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A Proposal for a Standardized Classification of Ovarian Development Classes in Teleost Fishes Luiz R. Barbieri1*, Nancy J. Brown-Peterson2, Melissa W. Jackson3, Susan K. Lowerre-Barbieri1, David L. Nieland4**, and David M. Wyanski5

1Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, FL 33701 USA 2Department of Coastal Sciences, University of Southern Mississippi, Ocean Springs, MS 39564 USA 3Department of Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL 32653 USA 4Coastal Fisheries Institute, Louisiana State University, Baton Rouge, LA, 70803 USA 5 Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC 29422 USA *Order of authorship is alphabetical. **Corresponding author: [email protected]

The scientific literature is replete with articles describing the progression of ovarian events that ultimately lead to spawning in fishes. Among teleost fishes there appears to be little variation in the ovarian structures observed; however, the sequence of oocyte development may be somewhat modified depending on the particular spawning strategy of the species in question: oviparous vs. ovoviviparous, nest guarder vs. broadcast spawner, serial spawner vs. isochronal spawner, usw. Differences between freshwater fishes and marine fishes are minor and may be mainly reflected in oocyte hydration in the latter, but not the former. Unfortunately, the differences among these spawning strategies have produced descriptions using an assortment of nomenclatural systems for the various classes of oocyte development. There is also little agreement in the numbers of developmental classes that should be acknowledged.

Recognizing the necessity for both consistency and comparability among investigations of fish reproduction, we present a classification system for teleost ovarian developmental classes based both on macroscopic and microscopic criteria. We believe that it is largely applicable to the situations presented in the spawning of most species in both fresh and salt water. Further recognizing that much of our research is structured to deliver estimates of various reproductive variables, the system is also amenable to the estimation of seasonality, age/size at maturity, diel periodicity of spawning, spawning frequency, and batch fecundity.

Our intent was to produce a classification system that is descriptive, but not overly arcane, with ease of both interpretation and use foremost in our minds. Table 1 represents eight ovarian development classes that are defined both with macroscopic and microscopic characteristics (see Figures 1-6 for examples). The macroscopic classification and its accompanying criteria are provided to those fishery biologists for whom histological examination of ovaries is either impractical or unnecessary; a great deal of useful information can be gleaned from informed observations of whole, fresh ovaries.

However, histological examination of ovarian tissues has been and remains the most accurate method to assess ovarian developmental classes. The criteria affixed to each 2 developmental class should be appropriate to the ovarian cycles of a great many teleosts; however, prior knowledge of the spawning strategy (multiple spawner, single spawner, hermaphrodite, live-bearer, etc.) of your particular species will be of great value in applying the classification. Further, the applicability of each class in estimating a reproductive variable is given after the description of the microscopic appearance. Thus, after cursory determination of ovarian class either by macroscopic or microscopic examination, several of the most important reproductive variables can be estimated.

We began working under the assumption that we should preserve, to some extent, the “traditional” nomenclature for ovarian development classes that has been historically applied in the literature (see Table 1). At a late stage in our discussions, one of us suggested a new format for the naming of the ovarian classes that is predicated on either the dominant oocyte stage or the most remarkable structure seen in the histological material; these are indicated in Table 1 in parentheses. We present both systems for your discussion.

Thanks to Harry Grier for comments and suggestions during discussions of this classification.

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Table I. Ovarian developmental classes for gonochoristic fishes with alternative name for each class included in parentheses. Macroscopic appearance refers to fresh ovaries. Reproductive variables for which each class may be useful in estimating are indicated in bold italics. POF = postovulatory follicles, FOM = final oocyte maturation.

Class Macroscopic Appearance Microscopic Appearance Immature Ovaries very small, translucent, ribbon-like. Only oogonia and primary growth oocytes present; no atresia; ovarian membrane thin. Incapable of spawning.

Developing Ovaries ranging from small to medium ( 25% of body cavity); light Only primary growth, cortical alveoli and a few early vitellogenic (Cortical alveoli) orange in color; no opaque (advanced vitellogenic) oocytes present. oocytes present; mature, but not yet capable of spawning. Start of spawning season.

Fully developed Ovaries large (50-75% of body cavity); pale yellow in color; opaque Primary growth, cortical alveoli, and advanced vitellogenic (Vitellogenic) oocytes prevalent and easily detected; little ovarian vascularization and oocytes present; atresia of vitellogenic oocytes may be evident, no signs of previous spawning. especially early in spawning season. Fully capable of spawning. Age and size at maturity.

Gravid Ovaries ranging from medium to very large (25-100% of body cavity); Primary growth to FOM oocytes or hydrated oocytes present; (FOM) clear (hydrated) oocytes visible amongst opaque oocytes, giving a hydrated oocytes un-ovulated. Degenerating POF from previous speckled appearance; late in season, ovaries may be smaller and reddish spawning event may be present; minor atresia of advanced due to an increase in the ratio of clear to opaque oocytes and ovarian vitellogenic oocytes may be observed.

spawners vascularization. Batch fecundity.

Spawning Ovaries ranging from medium to large (25-75% of body cavity); clear Ovulated ova and/or recent POF (< 0 hrs) present; primary

Serial (Ovulatory) oocytes have been ovulated and are visible as a collective clear strip growth to fully vitellogenic oocytes present; minor atresia of among the vitellogenic oocytes; some may have been extruded; advanced vitellogenic oocytes. occasionally no opaque oocytes present. Diel spawning periodicity and spawning site.

Postovulatory Ovaries somewhat flaccid, ranging from medium to small ( 30% of the POF (0 hrs < age < 24 hrs) present; primary growth to advanced (Postovulatory) body cavity); orangish in color due to increased ovarian vascularization. vitellogenic oocytes and occasionally remnant hydrated oocytes. Often a ‘ridge’ (a red area along the dorsal ovarian edge) is present. Minor atresia of advanced vitellogenic oocytes. Remnant hydrated oocytes may occur in the ‘ridge’ or at the posterior end Spawning frequency. of the ovaries. Spent Ovaries quite flaccid and small (< 20% of body cavity); mustard yellow to Vitellogenic oocytes in advanced stages of atresia; primary (Atretic) orange, occasionally maroon; often contain clear fluid; can detect a few growth and cortical alveoli oocytes present; degenerating POF opaque oocytes. from previous spawning may be present. End of spawning season.

Recovering Ovaries very small; dark orange to maroon in color; no opaque oocytes Oogonia and primary growth oocytes dominate; may have other (Primary growth) present; ovarian membrane thickened and more opaque than immature oocytes in late stages of atresia, especially immediately after fish. cessation of spawning; more follicular tissues than immature fish and thicker ovarian membrane; “muscle bundles” present. 4

Figure1. Developing (Cortical Alveoli) class in Figure 2. Fully developed (Vitellogenic) class in red striped mullet, Mugil cephalus, an isochronal drum, Sciaenops ocellatus, a serial spawner. spawner.

Figure 3. Fully developed (Vitellogenic) class in Figure 4. Gravid (FOM) class in spotted seatrout, striped mullet, Mugil cephalus, an isochronal Cynoscoin nebulosus, a serial spawner. spawner.

POF

Figure 5. Postovulaory (Postovulatory) class in Figure 6. Spent (Atretic) class in red drum, spotted seatrout, Cynoscion nebulosus, a serial Sciaenops ocellatus, a serial spawner. spawner. 5

Reproductive Classification of Teleosts: Consistent Terminology for Males and Females Nancy J. Brown-Peterson Dept. of Coastal Sciences, Univ. of Southern Mississippi, Ocean Springs, MS 39564 [email protected]

There is no standardization of classification terminology for teleost ovarian and testicular development. This proliferation of terminology is often due to the needs of various disciplines to describe reproductive processes on either the macroscopic or microscopic level. Furthermore, terminology used to describe ovarian development is often completely different from terminology used to describe a similar developmental class in testes. Thus, a common terminology, based on histological observations, that describes the progression of the gonadal developmental cycle for both males and females would substantially reduce confusion when trying to compare male and female gonadal development during the reproductive season. A suggested common terminology, modified from Brown-Peterson (2003), is presented in Table 1. Each reproductive class is necessarily a progression through gametogenesis and thus a range of oocyte stages or spermatogenesis can be present in a single class. Fig. 1A shows a female in the beginning of early maturation, with the initial appearance of cortical alveolar oocytes. Fig. 1B shows a female towards the end of the mid maturation class, with approximately equal numbers of cortical alveolar, yolk granule and yolk globular oocytes. Fig. 1C shows a multiple spawning species in late maturation. Fish in late maturation are capable of spawning, and multiple spawning species return to this class during the reproductive season after ovulation and spawning a batch of oocytes. Fig 1D shows a fish in the spawning class undergoing final oocyte maturation (FOM); all stages of FOM, as well as ovulation, are included in this class. Spent fish (Fig. 1E) are found at the end of the reproductive season and are characterized by widespread atresia. Fish in the regressed class can have late stage atresia present (Fig. 1F). Generally, the ovarian wall is thicker, there is more space between the lamellae and there are more blood vessels present than in immature fish. Muscle bundles, indicative of previous spawning, can also be present in regressed ovaries. Histological classification of male fish is based on the progression of spermatogenesis and the appearance of the germinal epithelium throughout the reproductive season. The presence of spermatozoa in the lumen of the lobules or ducts is not used for the histological classification, as, unlike females, males are capable of spawning in 4 reproductive classes (early, mid and late maturation as well as spent) and thus there is not a separate spawning class for males in the histological classification. The initiation of spermatogenesis with the appearance of spermatocysts and lumens in the lobules occurs in early maturation (Fig. 2A), and the germinal epithelium is always continuous in this class. All stages of spermatogenesis, as well as spawning, may occur during both early maturation (Fig. 2B) and mid maturation (Fig. 2C); these classes are differentiated by the appearance of a discontinuous germinal epithelium near the ducts in mid maturation. Spermatogenesis continues in late maturation with the occurrence of many spermatocysts along a discontinuous germinal epithelium throughout the testis, and spermatogonia are rare in this class. Males in the spent class often have lumens full of spermatozoa but active spermatogenesis has ceased and 6 spermatocysts are widely scattered along the discontinuous germinal epithelium (Fig. 2D). In some species, primary spermatogonia appear in the periphery of the testis in the spent class. Males in the regressed class are characterized by the absence of any spermatocysts (Fig. 2E), although residual spermatozoa is often found in the lumens and some males may be able to spawn in the early phases of this class. Primary spermatogonia begin to recolonize the testis and form a continuous germinal epithelium in the regressed class (Fig. 2F), and residual spermatozoa may remain in the lumen of the lobule although the fish are incapable of spawning.

