done by Hard (1942). It is important to have a consistent staging schema for the species. Data needs to be compared from one publication to the next based on a common staging schema. Staging schemas based on gross morphology are not detailed enough and do not provide the information necessary to detect whether the oocytes have truly entered into secondary growth or whether they are still immature. Histology provides the only means to determine oocyte developmental stages. External features of blue crabs in the Tampa Bay area are not clear indicators of the degree of gonadal maturity; for example,a large carapace width does not absolutely correlate to mature gonads (Table 9). Gonads in blue crab females may mature prior to the external features that are used as an indicator of sexual maturity. Using size alone to determine maturity would only provide a window of when the gonads are expected to be mature. Carapace width can overlap at each stage,providing an unreliable indicator of gonad stage (Fig. 31). Female crabs over 100 mm CW can be developing at different rates and therefore have ovarian lobes in different developmental stages. Sexual maturity should be determined by histological staging of the gonads. Additionally,the percentage of mature females in the different carapace-width ranges further suggests that carapace width is not a good indicator of gonadal development. Some females with smaller carapace widths in the 94124-mm CW range were found to be vitellogenic (Fig. 32). 38 Ta ble 11. Comparison of oocyte diameter ranges between this study and the study by Lee et al. (1996).

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Crabs with the larger carapace widths had ovaries in any of the developmental stages from primary growth to late secondary growth (Table 9). There are three main implications of these ndings. First,carapace width alone is not an adequate indicator of maturity. Second,some females in the 90124-mm CW range that had not yet molted into the adult instar showed obvious oocyte maturation and thus should be considered as maturing in analyses of size-at-maturity. Third,maturation must be determined histologically if female blue crabs have not yet molted into the adult instar. Some females with larger carapace widths were found to have ovarian lobes in earlier developmental stages. This result concurs with the nding that the ovaries in o vigerous females were in primary growth. Female blue crabs may begin a new developmental cycle after a batch of eggs has been fertilized,and that is why oocytes in larger females are found to be in early developmental stages. Ovigerous females with oocytes in primary growth were found not only in this study but also in studies by Lee et al. (1996) and Hinsch (1972). T ampa Bay blue crabs with oocytes in the late secondary growth stage occurred more frequently from December to March than at any other time of the year (see Fig. 30). Oocytes in the late secondary growth stage were also observed in specimens collected during July,August,and September. October was the only month during this study in which no vitellogenic crabs were observed. During April to June,oocytes in most females were undergoing primary growth or early secondary growth,and no oocytes in the late secondary growth stage were observed in any of the specimens. The only sponged females collected were found in November and December. Blue crabs with oocytes in early primary growth and juvenile crabs were found mostly during March, May,and June. Although sponged crabs were found only in November and December, sponged crabs may be found at other times of the year. A larger sample size would be useful in determining whether ovigerous females are present in other months of the year. There was no evidence that blue crabs in the Tampa Bay area may undergo a state of arrested development during the winter months (Johnson,1980). Temperature ranges in the sampling locations did not uctuate to extremes,and subtropical regions such as T ampa Bay provide favorable environmental conditions that allow year-round production of oocytes. The annual reproductive cycle of blue crabs in the Tampa Bay area may differ from the annual cycle of blue crabs in locations where water temperature ranges are cooler. As other studies corroborate (Steele and Bert,1994),Tampa Bay area blue crabs have a reproductive cycle that is continuous throughout the year. 39