Table 1. Histological characteristics of gonadal classes in female and male gonochoristic teleost fishes. CA—cortical alveolar oocytes; FOM—final oocyte maturation; POF—post ovulatory follicles; GE—germinal epithelium; SG— spermatogonia; SC—spermatocytes; ST—spermatids; SZ--spermatozoa Class Females Males Immature Primary growth oocytes only; Primary SG only. Lobules small, ovarian walls thin, few spaces many with no lumens between lamellae Early CA present. Some yolk granule Continuous GE throughout testis. All Maturation vitellogenic oocytes can be stages of spermatogenesis can be present. present, including SZ in lumen and ducts. Mid Dominated by CA and yolk Continuous GE in periphery of testis, Maturation granule oocytes; some yolk discontinuous GE in lobules near globular oocytes present. ducts. All stages of spermatogenesis Minimal atresia, no POFs present, with SZ in lumens and ducts. Late All oocyte stages present, but Discontinuous GE throughout testis. Maturation dominated by yolk globular, Numerous spermatocysts present, SZ vitellogenic oocytes. Some in lumens and ducts. SG rare. atresia present. POF present. Spawning Oocytes undergoing FOM or (Males in Early, Mid and Late ovulated. POF and atresia can Maturation, as well as spent classes, be present. CA and vitellogenic are all capable of spawning.) oocytes can be present Spent Widespread atresia of Discontinuous GE throughout testes, vitellogenic and some CA spermatocysts widely scattered, oocytes. POF can be present. containing only secondary SC, ST or SZ. Primary SG appear in periphery. SZ in lumen and ducts. Regressed Only primary growth oocytes. Continuous GE of only primary SG. Late stage atresia present. Residual SZ can be in lumen. Muscle bundles present.

References

Brown-Peterson, N.J. 2003. The reproductive biology of spotted seatrout. Pp.99-134 In Biology of the Spotted Seatrout, S.A. Bortone (ed). CRC Press, Boca Raton, FL. 7

PN A B

CN Y glob CA Early CA

Y gran

C D

Y gran GVM Y glob Y POF Y P

O g gCA F l r C

o a AE F

b Atresian Y glob Atresia

Figure 1. Reproductive classification of female teleosts. A. Early Maturation. B. Mid Maturation. C. Late Maturation. D. Spawning. E. Spent. F. Regressed. CA—cortical alveolar oocyte; CN—chromatin nucleolar oocyte; GVM—germinal vesicle migration; PN—perinucleolar oocyte; POF—post ovulatory follicle; Y glob—yolk globular oocyte; Y gran— yolk granular oocyte.

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Figure 2. Reproductive classification of male teleosts. A. Early Maturation (non spawning). B. Early Maturation (spawning). C. Mid Maturation. D. Spent. E. Regressed (spawning). F. Regressed (non spawning). 1 SC—primary spermatocyte; 2SC—secondary spermatocyte; 1SG—primary spermatogonia; 2SG—secondary spermatogonia; CY—spermatocysts; DGE— discontinuous germinal epithelium; L—lumen of lobule; ST—spermatid; SZ—spermatozoa.

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A Proposed “Straw Man” Reproductive Classification for Male Teleosts Nancy J. Brown-Peterson1 and David M. Wyanski 2 1Dept. Coastal Sciences, Univ. Southern Mississippi, Ocean Springs, MS 39564; 2Marine Resources Research Institute, S. Carolina Dept. Natural Resources, Charleston SC 29422; [email protected]

The reproductive classification of male teleosts has traditionally been of less concern to fisheries biologists than that of females, primarily because male reproductive potential is not considered in stock assessments. Often, the reproductive classification of testes is assessed macroscopically, rather then microscopically, and a bewildering array of terminology is represented in the literature. In an attempt to standardize this terminology, we present here a suggested macroscopic classification to identify the reproductive condition of males, as well as a microscopic classification based on histological observations that takes into account changes in spermatogenic activity during the course of the reproductive season. The morphology of teleost male testis can be divided into three separate types that correspond with basic phylogeny; these have been summarized by Parenti and Grier (2004). Anastomosing tubular testes, in which the germinal compartments do not terminate at the testis periphery but form highly branched, anastomosing loops or tubules are found in primitive teleosts (i.e., elopiforms, cypriniforms, salmoniforms, esociforms). In lobular testes, germinal compartments extend to the periphery of the testis where they terminate blindly. Restricted lobular testes, in which spermatogonia are only found at the distal ends of the lobules, are characteristic of atherinomorph fishes, and unrestricted lobular testis, in which spermatogonia are found throughout the testes, are characteristic of all higher teleosts. The macroscopic and microscopic descriptions suggested here apply to all male teleosts, regardless of testis type. The macroscopic classification (Table 1) is provided for fisheries biologists who desire to assess the spawning capability of male fish and are not concerned with the underlying spermatogenic activity of the testis. The Inactive class lumps all non-spawning males together, and includes immature and regressed/resting males. Males in the Developing class would indicate the initiation of the reproductive season, while those in the Spent class signal the end of the reproductive season.

Table 1. Macroscopic classification for male teleosts Class Description Inactive Testis small, thin, or ribbon-like, often clear or pinkish in color; no spermatozoa evident. Developing Testis small, but firm, white and often triangular shaped. Spermatozoa not released when testis is cut. Spawning Testis firm and white, often maximum size for species; spermatozoa released when pressure applied to abdomen or when testis is cut Spent Testis elongated and flaccid, often pinkish in color, no spermatozoa released

The microscopic, histological classification for males takes into account changes in spermatogenic activity relative to the germinal epithelium (GE) of the testis. This classification is based on histological descriptions of three species with an unrestricted lobular testis; common snook, Centropomus undecimalis (Grier and Taylor 1998), cobia, Rachycentron canadum (Brown-Peterson et al. 2002) and spotted seatrout, Cynoscion nebulosus (Brown-Peterson 2003), but also applies to swamp eel, Synbranchus marmoratus (Lo Nostro et al 2003) and the freshwater goby Padogobius bonelli (Cinquetti and Dramis 2003). The classification is based on the presence of a continuous GE (Fig. 1) or a discontinuous GE (Fig. 2). In both cases, the basement membrane underlies the Sertoli cells along the entire length of the lobules. A continuous GE is defined as each lobule completely surrounded by spermatocysts and/or primary spermatogonia in contact with the basement membrane. In contrast, a discontinuous GE is defined as areas of basement membrane not associated with spermatocysts interspersed along each lobule. The histological classification presented in Table 2 is not based on the presence or absence of spermatozoa in the lobules or the ducts, but rather on the progression of spermatogenesis in the 10 spermatocysts and the condition of the GE. Indeed, males can have sperm in the ducts, and be capable of spawning, in 4 of the 6 classes listed in Table 2. Thus, this classification is most useful in helping to determine the progression of the reproductive season (i.e., beginning, middle or end of season) rather than actual spawning capability. The transitional class is included for protandrous hermaphrodites that begin life as males and switch to females.

Table 2. Histological classification for male teleosts Class Description Immature Only primary spermatogonia present, lobules small or non-existent Early Continuous GE in all lobules, extending from the ducts to the periphery. All Developing stages of spermatogenesis present, lobules and ducts can contain spermatozoa. Mid Continuous GE in lobules in the periphery of the testis, discontinuous GE in Developing lobules near the ducts. All stages of spermatogenesis present, spermatozoa in lobules and ducts. Late Discontinuous GE in lobules throughout testis. Numerous spermatocysts Developing containing mostly secondary spermatocytes and spermatids; spermatogonia rare. Spermatozoa in lobules and ducts. Regressing Discontinuous GE in all lobules; spermatocysts widely scattered; no secondary spermatogonia or primary spermatocytes present. Primary spermatogonia can be present. Spermatozoa in lobules and ducts. Recovering Only primary spermatogonia present as a continuous GE; lobules small; residual spermatozoa may be in lobules or ducts. Transitional Discontinuous GE, reduced spermatogenesis, primary oocytes present, development of lamellae.

Thanks to Harry Grier for comments and suggestions during the “straw man” discussions.

References

Brown-Peterson, N.J. 2003. The reproductive biology of spotted seatrout. In S. Bortone (ed), Biology of the Spotted Seatrout, pp. 99-134. CRC Press, Boca Raton, Florida.

Brown-Peterson, N., H.J. Grier and R. Overstreet. 2002. Annual changesin the germinal epithelium determine reproductive classes in male cobia, Rachycentron canadum. J. Fish Biol. 60:178-202.

Cinquetti, R. and L. Dramis. 2003. Histological, histochemical, enzyme histochemical and ultrastructural investigations of the testis of Padogobius martensi between annual breeding seasons. J. Fish. Biol. 63:1402-1428.

Grier, H.J. and R.G. Taylor. 1998. Testicular maturation and regressin on the common snook. J. Fish. Biol. 53:521-542.

Lo Nostro, F., H.J. Grier, L. Andreone and G.A. Guerrero. 2003. Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel, Synbranchus marmoratus Bloch, 1795 (Teleostei, Synbranchidae). J. Morph. 258:107-126.

Parenti, L.R. and H.J. Grier. 2004. Evolution and phylogeny of gonad morphology in bony fishes. Integr. Comp. Biol. 44:333-348.

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Figure 1. Continuous germinal epithelium from male cobia. Typical of a fish in the early or mid developing classes. BM—basement membrane; CY—spermatocysts; 1SC—primary spermatocysts; 2SC—secondary spermatocytes; 1 SG—primary spermatogonia; ST— spermatids; SZ—spermatozoa.

Figure 2. Discontinuous germinal epithelium from male cobia. Note widely scattered spermatocysts. Typical of a fish in the spent class. CY—spermatocysts; SZ--spermatozoa

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Reproductive classification of protogynous groupers from the southern Gulf of Mexico Thierry Brulé and Teresa Colás-Marrufo Centro de Investigación y de Estudios Avanzados del IPN, Unidad Mérida, Antigua Carretera a Progreso Km. 6, A.P. 93 Cordemex, C.P. 97310, Mérida, Yucatán, México [email protected]

Groupers are perciform teleosts of the family Serranidae which includes species of great abundance and of commercial fishery importance (Heemstra and Randall, 1993). In fisheries study, the particular event of interest in the reproductive cycle of a species is the time of spawning, when fully developed gametes are released (King, 1995). For this purpose, ovaries and testes are examined microscopically and individuals are generally grouped into various developmental or seasonal classes (Moe, 1969). The remarkable feature of grouper reproduction is protogynous hermaphroditism: individuals mature first as females and then later become males (Shapiro, 1987). In these fishes, the configuration of germinal tissues falls into an undelimited type and the process of sex change involves one tissue expanding and infiltrating to replace the other (Smith, 1965, 1967; Sadovy and Shapiro, 1987). The histological changes in the gonads during maturation and sex change processes have been described for a number of West Atlantic groupers. But terminology and reproductive classifications used in these studies were variable by author. This work presents a classification system for protogynous fishes as a contribution to the development of a universal gonadal classification for fisheries reproductive studies. Red grouper Epinephelus morio, black grouper Mycteroperca bonaci and gag M. microlepis from the continental shelf of the Yucatan Peninsula, Mexico (Campeche Bank) were analyzed in the present study. These species are monandric protogynous fishes. Their gonads were preserved in Bouin’s fluid, embedded in paraffin, thin sectioned at 6 μm and stained in Gabe and Martoja's triple stain for light microscopy (Gabe, 1968). Following Taylor et al. (1998), “stage” and “class” terms were used for gamete and gonad development, respectively. Histological descriptions of female and male germ cells (stages) followed Wallace and Selman (1981) and Moe (1969), respectively. Thirteen gonad reproductive classes were defined using criteria based on Smith (1965), Moe (1969), Sadovy and Shapiro (1987), Grier and Taylor (1998), Taylor et al. (1998), Brown-Peterson et al. (2002) and Lo Nostro et al. (2003) (Table 1; Figures 1 and 2).

Brown-Peterson, N.J., H.J. Grier, and R.M. Overstreet. 2002. Annual changes in germinal epithelium determine male reproductive classes of the cobia. Journal of Fish Biology 60:178-202. Gabe, M. 1968. Techniques histologiques. Masson, Paris. Grier, H.J., and R.G. Taylor. 1998. Testicular maturation and regression in the common snook, Journal of Fish Biology 53:521-542. King, M. 1995. Fisheries biology, assessment and management. Fishing News Books, Blackwell Science, Oxford. Lo Nostro F., H.J. Grier, L. Andeone, and G.A. Guerro. 2003. Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel, Synbranchus marmoratus Bloch 1795 (Teleostei, Synbranchidae). Journal of Morphology 257:107-126. Heemstra, P. C., and J. E. Randall. 1993. FAO species catalogue. Vol. 16. Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date. FAO Fisheries Synopsis 125, FAO, Rome, 382 p. Moe, M. A. 1969. Biology of the red grouper Epinephelus morio (Valenciennes) from the eastern Gulf of Mexico. Florida Department of Natural Resources, Marine Research Laboratory,Professional Papers Series 10, Florida, 95 p. 13

Sadovy, Y., and D. Y. Shapiro. 1987. Criteria for the diagnosis of hermaphroditism in fishes. Copeia 1:136-156. Shapiro, D. Y. 1987. Reproduction in groupers. In J. J. Polovina and S. Ralston (eds), Tropical Snappers and Groupers: Biology and Fisheries Management, p. 295-327. Westview Press, Boulder, CO. Smith, C. L. 1965. The patterns of sexuality and the classification of serranid fishes. American Museum Novitates 2207:1-20. Smith, C. L. 1967. Contribution to a theory of hermaphroditism. Journal of Theoretical Biology 17:76-90. Taylor R.G., H.J. Grier, and J.A. Whittington. 1998. Spawning rhythms of common snook in Florida. Journal of Fish Biology 53:502-520. Wallace R.A. and K. Selman. 1981. Cellular and dynamic aspects of oocyte growth in Teleosts. Amer. Zool. 21: 325- 343.

Table.1 Reproductive classification system for protogynous fishes defined by microscopic characteristic of groupers gonads (Epinephelus morio, Mycteroperca bonaci and M. microlepis).

Reproductive class Histological characteristics Female 1- Immature Chromatin nucleolar oocytes (CO) Perinucleolar oocytes (PO) 2- Regressed CO + PO Muscular bundles, connective tissue and blood vessels in the center of ovarian lamellae. 3- Early maturation CO + PO Yolk vesicle (cortical alveoli) oocytes (YVO) 4- Mid maturation CO + PO + YVO Yolk globule oocytes (YGO) 5- Late maturation CO + PO + YVO +YGO, some of them with germinal vesicle migration and/or fusion of yolk and oil globule (FOM) and/or Hyaline oocytes (HO) and/or Postovulatory follicles (POF) 6- Regressing CO + PO Residual YVO and YGO, most of them in  and/or  stage atresia Transitional 7- Sex-transition CO + PO YVO sometimes present, degenerating or not Few clusters of spermatogonia (SG) and spermatocytes (SC) in lamellae Male 8- Immaturea Not assessed 9- Regresseda SG Residual spermatozoa (RSP) in lobules are scarce 10- Early maturationa Spermatocysts of SG, SC and spermatids (ST) 11- Mid maturationa SG + spermatocysts of SC, ST Spermatozoa (SP) fill lobules In the center of lamellae, some lobules anastomose in newly formed intralobular sinuses 12- Late maturationa Some SG and spermatocysts of SC and ST SP fill intralobular sinuses 13- Regressinga SG + RSP in lumen of lobules a In some testes, CO, PO and YVO can be also observed with spermatogenesis stages. 14

Figure 1. Histological sections of female grouper gonads in the: 1) immature class (M. microlepis; 845 mm FL); 2) regressed class (E. morio; 720 mm FL); 3) early maturation class (M. microlepis; 930 mm FL); 4) mid maturation class (M. microlepis; 730 mm FL); 5) late maturation class showing (a) oil globule fusion and germinal vesicle migration in YGO and POF (M. microlepis, 1035 mm FL), and (b) hyaline oocytes (M. microlepis; 880 mm FL) and 6) regressing class (M. microlepis; 770 mm FL). A- atresia, BV- blood vessel, CO- chromatin nuclear oocyte, GV- germinal vesicle, HO- hyaline oocyte, MB- muscular bundle, OC- ovarian cavity, OL- ovarian lamellae, PO- perinucleolar oocyte, POF- postovulatory follicle, YG- yolk globule, YGO- yolk globule oocyte, YV- yolk vesicle, YVO- Yolk vesicle oocyte, ZR-zona radiata. Scale bars= 200 microns. 15

Figure 2. Histological sections of transitional and male grouper gonads in the: 1) sex-transition class (E. morio; 421 mm FL); 2) regressed class (M. microlepis; 982 mm FL); 3) early maturation class (M. microlepis; 1070 mm FL); 4) mid maturation class (M. microlepis; 1065 mm FL); 5) late maturation class (M. bonaci; 1160 mm FL) and 6) regressing class (M. bonaci; 1180 mm FL). A- atresia, BV- blood vessel, CO- chromatin nuclear oocyte, FOC- former ovarian cavity, LA- lobule anastomose, LL- lobule lumen, MMC- melanomacrophage center, RSP- residual spermatozoa, SC- spermatocytes, SG- spermatogonia, SP- spermatozoa, ST- spermatids, TL- testicular lamellae (former ovarian lamellae). Scale bars= 50 microns. 16

The Gametogenic Cycle of Pagellus bogaraveo in Strait of Gibraltar (Cádiz, SW Spain) Bruzón, M.A.¹; San Martín, M.²; Rodríguez-Rúa, A.¹; Jiménez-Tenorio, N.²; García- Pacheco, M.²; Pozuelo, I.² ¹ IFAPA. Consejería de Innovación, Ciencia y Empresa, CIFPA "El Toruño". Ctra. Nacional IV Km 654, 11500 El Puerto de Santa María, Cádiz, Spain. [email protected] ² DAP. Consejería de Agricultura Pesca y Alimentación. c/ Bergantin 39, 41012 Sevilla, Spain

Introduction Pagellus bogaraveo (Brünich, 1768) (Osteichthyes: Sparidae) is distributed in the Westearn Mediterranean Sea and the Eastern Atlantic Ocean, from Norway to White Cape, Madeira and Canaries Islands. It inhabits bottoms from 400 m (Mediterranean Sea) to 700 m deep (Atlantic Ocean). P. bogaraveo is a very important species and it reaches high commercial values in fish markets.