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40 CONCLUSION This study gives a signicantly detailed description of the ovarian structure of the species. In blue crab,the ovaries are enclosed within distinct connective tissue that makes up the ovarian lobes. Inside these units,all oocytes are in a similar developmental stage. Ovarian pouches have a germinal zone,also referred to as the germaria,which is located at or around the center of the ovarian pouches and which contains the oogonia and the early primary growth oocytes. Oocytes in more advanced developmental states move to the periphery of the ovarian pouch. This distribution of cells from the center of the ov arian pouch to the periphery illustrates the advance of oogenesis. A new staging schema for the species is introduced with this study and is based on the histological morphology of the ovaries. The new staging schema,based on highresolution light microscopy,is important for the analysis of data sets that involve oogenesis. Developmental stages of blue crabs in this study progress from oogonia to primary growth,which is divided into early primary growth and late primary growth,and then to secondary growth. Secondary growth consists of early secondary growth to midsecondary growth and late secondary growth. Oogonia were studied and found to be clear with scant ooplasm and with interphase chromosomes in the periphery. Primary growth involves the formation of a perinuclear yolk complex (i.e.,PAS positive body),which disperses its content into the ooplasm. After the perinuclear yolk complex disperses, observable morphological characteristics such as lampbrush chromosomes,cortical alveoli,and lipids deposit are noted. Therefore,during primary growth,the oocyte is basophilic,and vesicles containing oil droplets were seen as clear (i.e.,empty,because of the slide preparation process) vesicles. In the nucleus of primary growth oocytes, laments were observed. These laments are indicative of lampbrush chromosomes and could signify that preprimary growth oocytes have entered into arrested meiosis I. The developmental stages continue into secondary growth (vitellogenesis),the onset of which is signied by the formation of yolk globules. During secondary growth, oocytes become more acidophilic and yolk globules increase in volume and numbers. Full-grown oocytes have a uid ooplasm where the yolk globules have coalesced. After fertilization,the female crab carries fertilized eggs in a sponge outside her abdomen. While a female blue crab carries the sponge eggs,ovaries have only primary growth oocytes. Throughout development,follicular cells within the ovaries can be observed. They have an elongated nucleus and surround the oocytes. These cells do not multiply; they remain as a single layer of squamous cells throughout oogenesis. Basal lamina and somatic cells are also described. Neither of these cells had previously been described in studies of the species. It was noted that follicle cells are always inside the germinal compartment and that somatic cells are outside the germinal compartment. This study

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41 corroborates studies of different species of the family Portunidae,that the germinal zone is found in the center of the ovarian lobe and that the oocytes,as they develop,move outwards to the periphery of the ovarian lobe. A concise description of the process of oogenesis by describing development from preprimary growth to full-grown is given. The reproductive cycle of the blue crab in the Tampa Bay area showed that female blue crabs undergoing late secondary growth were found mostly from December to March. Blue crabs in the Tampa Bay area have secondary growth oocytes during the winter months,and spawning occurs from winter to summer. Some specimens in the late secondary growth stage were also observed during July,August,and September. During the April to June,no females undergoing late secondary growth were observed. Most of the females during those months were undergoing primary growth or early secondary growth. The only sponged females collected were found in November and December. Blue crabs with oocytes in early primary growth and juvenile crabs were noted mostly during the March,May,and June. Blue crabs in the Tampa bay area do not have an arrested secondary growth (vitellogenesis) during the winter months. Not all females in the population are at the same developmental stage,and therefore there is a wide spread between oocyte diameters per month,as expected. The carapace width of female blue crabs in the different developmental stages overlapped and was not a good indicator of maturity or developmental stage. Females that exhibit immature external features could show internal features of maturity. It is known in published studies of the species that blue crabs reach adult size at 130139 mm CW. However,this study nds that blue crabs with a smaller carapace width can be undergoing secondary growth. Furthermore, animals with large carapace widths were found that they could be in all developmental stages. Some females in the 90124-mm CW range that had not yet molted in the adult instar showed obvious oocyte maturation and thus should be considered as maturing in analyses of size at maturity. Size at maturity of female blue crabs should be determined by histological means if the female has not yet molted into the adult instar.

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42 REFERENCES Ando,H.,and T. Makioka. 1999. Structure of the ovary and mode of oogenesis in a freshwater crab Po tamon dehaani. Journal of Morphology 239(1):107114. Armstrong,J. H. 1988. Reproduction in the paddle crab Ovalipes catharus (Decapoda: Portunidae) from Blueskin Bay,Otago,New Zealand. New Zealand Journal of Marine and Freshwater Research 22:529536. Azevedo,C. 1984. Development and ultrastructural autoradiographic studies of nucleolus-like bodies (nuages) in oocytes of a viviparous teleost ( Xiphophorus hellen ). Cell & Tissue Research 238:121128. Daugherty,F. M.,Jr. 1952. The blue crab investigation,19491950. Texas Journal of Science 4:7784. Fischler,K. J. 1959. Occurrence of extremely small ovigerous crabs ( Callinectes sp.) in coastal North Carolina. Ecology 40(4):720. Florida Fish and Wildlife Conservation Commission,Fish and Wildlife Research Institute. 2006. The life cycle of a blue crab in Florida. Available at http://research.myfwc.com/features/view_article.asp?id=25786. Florida Fish and Wildlife Conservation Commission,Fish and Wildlife Research Institute. 2008. Commercial sheries landings in Florida. Available at http://research.myfwc.com/features/view_article.asp?id=19224. Gerhart,S. D.,and T. M. Bert. 2008. Life-history aspects of stone crabs (Genus Menippe ):size at maturity,growth,and age. Journal of Crustacean Biology 28(2): 252261. GŸlsoy,N. 2007. Development of the yolk nucleus of previtellogenic oocytes in rainbow trout, Oncorhynchus mykiss, studied by light microscopy. Journal of Applied Biological Sciences 1(2):3335. Hamasaki,K.,K. Fukunaga,and S. Kitada. 2006. Batch fecundity of the swimming crab Po rtunus trituberculatus (Brachyura:Portunidae). Aquaculture 253:359365.