Material and methods A total of 700 specimens were studied, collected between October 2003 and October 2004. Total length (cm) and total weight (g) for each individual were measured. Sections of gonads were fixed in formol buffered to 4% or in Bouin's liquid, and transferred into 70º alcohol for their conservation. Samples were dehydrated and embedding in paraffin with a vacuum and pressure tissue processor. A paraffin dispenser, a cold plate for inclusion and a heating plate with rotating base were used in the process of the blocks formation. Sections of 3 µm were obtained with a rotary microtome.

Sections were stained with Haematoxylin-V.O.F (light green, orange G and acid fucsin, Gutiérrez, 1967) and with Haematoxylin-Eosin (Martoja and Martoja-Pierson, 1970), and then mounted on cover slips, using Eukitt as a preserving medium.

Results Gametogenic Cycle: P. bogaraveo is a proterandric hermaphrodite species. Its reproduction season, in the Strait of Gibraltar, runs from October until April in males and from January until March in females. We distinguished five stages for male (Micale et al., 2002): Immature (figure A), Developing (figure B), Maturing, Spawning (figure C) and Recovering (figure D). We also distinguished five phases for female (Wallace and Selman, 1981; Sarasquete et al., 2002): Previtellogenesis (figure E), Vitellogenesis (figure F), Maturation (figure G), Spawning and Postspawning (figure H). Sex reversal occurs from 30 to 35 cm. Hermaphrodite with immature testicle and ovary (figure K). Hermaphrodite with testicle in development (figure L).

References Gutiérrez, M. (1967). Coloración histológica para ovarios de peces, crustáceos y moluscos. Inv. pesq. 31 (2): 265-271. Martoja, R. y Martoja-Pierson, M. (1970). Técnicas de histología animal. Toray-Masson, S.A. Barcelona, 350 pp. Micale, V., Maricchiolo, G. y Genovese, L. 2002. The reproductive biology of blackspot sea bream Pagellus bogaraveo in captivity. I. Gonadal development, maturation and hermaphroditism. J. Appl. Ichthyol. 18: 172-176. 17

Sarasquete, C., Cárdenas, S., González de Canales, M.L., Pascual, E. (2002). Oogenesis in the bluefin tuna, Thunnus thynnus L.: A histological and histochemicas study. Histol. Histopathol. 17: 775-788. Wallace, R.A., Selman, K. (1981). Cellular and dynamic aspects of oocyte growth in teleosts. Amer. Zool. 21: 325-343.

Acknowledgements This study was supported by IFOP project.

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Figure 1. Percentage of the different stages of gonad development during an annual cycle in male.

A B

75 µm 150 µm

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Figure 2. A: Immature, B: Developing, C: Maturing, D: Spawning, E: Recovering. F: Hermaphrodite with testicle in development.

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1 2 3 4 5

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Figure 3. Percentage of the different stages of gonad development during an annual cycle in female.

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Figure 4. A: Previtellogenesis, B: Vitellogenesis, C: Maturation, D: Spawing, E: Postspawning. F: Hermaphrodite with immature testicle and ovary. 20

GONADAL MORPHOLOGY AND TERMINOLOGY OF GOBIID FISHES Dr. Kathleen S. Cole, Department of Zoology, University of Hawaii at Manoa, 2538 McCarthy Mall, Edmondson 152, Honolulu, HI 96822 (e-mail: [email protected])

Gobiidae comprises the largest family of marine fishes (Nelson 2006). Hermaphroditism is currently documented for 14 of 210 described genera (Cole unpublished). The study of hermaphroditism in gobiids has revealed several issues associated with gonad morphology that underscore the importance of carrying out detailed histology and applying appropriate terminology in order to accurately characterize gonadal features, establish the origin and function(s) of reproductive characters, and correctly assess reproductive state. Within the Gobiidae, all reported instances of hermaphroditism occur within one subfamily, the Gobiinae (Cole, unpublished data) and are predominantly found within one clade, the Priolepis group (sensu Birdsong et al 1988). Given their close phylogenetic relationship, one might expect that features of gonad morphology among hermaphroditic gobiines would show a strong similarity. In fact, ovarian, ovotestis and secondary testis features vary considerably among these closely related genera. Consequently, the diversity of gonad form among hermaphroditic gobiids is instructive in that it indicates within-taxon conformity of hermaphroditic gonad morphology in other taxa cannot be assumed, although it may generally be observed. Testis morphology among all gobiids is distinguished by the universal presence of various reproductive-associated non-germinal tissues and structures. These features greatly increase the complexity of the gobiid male reproductive system. In order to correctly characterize how gonadal features relate across hermaphroditic gobiid species and taxa (i.e. to identify homologies) requires a thorough understanding of how the gonad forms, and transforms. And in order to accurately depict the type of gonadal function that is most likely associated with a particular gonad morphology, accurate and unambiguous descriptors are required. To these ends, the use of appropriate terminology is critical. Among hermaphroditic gobiids, gonad morphology is generally conserved among congeneric species. However, a comparison of gonad morphology across all known hermaphroditic genera demonstrates that there is no ‘typical’ gonadal morphotype. In terms of the presence and distribution of germinal tissues, in some genera, the adult ovary has no pre-formed testicular tissue (Coryphopterus, Lophogobius, Rhinogobiops and Fusigobius). In other genera, spermatogenic tissue is visible and identifiable as such, but its’ distribution shows genus-specific differences. In Eviota, Gobiodon and Paragobiodon, spermatocysts are evident throughout the ovigerous tissue and gonadal transformation is characterized by the proliferation of spermatogenic tissue and concurrent reduction in ovigerous tissue in which remnant early-stage oocytes are the last visible manifestations. In contrast, in other genera such as Trimma and Priolepis, the gonad is partitioned into a region of ovigerous tissue and a separate membrane-bound region of spermatogenic tissue resulting in an ovotestis having discrete testicular and ovarian regions. Gonadal transformation in these species is characterized by an expansion of the testicular portion and reduction of the ovarian portion. 21

As with most gobioids, the gobiid testis is characterized by the presence of elaborations of secretory tissue in the form of accessory gonadal structures (AGS). In most hermaphroditic species so far described, these structures become fully differentiated and functional at the time of sex change from an adult female to a secondary male. However, how this transformation occurs varies considerably across species. In addition, gonadal morphology of adult females shows considerable variability at the genus-specific level, as does secondary testis morphology. For example, the origins of the AGS varies considerably across protogynous gobiines. In several genera, including Coryphopterus, Lophogobius, Rhinogobiops and Fusigobius, the AGS arises from an undifferentiated tissue mass associated with the ovarian wall. In contrast, among species of Eviota and Priolepis, the accessory gonadal structure is partially differentiated in functional females, and in Gobiodon, adult females exhibit fully formed and secreting accessory gonadal structures. Gobiodon and it’s sister genus, Paragobiodon, are unusual among gobioids in having what appear to be two sets of gonadal accessory structures. In two species of Gobiodon so far examined, both pairs of secretory structures are fully formed and actively secreting in both male and female adults. The lobes if one pair of AGS are continuous with the posterior region of the gonadal lobe, while the lobes of the other pair are associated with the gonadal duct. In the latter case, were the occurrence of these structures restricted to males, they could be termed sperm duct glands, sensu Miller (1984). However, as they are also present among females and there are associated with the oviduct, a more appropriate term would be ‘gonadal’ - rather than ‘sperm’ - duct accessory structure In summary, the remarkable diversity of gonad morphology among hermaphroditic gobies can only be understood through the provision of detailed and accurate descriptions of morphological features and by understanding the context in which these features are expressed. Consequently, the application of accurate and informative terminology is critical. In the case of accessory gonadal structures, the use of terminology that reflects function, activity state and ontogenetic origin is essential to recognize homology, where it exists, and/or the presence of recently evolved novel features. In the case of the gonad, accurate descriptive terminology is a prerequisite for proper assessment of gonadal state and function. And making a valid characterization of sexual pattern necessitates distinguishing between unidirectional, irreversible sex change that occurs in some fish taxa, and labile sexual function through re-allocation of resources to male or female gamete production the characterizes others.

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Maturity Stage Key for Hake (Merluccius merluccius) based on histology Patrícia Gonçalves* and Cristina Morgado *Email: [email protected] IPIMAR – DRM Av. Brasília 1449 -006 Lisboa PORTUGAL

Introduction The European hake, Merluccius merluccius, is widely distributed in Northeast Atlantic and in Mediterranean (Goñi et al., 2004). The spawning area of the hake population in Atlantic waters extends all along the western margin of Europe from Portugal to North Scotland (Alvarez et al., 2001). Hake spawn throughout the year, peaks have been reported closer to winter months (February-March) in the Portuguese Coast (Alvarez et al., 2001). Hake is a multiple spawner with an indeterminate annual fecundity, which means that potential annual fecundity is not fixed before the onset of spawning and previtellogenic oocytes develop and became recruited into the yolked oocyte stock at any time during the spawning season (Hunter et al., 1992). The fishery management requires knowledge of different aspects of the reproductive biology, like age or length at maturity, fecundity and spawning frequency. Annual changes of these variables could affect the stock productivity and produce variability on the recruitment of fishes (Macchi et al., 2004). In order to determine the individual stage of sexual maturation, macroscopic staging of reproductive organs is regularly applied. However, the subjectivity and ambiguity of the visual inspection method may lead to severe misclassification of the fish reproductive status (Vitale et al., 2006). The histological analysis of gonadal development is the most accurate methodology to determine the individual stage of sexual maturation. The aim of this work is describe a maturity stage key for hake females based on histological analysis of ovaries and to make the relation between this and a macrocospic scale more objective as possible.

Material and Methods A total of 200 females gonads were collected and macroscopically classified according a four stages maturity scale (Immature/Resting, Development, Spawning, Post- spawning). After sampling procedure gonads were transferred to a 70% alcohol and two days later were fixed in 10% formol. Photographs of whole ovaries were taken prior to fixation in formol. Ovaries were weighted and a portion of sample (2.0 g) was removed from each gonad. Ovaries samples were dehydrated through ascending concentrations of ethanol, cleared in xylol and embedded in parafinn using standard routine histology procedures. From each gonad serial 5 µm sections were cut in a rotating microtome and stained with Harri's haematoxylin and eosin. The slides are mounted using Entellan® with a coverslip on surface. The slides are observed by means of a binocular microscope under transmitted light at different magnifications. Histological staging of ovaries was based on the stage of oocyte development and on the occurrence of the postovulatory follicles and atretic oocytes. The reproductive status was based in only the larger and more advanced oocytes.

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Results and Conclusions The microscopic slides observation permits to classified the reproductive status of each female. In Table I is described the microscopic characteristics for hake females maturity stages. The morphological changes observed in the different oocytes growth phases were similar to those established for other species, and the terminology used was adapted from Hunter and Macewicz (1985) and Vitale et al. (2006). In order to guarantee that macrocospic scale is more objective a new scale has been created with four stages shown in Table II.

Maturity Stages Microscopic Characteristics Immature (Figure 1) Unyolked oocyte (23 – 87 µm) Resting (Figure 1) Unyolked oocyte; perinucleolar stage oocytes and atretic oocytes (141-341 µm) Development Cortical alveoli oocytes (105 – 319 µm) and atretic oocytes (191-331µm) (Figure 2) Spawning Migratory nucleus stage oocytes (542-684µm); hydrated oocytes (317-828µm) (Figure 3) Post-Spawning Post-ovulatory follicules (86-375µm) and atretic oocytes (144-414µ) (Figure 4)

Table I - Microscopic maturity stage key for female hake with the development oocytes stages present in each one of the four maturity stages and with the minimum and maximum diameter (µm).

Maturity Size Shape Colour Opaque Hyaline Macroscopic Stages OocytesA OocytesB Aspect Immature/ Small Elongated Pink/transparent Absents Absents Resting Development Medium/ Cylindrical Pink/orange Presents Absents Without stepped Large on areas or hurt areas Spawning Large Cylindrical Pink Presents Presents Gonad is fine; hydrated oocytes flow under pressure in the abdomen Post- Large Shrunken Pink dark Presents Absents Flaccid spawning

Table II - Macroscopic maturity stage key for female hake based on histological classification.A – Vitellogenic oocytes; B – Hydrated oocytes.

However, the macroscopic staging method continues to deal with some constraints specially related to the following stages: immature/resting, development/post-spawning. In indeterminate species the oocyte maturation development is like a continuous cycle, what explains the macroscopic misclassification between development and post- spawning. The misclassification of immature/resting could lead to an underestimation of the spawning stock biomass and conducted to inadequate management options. The use of histological maturity keys becomes usefully to implement accurate and objective maturity determinations.

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References Alvarez, P., Motos, L., Uriarte, A., Egaña, J. 2001. Spatial and temporal distribuition of European hake Merluccius merluccius (L.), eggs and larvae in relation to hydrographical conditions in the Bay of Biscay. Fisheries Research 50:111-128 Goñi, R., Alderstein, S., Alvarez, F., Gárcia, M., Sánchez, P. Sbrana, M., Maynou, F. and Viva, C. 2004. Recruitment indices of European Hake Merluccius merluccius (Linnaeus, 1758) in the Northwest Mediterranean based on landings from bottom- trawl multispecies fisheries. ICES – Journal of Marine Science, 61: 760-773 Hunter, J. R. and Macewicz, B. J. 1985. Measurement of spawning frequency in multiple spawning fishes. Lasker - An egg production method for estimating spawning biomass of pelagic spawning fish: Application to the Northern Anchovy, Engraulis mordax – NOAA Technical Report NMFS 36:67-77 Hunter, J. R., Macewicz, N. C. H- Lo, and Kimbrell, C. A. 1992. Fecundity, spawning, and maturity of female Dover sole, Microstomus pacificus, with an evaluation of assumptions and precision. Fish. Bull., U.S. 90: 101-128 Vitale, F., Svedang, H. and Cardinale, M. 2006. Histological analysis invalidates macroscopically determined maturity ogives of the Kattegat cod (Gadus morhua) and suggests new proxies for estimating maturity status of individual fish. ICES – Journal of Marine Science, 63: 485 – 492

Figure 1- Histology sections of immature (left photos) and resting (right photo) ovaries of hake, U. - unyolked oocytes; P. - perinucleolar oocytes.

Figure 2 - Histology sectins of development ovaries of hake, U. - unyolked oocytes; C.A. - cortical alveoli oocytes; MN- migratory nucleus stage oocyte; A. - atretic oocyte.

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Atretic process in European hake ovary in relation to reproductive stages *Korta, M., ^Dominguez, R., *Murua, H. and ^F. Saborido-Rey. *Azti-Tecnalia, Herrera Kaia-Portu aldea z/g, 20110 Pasaia, Spain. [email protected] ^Instituto de Investigaciones Marinas, c/Educado Cabello 6, 36802 Vigo, Spain.

Atresia etymologically means the absence of perforation, and in the particular case of fish ovaries, it refers to oocytes that remain occluded inside the follicular layer. When that happens, vitellogenic oocytes undergo into a degenerative process that individual fish use to reabsorb the energy of yolk stored for the further egg nutrition. The degenerative process starts with the break down of the chorion and finishes with the invasion of hypertrophied Granulosa layer into oocyte core (Fig.1).

Fish oocyte atretic process has proved to be associated to fish poor nutrition condition, lack of appropriate environmental conditions or exposure to pollutants (Rideout et al., 2005). However, it is also a natural process that happens throughout the life of a mature fish. During the spawning period, i.e. pre-spawning, spawning and post-spawning season, atretic oocytes appear in the ovaries at different intensity levels. Moreover, the variation of atresia intensity or oocyte atresia pattern may be related to the reproductive strategy of fish.

The difference in ovarian atretic oocyte levels during the spawning season in European hake, Merluccius merluccius, is used here to intent to identify the particular state of the fish along the spawning season. The ovary of European hake has two elongated lobes oriented longitudinally joined by an oviduct that ends in a cloacae. This ovary is cyst- ovarian type (Hoar, 1969); the ovary is a hollow organ in which numerous lamellae containing oogonium are projected into the central lumen. The European hake is considered to be a batch spawner species of indeterminate fecundity (Murua et al., 2006 in press)

Out of the spawning season the ovary shows squeezed smallest primary growth oocytes developed through folliculogenesis from oogonium. They show the characteristic intensely staining basophilic cytoplasm and multiple nucleoli nucleus. At this ovary stage is not easy to distinguish immature individuals from the ones that they have already spawned at least one time in their life (Fig.2).

Prior to the beginning of the spawning season, oocytes mature through vitellogenesis asynchronously (Marza, 1938; Wallace and Shelman, 1981). Thus, all stages of oocytes are found in the ovary without any dominant population and they are distributed homogeneously within the lobes (Alheit et al., 1983; Murua et al., 2003). Ripe European hake store vitellogenin and lipids in concentric layers around the nucleus of spherical oocytes, mainly dyed with eosin (Fig.3). Atresia is nonexistent.

During the spawning season oocytes matured to hydration in batches and once the ovulation occurs, oocytes break down the follicular layer and drop into the ovarian lumen to be released (Holden and Raitt, 1974). At the same time, during spawning season, new vitellogenic oocytes are developed continually by the “de novo” 26 vitellogenesis (Hunter and Goldberg, 1980). Few atretic advanced vitellogenic oocytes appear scattered among wealthy oocytes and postovulatory follicles in still reproductive females (Fig.4).

When an individual enters in a post-spawning period, the stock of largest vitellogenic oocytes, that have been not shed, become atretic. A post-spawning ovary presents, at a first state, more than half of the advanced vitellogenic atretic oocytes (Murua et al., 2006 in press). Some hydrated oocytes from last batch do not drop into the ovarian lumen and get also trapped in the estroma (Fig.5). In the next step of reabsorption, the atretic oocyte of large vitellogenic oocytes reach the 100 % and early vitellogenic oocytes become atretic too. All vitellogenic oocytes are atretic afterwards (Fig.6).

Post-spawning is followed by a recovery stage which is considered as a non- reproductive recovery season. In that stage, vitellogenic oocytes are at late atretic stage or almost disappeared. Cortical alveoli oocytes degenerate and the primary growth oocytes constitute the reproductive cell population reservoir for the next spawning season. There are signs of haemorrhage. Old beta atretic oocytes remain together with late postovulatory follicles before they disappear completely (Fig.7). Recovered ovaries and immature ones present no difference between each other. However, both stages can be sometimes differentiating due to a thicker ovarian wall in recovering fishes (Fig.8).

On the other hand, there are found rare ovary stages where cortical alveoli are atretic but no past spawning activity is evidenced, indicating a pause in oocyte maturation (Fig.9) which can be interpreted as skip spawning. In any case, this is not a very common process in European hake.

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Fig.1. Alpha atretic advanced vitellogenic oocyte. Fig.2. Ovary constituted of primary growth oocytes. Fig.3. Ripe ovary with all oocyte stages developed through vitellogenesis. Fig.4. Ripe ovary involved in spawning process. Fig.5. Early post-spawning ovary. Fig.6. Advanced post-spawning ovary.

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Fig.7. Recovering ovary.

Fig.8. Thick ovarian wall in recovering ovary.

Fig.9. Ovary with oocyte maturation in pause

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Early gonadal development in the orange-spotted grouper, Epinephelus coioides (Serranidae, Epinephelinae) Min Liu & Yvonne Sadovy (The Swire Institute of Marine Science, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China)

Sexual patterns in groupers (Serranidae, Epinephelinae) include both gonochorism and protogynous hermaphroditism, according to a perspective of reproductive function expressed in individuals during their lifetimes. Most histological studies of protogynous groupers have focused on changes in gonadal morphology undergoing sex change from functional females to males rather than on gonadal development prior to first sexual maturation. Protogyny can include one or two male pathways of sexual development. While in some families, these two different pathways might be reflected in different gonad structure, this does not appear to be the case in serranids wherein all males, irrespective of gonadal ontogeny appear to have a similar testicular structure. Early sexual differentiation and diandry have been little studied in the Serranidae and more work is needed to understand the cellular and morphological changes that occur during sexual differentiation and development to understand the developmental basis of different pathways of male development.