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43 Hard,W. L. 1942. Ovarian growth and ovulation in the mature blue crab, Callinectes sapidus Rathbun. Maryland Department of Natural Resources,Board of Research and Education Publication 46:117. Hayat,M.A. 1981. Fixation for Electron Microscopy. Academic Press,New York. 500 p. Hinsch,G. W. 1972. Some factors controlling reproduction in the spider crab, Libinia emarginata. Biological Bulletin 143:358366. Humason,G. L. 1972. Animal Tissue Techniques. 3rd ed. W. H. Freeman,San Francisco, California. 641 p. Johnson,P. T. 1980. Histology of the Blue Crab, Callinectes sapidus, a Model for the Decapoda. Praeger Publishers,New York. 440 p. K omm,B. S.,and G. W. Hinsch. 2005. Oogenesis in the terrestrial hermit crab, Coenobita clypeatus (Decapoda:Anomura):II. Vitellogenesis. Journal of Morphology 192(3):269277. Lee,C.-Y.,H. R. Umphrey,R. D. Watson. 1996. Developmental changes in the level of vitellin-immunoreactive proteins in hemolymph and tissues of the blue crab Callinectes sapidus: relation to vitellogenesis. Journal of Crustacean Biology 16(1):19. Mense,D. J.,and E. L. Wenner. 1989. Distribution and abundance of early life history stages of the blue crab, Callinectes sapidus, in tidal marsh creeks near Charleston,South Carolina. Estuaries 12:157168. Millikin,M. R.,and A. B. Williams. 1984. Synopsis of biological data on the blue crab, Callinectes sapidus Rathbun. FAO Fisheries Synopsis No. 138,NOAA Technical Report NMF 51:138. Minagawa,M.,J.-R. Chiu,F. Kudo,F. Ito,and F. Takashima. 1993. Female reproductive biology and oocyte development of the red frog crab, Ranina ranina, off Hachijojima,Izu Islands,Japan. Marine Biology 115(4):613623. Nagaraju,G. P. C.,P. R. Reddy,and P. S. Reddy. 2006. In vitro methyl farnesoate secretion by mandibular organs isolated from different molt and reproductive stages of the crab Oziotelphusa senex senex. Fisheries Science 72 (2):410-414. Nayyar,R. P. 1964. The yolk nucleus of sh oocytes. Quarterly Journal of Microscopical Science 105(3):353358.

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44 Oesterling,M. J. 1976. Reproduction,growth and migration of blue crabs along Floridas Gulf coast. State University System of Florida Sea Grant. Report SUSF-SG-76003. 19 p. Perry,H. M. 1975. The blue crab shery in Mississippi. Gulf Research Reports 5(1): 39. Quintero-Hunter,I.,H. G. Grier,and M. Muscato. 1991. Enhancement of histological detail using metanil yellow as counterstain in periodic acid Schiff's hematoxylin staining of glycol methacrylate tissue sections. Biotechnic & Histochemistry 66(4):169. Sastry,A. N. 1983. Ecological aspects of reproduction. Pp. 179 in F. J. Vernberg and W. B. Vernberg,eds. The Biology of Crustacea. Environmental Adaptations. Academic Press,New York. Steele,P. 1982. A synopsis of the biology of the blue crab Callinectes sapidus Rathbun in Florida. Pp. 29 in H. M. Perry and W. A. Van Engel,eds. Proceedings of the blue crab colloquium,October 16,1979,Biloxi,Mississippi. Gulf States Marine Fisheries Commission Information Series No. 7. Steele,P.,and T. Bert. 1994. Population ecology of the blue crab, Callinectes sapidus Rathbun,in a subtropical estuary:population structure,aspects of reproduction, and habitat partitioning. Florida Marine Research Publications No. 51. 24 p. Swiney,K. M.,and T. C. Shirley. 2001. Gonad development of southeastern Alaskan dungeness crab, Cancer magister, under laboratory conditions. Journal of Crustacean Biology 21(4):897. Ta gatz,M. E. 1968. Biology of the blue crab, Callinectes sapidus Rathbun,in the St. Johns River,Florida. U. S. Fish and Wildlife Service Fishery Bulletin 67(1): 17. W illiams,A. B. 1965. Marine decapod crustaceans of the Carolinas. Fishery Bulletin, U.S. 65:1. Zar,J. H. 1996. Biostatistical Analysis. Prentice-Hall,Upper Saddle River,New Jersey. Zinski,S. C. 2006. The Blue Crab Archives. Available at http://www.bluecrab.info and http://www.bluecrab.info/taxonomy.html