The orange-spotted grouper Epinephelus coioides (misidentified as ‘E. suillus’ or ‘E. tauvina’ in early publications), widely distributed in Indo-West Pacific, is protogynous hermaphroditic, a common sexual pattern in the genus. Diandry was suggested on the discovery of a few small males (2-3 years old and <55 cm SL, similar to females), indicating that not all males pass through a functional female phase. However, there is no detailed gonadal description of sexual differentiation in this species, despite its importance in mariculture in Southeast Asia. The aim of this study was to describe early gonadal development of E. coioides, from the larval phase (week-1 after hatched) to first sexual maturation (about 2-3 years old), using paraffin- and resin-embedding techniques, to examine primary male development and for a better understanding of the sexual pattern of this species.

Specimens were collected between October 2004 and June 2006, and will continue to be collected until October 2006. To date, 680 specimens, all from captive breeding, between week-1 and week-117 after hatching, and ranging between 2.8 mm notochord length (NL) and 37.5 cm standard length (SL), have been collected every 2-5 weeks. The results are summarized here. Paired gonadal ridges were first detected at week-3 with a few somatic cells (SC) (Plate Ia); blood vessels (BV) and a gonium (G) first appeared in the ridges at week-7 (Plate Ib) and week-12 (Plate Ic), respectively. All gonads developed an ovarian lumen, completed as early as week-16, with a steady increase in gonium number (Plate Id). Meiotic division of gonia (meG) was first detected at week-30 (Plate Ie), concomitantly with the appearance of primary-growth stage oocytes (O1) (Plate If). Nuage (Nu) in gonia with basement membrane (BM) surrounded were detected by TEM (Plate IIa & b). The ovarian-phase gonads did not change fundamentally between week-30 to week-82; primary-growth stage oocytes (O1) did not exceed 110 µm and ovarian lumen formation is completed (Plate IIc & d). Bisexual 30 gonads (with both male and female germ cells) were first noted at week-86; spermatogenetic tissue (ST) was scattered along the germinal epithelia of the lamellae, together with primary-growth stage oocytes (O1) (Plate IIe & f). From this bisexual phase, some individuals developed into functional females; female sexual maturation was first found at week-113 (May 2006 specimens), with vitellogenic stage oocytes.

Although male development has not been detected in all specimens collected, bisexual gonad development prior to first sexual maturation probably reflects sexual plasticity in E. coioides (as already described for Cephalopholis boenak). Juvenile sexual differentiation by social control is being conducted in captive animals to determine whether social factors influence juvenile sexual differentiation of this species (as shown in C. boenak).

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Plate I. (a) Week-3 larva (0.61 cm standard length, SL) with a few somatic cells (SC) in paired gonadal ridges; (b) Week- 7 juvenile (1.9 cm SL) with more somatic cells in left, gonadal ridge and blood vessel (BV) appear; (c) Week-12 juvenile (2.7 cm SL) with a single gonium (G, arrow) in right, gonadal ridge; (d) Week-16 juvenile (8.2 cm SL) with two gonia (G, arrows) in a section; (e) Week-30 juvenile (13.0 cm SL) with gonia in meiotic division (meG, arrow). No primary-growth stage oocytes (O1) in this stage; (f) Week-38 juvenile (19.0 cm SL) with meG (arrow) and primary-growth stage oocytes (O1). (c) and (d) with resin embedding; the others with paraffin embedding. 32

Plate II. (a) Week-38 juvenile (17.5 cm SL) with three gonia (G) together. Nu, nuage; (b) Higher magnification of (a) with basement membrane (BM); (c) Week-69 juvenile (23.2 cm SL) with primary-growth stage oocytes (O1) and ovarian lumen (L); (d) Week-82 juvenile (27.5 cm SL) with primary-growth stage oocytes (O1) and ovarian lumen (L); (e) Week-86 juvenile (31.5 cm SL) with primary (O1), ovarian lumen (L) and spermatogenic tissue (ST); (f) Week-91 juvenile (25.8 cm SL) with primary (O1), ovarian lumen (L) and spermatogenic tissue (ST). (a) and (b) with transverse electronic microscope; the others with paraffin embedding.

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Ovarian classification—standardization of the nomenclature By Dr. Susan K. Lowerre-Barbieri and Dr. Luiz Barbieri F.W.R.I./F.W.C. 100 8th Ave. SE; St. Petersburg, FL 33704. [email protected] and [email protected]

As fisheries management shifts towards an ecosystem approach, there will be an increased need for reproductive data over large spatial and temporal scales. Because of the variety of methodologies and nomenclature currently being used in fisheries reproductive research, it is not currently possible to combine data sets over the necessary scales (e.g., Bromley 2001). The foundation of many fisheries reproductive studies is the ovarian classification scheme (also known as grading keys or systems) because these gonad classes are used to determine such reproductive parameters as: size at maturity, duration of spawning season, diel periodicity, spawning location, and fecundity. Pollard (1972) suggested that most species could be classified based on the Hjort (1910) scheme (from Hay, 1985): (1) undeveloped (virgin); (2) starting; (3) developing; (4) maturing; (5) mature; (6) ripe; (7) spent; (8) recovering. And in fact, most ovarian classification schemes include an immature class, one or more maturing/developing classes, one class for ovaries capable of spawning (i.e., fully yolked), 1-2 classes for hydrated ovaries (gravid and running ripe) and various stages for “spent” ovaries (Hunter and Macewicz (1985). However, the names or terms to describe ovarian classes are highly varied and sometimes the same ovarian classification term can be used to mean different things such as the term, “ripe” or “developing”. Ovarian classification nomenclature appears to be derived from terms used before histological analysis became commonplace. More recent reproductive studies, which have incorporated histological analysis, continue to use these terms. Gonad class names apparently are chosen based on either the frequency with which they are used in the literature or how descriptive they are of the process being identified (i.e., developing, spawning, regressing) and thus are somewhat subjective. Developing a clearly-defined naming procedure might remove much of the confusion currently associated with gonad classes. We propose naming the classes based on the histological criterion used to define them (regardless of what classes are chosen). To demonstrate our proposed nomenclature, we chose to apply it to the ovarian classes associated with a multiple spawner with indeterminate fecundity. We chose this as the example, as it is both prevalent and fairly complex. We looked at the histological definition for eight commonly used classes (Table 1), all but one of which can easily be named using the histological criterion (Table 2). The only classes which can not be easily distinguished, based on clear histological criterion, are those of immature females versus those of females outside of the spawning season. Hunter and Macewicz (2001) found no histological criterion that could be used to distinguish these two ovarian classes in Microstomus pacificus and suggest that the best way to reduce bias associated with incorrectly assigning these classes is to sample in a period just before the onset of spawning, when post spawning females would be expected to be uncommon. However, Wyanski and Pashuk (2001) described the appearance of “muscle bundles” in the ovigerous lamellae as a means of distinguishing immature females and others (Morrison 1990) have used the thickness of the ovarian wall as a distinguishing 34 characteristic. Thus, as a group we are left with the decision as to whether immature ovaries can truly be assigned to an ovarian class or whether assigning maturity should be considered a process outside of ovarian classification and addressed separately. If we do keep immature as a separate class, then we suggest primary growth/immature and primary growth/mature as the names for these classes. In the literature there are examples of other attempts at standardizing the ovarian classification scheme. Hilge (1977) addresses many of the same issues we are addressing in the year 2006, including the contentious issue of naming a class “resting”, which apparently was first raised by Franz in 1910. “Who is able to say, when the ovary is more active, during the processes of resorption and regeneration, or at the growth period of the eggs?” Which brings to the forefront the issue of how can we interest other scientists in using a standardized nomenclature?

Literature cited Bromley, P.J. 2001. Progress towards a common gonad grading key for estimating the maturity of North Sea Plaice. In Kjesbu, O.S., Hunter, J.R., and P.R. Witthames (editors), Report on the working group on Modern approaches to assess maturity and fecundity of warm- and cold-water fish and squids. Institute of Marine Research. Bergen, Norway.

Casselman, J.M. 1987. Determination of age and growth. Pages 209-242, Chapter 7 in A.H. Weatherley and H.S. Gill. The biology of fish growth. Academic Press, London. 443 pp.

Hay, D.E. 1985. Reproductive biology of Pacific herring (Clupea harengus pallasi). Can. J. Fish. Aquat. Sci. 42 (Supplement 1):111-126.

Hilge, V. 1977. On the determination of the stages of gonad ripeness in female bony fishes. Meeresforschung (25) 149-155.

Hunter, J.R. and B.J. Macewicz. 1985. Measurement of spawning frequency in multiple spawning fishes. In R. Lasker (editor), An egg production method for estimating spawning biomass of pelagic fishes: application to the northern anchovy, Engraulis mordax, NOAA Technical Report NMFS 36, 79-94.

Hunter, J.R. and B.J. Macewicz. 2001. Improving the accuracy and precision of reproductive information used in fisheries. In Kjesbu, O.S., Hunter, J.R., and P.R. Witthames (editors), Report on the working group on Modern approaches to assess maturity and fecundity of warm- and cold-water fish and squids. Institute of Marine Research. Bergen, Norway.

Morrison, C.M. 1990. Histology of the Atlantic cod, Gadus morhua: an atlas. Part three. Reproductive tract. Can. Spec. Publ. Fish. Aquat. Sci. 110: 177 p.

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Pollard, D.A. 1972. The biology of a landlocked form of the normally catadromous salmoniform fish, Galaxias maculatus (Jenyns). III. Structure of the gonads. Australian Journal of Marine and Freshwater Research 23, 17-38.

Wyanski, D. M. and O. Pashuk. 2001. A histological potpourri: Characters to distinguish immature and resting reproductive classes in females, sex transition in serranids and sparids and post-mortem artifacts. 2001 AFS Southern Division histology workshop.

Table 1. Histological criterion used to define the ovarian classes for a gonochoristic multiple spawning fish and what this class indicates.

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Table 2. Potential nomenclature for ovarian classes based on the histological criterion used to define them compared to the various names used for these classes in the literature.

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Histological classifications of marine fish ovaries and its use in assessment of maturity, spawning and fecundity; insights gained from analysis of nine eastern Pacific Ocean teleost species that spawn pelagic eggs and a squid. Beverly J. Macewicz Southwest Fisheries Science Center, National Marine Fisheries Service, NOAA 8604 La Jolla Shores Drive, La Jolla, CA 92037-1508 E-mail address: [email protected]

Background: It is important for stock assessment and to managers of a fishery to have data on reproductive state of the fish population. They want to know spawning season, maturity schedules or ogives, fecundity, and spawning rates. Histology of the gonads can provide finer and more accurate information necessary to examine or estimate all of these parameters. My analyses have concentrated on female marine fish (spawning pelagic eggs) and their ovaries because the data is critical either for the estimation of spawning biomass, by the daily egg production method (DEPM, Northern anchovy (Engraulis mordax), Peruvian/Chilean anchoveta (Engraulis ringens), Pacific sardine (Sardinops sagax)) or by the fecundity reduction method (Dover sole (Microstomus pacificus) and sablefish (Anoplopoma fimbria)), or for stock assessment (Pacific mackerel (Scomber japonicus), jack mackerel (Trachusus symmtricus), skipjack tuna (Katsuwonus pelamis), hake (Merluccius productus)).

Using histological characters to describe reproduction: Characters (structures) may be evident in the ovary for different periods of time. Evidence may depend on temperature, food supply, the amount of time to or elapsed after peak daily spawning, or even availability of the female to capture (migration, sampling gear). Fecundity either batch (number released in one spawning) or potential total (number released during whole season or year) requires counting the proper oocyte stage. In addition to counting hydrated oocytes (and no new postovulatory follicles present), oocytes in stages as early as beginning migratory nucleus can be used to estimate the batch if in whole oocyte preparations they are distinct from other yolk oocytes (T. symmetricus). Misidentification as indeterminate for a species that fixes total fecundity may occur if females are collected only during spawning because ovarian histology may show numerous batches developing and postovulatory follicles (M. pacificus). Histology can identify advanced yolk oocytes in ovaries but total fecundity will be inaccurate unless the oocytes are measured and verified to be separate from other yolking oocytes (A. fimbria). Postovulatory follicles (POFs) are usually distinct 24 hours after spawning. Subsequent degeneration varies by species and water temperature: 24 hours after spawning degeneration is more at 24°C (K. pelamis) than at 16°C (S. japonicus). More than one spawning event can be detected (Fig. 2) and duration estimated either by later migratory nucleus/hydrating oocytes (if <30h before spawning) and aged POFs or by 2 or more ages of POFs (i.e. S. japonicus, T. Symmetricus, M. productus). Prevalence of spawning females in a population [any imminent (hydrating ooctes present) or recently (<60h old POFs present)] is not the same as the daily spawning fraction of mature females needed by DEPM to estimate spawning biomass. Daily 38 spawning fraction requires identifying characters (hydrated oocyte, POF) that are present within 24 hour periods; more than one period can be averaged. Individual female spawning frequency (S. japonicus every 1-2 days) may be different than the population average (about every 12 days) if spatial separation or differential movements by spawning or inactive postspawning females occur. A representative sample of the population is necessary but may be difficult to obtain. Identification of atresia (degeneration and absorption of an oocyte and its follicle) and duration of stages (Fig. 2) is necessary to ensure postovulatory follicles are identified and aged correctly, separate postspawning mature females with regressed ovaries from immatures (although some may become indistinguishable, i. e. M. pacificus), and a key histological marker for the cessation of spawning (high prevalence of high intensity of atresia). Ovarian activation is not sexually maturity (except for the very first time). When setting criteria for classification of maturity, in addition to the oocyte development stage, criteria indicating evidence of spawning (postovulatroy follicles), possible past spawning (beta stage atretic structures), or possible past developement (alpha stages of any oocyte) must be considered and most likely included.

Recommendations for initial histological analysis of a new species:  conduct temperature specific laboratory maturation, spawning, and starvation studies...... or  sample the population near the peak or within the spawning season and at various hours of the day, record temperature  preserve ovaries immediately after capture and death, or within 1-2 hrs, and do not freeze or heat (ooctyes may appear abnormal (artifact); stress may induce early spawning and/or alpha stage atresia in yolked oocytes, e.g. skipjack tuna)  obtain good histological serial sections <6 microns thick and stained well  record all histological characteristics of the ovary before attempting to assess state - development of oocytes present and indication of size or intensity: the most advanced and any other stages (oogonia, primary, cortical alveoli, lipoid vesicle (droplet), vitellogenic (yolking.....advanced yolk), migratory nucleus, and hydrating/ed) - postovulatory follicles - indicate amount of degeneration - atresia – alpha stage: any degenerating oocyte (with/without yolk), note intensity; beta stage, gamma/delta stages - ovary wall thickness (measured or qualitative) and note if oocytes are in lamellae contiguous with whole wall (e.g. N. anchovy), partial (e.g. Pacific sardine), or at one spot (e.g. Sebastolobus sp.) - parasites or abnormal cells

Universal ovarian classification: Universal names can be assigned for the histological structures in marine oviparous ovaries. Most criteria (i.e. hydrated oocytes present and no degenerating POFs) indicate obvious reproductive states and we can develop universal classes (i.e. mature – ovulation). Some criteria may indicate more than one state (uncertain maturity) and a flexible class(es) may be needed (?species dependent).

39

A A

B B

C C

Figure 1. Jack mackerel ovaries (histological sections and whole oocyte preparations and whole oocyte size distributions) indicating final oocyte maturation stages from early (A) and advanced (B) migratory- nucleus stages to hydration stage. Batch fecundity can only be estimated from females B and C. Although early migratory-nucleus stage oocytes are identifiable in histology section of the ovary of female A, they are not visually distinct to separate in whole oocyte preparations or by size distributions. Bars are 0.5 mm.

40

A

B

C

Figure 2. Top. left, Conceptual diagram showing when various histological stages could be identified in an ovary of a S. japonicus female that spawned every day and right. Bottom. left, Duration of various atresia stages during resorption and recovery in E. mordax ovaries and E. mordax atretic state classification. Top. right, Fecundity estimation during a hypothetical cycle of oocyte maturatrion and spawning for a A. fimbria female. Bottom. right, Dover sole ovaries showing: recruitment (A), within maturity window for fecundity (B), and > 5 spawning events (C; POFs and oocyte stages).

41

Histological classifications of the Pacific sardine (Sardinops sagax) ovary: adaptaions to a basic system.

Beverly J. Macewicz Southwest Fisheries Science Center, National Marine Fisheries Service, NOAA 8604 La Jolla Shores Drive, La Jolla, CA 92037-1508 E-mail address: [email protected]

Histological analysis identifies female maturity, activity (atretic states), imminent or recent spawning (spawning fraction), and those for use in estimation of batch fecundity. Sardinops sagax ovarian histological analyses and reproductive classification has as its basis the classification of Northern anchovy (Engraulis mordax) ovaries. Differences exist in Sardinops sagax ovaries and those seen include: lamellae only present on 2/3 of the ovarian wall, the presence of lipid globules (a.k.a. oil droplets) in developing oocytes, granulosa follicle cells that enlarge to a highly elongated columnar shape in pre- hydration oocytes (migratory-nucleus stages, MN), larger postovulatory follicles (POF) and recognizable longer (possible longer duration), beta stage atresia has lipid vacuoles, more parasites, and evidence of remnant, disintegrating hydrating oocytes. Therefore the data sheet used to record the identified structures present in the histological sections of the ovary of each female E. mordax in a trawl sample (Figure 1A) was modified for recording S. sagax ovarian histology identifications (Figure 1B). The data for each female entered into electronic (computer) databases.

Various combinations of the data are used to analyze adult female reproductive parameters needed for spawning biomass estimation by the daily egg production method (DEPM). Combinations may vary depending on parameter estimated. We also use knowledge of: time of daily peak spawning, when the female was captured and died (time of day and if between seasons or during what part of the spawning season), what was the recent water temperature encountered, and possible duration of criteria (oocyte stages, atresia, or degeneration of postovulatory follicles).

S. sagax female maturity (Table 1). If captured during the spawning season, a mature S. sagax females has spawned before (hours to months ago) or is capable of spawning its first batch within one to two weeks of capture. Hence, immature females have never spawned in their lifetime and may include ovaries with early yolk oocytes if the duration of further maturation (continued vitellogenesis and hydration) will take longer than a couple of weeks. An active female is a mature female that is capable of spawning (in a few hours to two weeks) or had spawned in the last 70 hours; active females are indicated histologically by presence of yolked oocytes (includes migratory nucleus or hydrated), any α atresia is < 50% of yolked oocytes, and postovulatory follicles (POF < 70 hours) may be present (codes 108-115).

Identification of spawning for estimation of spawning fraction. Our sampling of S. sagax occurs during the night between 18:00 and 06:00. During these hours, we can determine 4 spawning events (nights) based on histological criteria: A) spawn tomorrow night, advanced stage MN oocytes; B) spawning the night of capture, hydrated ooctes and/or POF without deterioration or barely begun degeneration; C) spawned last night, POF aged about 18-30 hours old; and D) spawned 2 nights before capture, POF aged about 42-56 hrs old. The postovulatory follicle of S. sagax has large granulosa cells that degenerate over a longer time period. This fact with a thicker theca-connective cell layer has allowed better identification of older POFs (42-56 hrs after spawning). POFs > 66 hrs are less evident (number and size), may be confused with late stage β atresia, and are not used to estimate a spawning event.