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45 APPENDIX

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46 Ta ble A-1.20% Paraformaldehyde stock solution (Hayat,1981) Pa raformaldehyde500 g Distilled water2090 mL PMFA is dissolved in distilled water and heated to 60¡C under the hood. 1N NaOH is added to depolymerize the solution,approximately 20 mL. The solution needs to cool down to room temperature,and then the nal volume is adjusted to 2500 mL. The solution is ltered and refrigerated. Ta ble A-2.5% Paraformaldehyde (PFMA) 0.1 molar phosphate buffer (Humason,1972) 20% PFMA1000 mL Distilled water1000 mL 0.2 M phosphate buffer2000 mL Dilute 20% PFMA stock solution 1:1 with distilled water. Dilute that 1:1 with 0.2 M phosphate buffer,pH 7.4. Ta ble A-3.Inltration Routine 70% EtOH95% EtOH50% JB-4100% JB-4Activated JB-4Sample is embedded Sample can Two to Four daysFour daysFour days remain three hours indenitelydepending on tissue size and thickness APPENDIX

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47 W eigert's Solution B 29% Aqueous ferric chloride40 mL Distilled water950 mL HCl10 mL W eigert's Working Solution Solution A150 mL Solution B150 mL APPENDIX (Continued) Ta ble A-4. Hematoxylin and Eosin (H&E) W eigert's Solution A:1% Hematoxylin in 95% Ethanol 5% Hematoxylin stock solution100 mL 95% Ethanol400 mL W eigert's Hematoxylin:5% Hematoxylin stock solution Hematoxylin25 g 95% Ethanol500 mL

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48 Ta ble A-5.Periodic Acid Schiff's/Metanil Yellow (PAS/MY) 0.02% Metanil Yellow in 0.25% in Acetic Acid,Working Solution Stock Solution A50 mL Distilled water75 mL Stock Solution B125 mL 1% Periodic Acid Periodic acid2.5 mL Distilled water250 mL Schiff's Reagent Distilled water900 mL Basic fuchsin5 g 1N hydrochloric acid100 mL Potassium metabisulte10 g Metanil Yellow Counterstain Stock Solution A:0.1% aqueous metanil yellow Metanil yellow0.5 g Distilled water500 mL Stock Solution B:0.5% glacial acetic acid Distilled water995 mL Glacial acetic acid5 mL W eigert's Working Solution Solution A150 mL Solution B150 mL APPENDIX (Continued)


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Ovarian morphology, oogenesis, and changes through the annual reproductive cycle of the female blue crab, Callinectis sapidus Rathbun, in Tampa Bay
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ABSTRACT: The blue crab, Callinectes sapidus Rathbun, 1896, was studied because of its high dollar value to Florida's commercial and recreational fisheries. The purpose of this study was to describe the structure of the ovary and oogenesis in the blue crab and the morphological changes in the female reproductive developmental stages over time. Histological techniques for high-resolution light microscopy were used to determine sexual maturity of female blue crabs. The ovarian morphology, oogenesis, and changes through the annual reproductive cycle of blue crabs in Tampa Bay were investigated for a period of two years, from January 2005 to January 2007. Ovarian structure was assessed by analyzing histological sections embedded in plastic epoxy resin, which provided a higher resolution than any other embedding material previously used in research on blue crab reproduction.Qualitative analyses of female gonads were made by describing the structure of the oocytes and determining the developmental stage of the oocytes from oogonia to full-grown oocytes. This study developed and introduced a new reproductive staging criteria for the species. Morphological characteristics of ovarian tissues and oocytes were determined to develop a classification for oocyte maturation stages. Morphological changes in the oocytes are well defined, and these were used to develop the staging schema. In this study, it was found that carapace width is not a good indicator of maturity or developmental stage. Examination of the annual reproductive cycle indicates that late secondary growth occurs from July to March, and gravid crabs were found during November and December. Histological examination of ovarian tissue is essential for determining maturity in female blue crabs.By observing ovarian characteristics and by establishing the length of secondary growth during oogenesis in blue crabs of Tampa Bay, a more thorough understanding of the cyclic reproductive aspects of this species was obtained and specifically that animals at a carapace width between 100 mm and 125 mm may have mature oocytes, yet external features may not indicate that they are mature.
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