Batch fecundity, the number of oocytes to be released during the next spawning. Estimation of batch fecundity is by the gravimetric method using whole oocyte preparations from females that have not begun releasing that batch. Hydrated oocytes can be counted with confidence because they provide the best visual identification (fluid uptake and yolk coalescence results in larger and translucent appearing oocytes). Histological preparation can identify the migrating nucleus and lipid droplet coalescence earlier than can be seen visually in whole oocyte preparations (blocked by the dense yolk globules). The elongated granulosa cell in S. sagax follicles have provided an extra clue that can be seen in whole preps. Hence, we can increase the number of females we use for batch fecundity estimation.

42

A

B

Figure 1. Portions of histological ovarian data recording sheets for E. mordax (A) and S. sagax (B).

Table 1. Ovarian criteria for each code and indication of female maturity. Oocyte class Atresia ♀ Maturityb Spawna ad- α α α anytime within Code no early soon or vance δ no e a r l y β a d v . absolute season yolk yolk recent c yolk y o l k y o l k y o l k I and M + other 101 + I I 102 + - + U most I 103 + - - + U M 104 + - + + U M 105 - + - - U most I 106 - + - - + U most I 107 - + - - - + U M 108 - - + - - - - + ≥ 50% M M 109 - - + - - - - + ≥ 50% + M M 110 - + - - + + + M M 111 - + - - - + M M 112 - - + - - - - M M 113 - - + - - - - + <50% M M 114 - - + - - - - + M M 115 - - + - - - - + <50% + M M blank = not present; - = may be present; + = must be present

a spawn = presence of hydrated oocytes or recent POF, degeneration <70 hours after spawning) b female: I = immature, U = uncertain (add criteria or other information to determine), M = mature c other data used during season: β atresia indicate mature; ovaries of immatures (some may have begun developing) have no δ atresia and thin-medium ovary walls, while ovaries of previous mature females have δ and thick walls in these classes, hence 102, 105, 106 will be split.

43

Classification schemes for monandric, protogynous fishes: a review and extension of Moe’s (1969) model Richard S. McBride, Jen M. Funk1, and Angela B. Collins Fish & Wildlife Research Institute, Florida Fish & Wildlife Conservation Commission 1Eckerd College, St. Petersburg, Florida [email protected]; [email protected]; [email protected]

Progress in science requires formulation of clearly-stated models and supporting terminology for the development of such models. In this sense, Martin Moe’s (1969) investigation of the biology of red grouper (Serranidae: Epinephelus morio) stands as a seminal paper for diagnosing and classifying the reproductive processes of a monandric, post-maturational, and protogynous hermaphrodite. While others had set standards for identifying either the ontogenetic or the seasonal reproductive development of hermaphrodites, Moe (1969) synthesized a model that incorporated both of these aspects of maturation (Figure 1). In addition, he correctly used the terms “stage” and “class”, which were in the literature to characterize the development of germ cells vs. the gonad (= individual), respectively.

According to the “Web of Science”, 130 peer-reviewed papers have cited Moe (1969), many citing his reproductive model specifically. We review these citations, noting how nearly all of these studies have accepted Moe’s scheme of 10 classes without modification. In a recent, unpublished investigation of hogfish (Labridae: Lachnolaimus maximus) reproductive biology we found it useful to break up and reorder some of the transitional and male classes (Figure 2). In particular, we have added classes that allow tracking of the rate of sex change, which in L. maximus occurs over several months spanning the post-spawning season. The main criteria for these new classes rest on the pace of oocyte degradation in ovo-testes, a process that Moe (1969) foresaw when he noted (p. 27): “Males are also broadly divided as early and old males, based on the presence or absence of early oocytes (oocyte Stage 2) in the matrix of testicular tissue. Early males contain great numbers of Stage 2 oocytes held over from the female phase of sexuality. These early oocytes diminish in size and number as the male ages and eventually disappear from the testis.”

We postulate that while Moe’s (1969) model for monandric, protogynous fishes is fundamentally sound, closer scrutiny and further tests with histological data will probably find variation on the basic theme; and these variations themselves will add insight into the reproductive biology of hermaphroditic fishes.

To find: “Moe, M. A., Jr. (1969) Biology of the red grouper Epinephelus morio (Valenciennes) from the eastern Gulf of Mexico. Florida Department of Natural Resources Professional Papers Series Number 10: 1-95,” use http://www.floridamarine.org/publications/search.asp; search for ‘Moe’ as an author

44

1 First maturation 1 First maturation

2 2 Recrudesence Recrudesence Final Final 4 Non-spawning Spawning Oocyte Non -spawning Spawning Oocyte 3 3 Period Period Maturation Period Period Maturation Regression 5 Regression 4 5 6 Sex change Sex change Second maturation 7* Second maturation Recrudesence 6 8* First-spawning as a male 9* 10 Non-spawning Period 7 8 Spawning Period 9

9 Spawning Period 8 Non-spawning Period Regression Recrudesence 7 10 Recrudesence Regression

Figure 1 – Moe’s (1969) model for sexual Figure 2 – The sexual development development of red grouper, Epinephelus of hogfish, Lachnolaimus maximus, a morio , a monandric, protogynous monandric, post-maturational, hermaphrodite. “[T]he seasonal and protogynous hermaphrodite. ontogenetic changes in gonad development Reproductive Classes 1-4 are female in red grouper have been divided into ten and Classes 7-10 are male. Classes classes which form the basis for population 5 -9* represent transitional, and seasonal analyses of sex distribution immature, and first-year males. and spawning. These are frequently grouped From: McBride, R.S. and M. in later analyses into developmental Johnson. unpubl. ms. Sexual and (ontogenetic) classes and seasonal classes reproductive development of hogfish in the reproductive cycle of mature fish. In (Labridae: Lachnolaimus maximus), females, Class 1 is immature and Classes 2, a hermaphroditic reef fish. For 3, and 4 are seasonal [classes] in the mature Biological Bulletin. females. Classes 5 and 6 are developmental classes, transitional and immature male, and Classes 7, 8, 9, and 10 are seasonal classes Major life events are indicated in italics. in the mature male.” (p. 27)

45

Male reproductive classification in gonochoristic marine fishes H. Scott Meister, David M. Wyanski, and Oleg Pashuk South Carolina Department of Natural Resources Marine Resources Research Institute 217 Ft. Johnson Road, PO Box 12559 Charleston, South Carolina 29422-2559

The Marine Resources Monitoring Assessment and Prediction project (MARMAP), a cooperative effort of the South Carolina Department of Natural Resources (SCDNR) and National Marine Fisheries Service (NMFS), employs fishery- independent sampling techniques to investigate the life history and abundance of economically important reef fishes off the Atlantic coast of the southeastern U.S. Additional samples for life-history studies are collected using fishery-dependent sampling techniques. Targeted species are captured using a variety of gear types deployed at stations along the continental shelf from Cape Lookout, North Carolina to West Palm Beach, Florida. To assess the sex and reproductive class of priority species, a transverse section from the posterior portion of the gonads is removed and fixed in 11% formalin. After a minimum fixation period of two weeks, the tissue is transferred to 50% isopropanol at least two weeks prior to histological processing. Samples are then vacuum infiltrated, blocked in paraffin, and sectioned to a thickness of 7 μ using a rotary microtome. Three sections from each sample are mounted on a glass slide, stained with double-strength Gill’s hematoxylin, and counter-stained with eosin-Y. Prepared slides are interpreted on a compound microscope by two readers working independently without knowledge of date of capture, length, or age of specimens. Specimens are first assigned a sex code from 0 – 9 (see “Sex transition in serranids and sparids”; Wyanski and Pashuk, 2005 Gonad Histology Workshop), followed by a reproduction class. In the case of male gonochorists, a sex code of “1” is assigned indicating the gonad is entirely testicular, and reproduction classes from “0– 5” are assigned based upon the degree of testicular development. A class of “0” is used to represent reproductively inactive specimens of known sex but unknown maturity. Males of the gonochoristic species we investigate, like the majority of teleosts, have lobular testicular structure. In this type of testes the gonad is composed of numerous lobules, each ending at the periphery of the testes, and separated from one another by a network of connective tissue (Grier, 1993). Within these lobules, primary spermatogonia undergo mitotic division becoming secondary spermatogonia enclosed within a developing spermatocyst. In a progression of cell development known as spermatogenesis, the secondary spermatogonia within the spermatocysts meiotically divide to form spermatocytes. These spermatocytes then meiotically divide to become spermatids. In a process known as spermiogenesis, the spermatids develop a flagellum and midpiece, and become known as spermatozoa, or active sperm (Takashima and Hibiya, 1995). The increasingly developed germ cells cause the spermatogenic cysts to grow and eventually rupture, releasing sperm that flows from the lobular lumina into the duct system (efferent ducts and the main testicular duct) of each testis, a process known as spermiation (Nagahama, 1983). The main ducts join together to form the 46 common sperm duct, through which the male gametes (active sperm) are transported from the testes to outside the body.

Gonochoristic males are assigned to a reproductive class using the following criteria based on Wenner et al. (1986):

 Class 0 – Uncertain maturity: Inactive testes; unable to assess maturity (i.e., specimen is either immature or regressed) (Figs. 2e, 2f).

 Class 1 – Immature: Small transverse section compared to regressed male; spermatogonia and little or no spermatocyte development; lobules not evident (Figs. 1a, 1b).

 Class 2 – Developing: Development of cysts containing primary and secondary spermatocytes through some accumulation of spermatozoa in lobular lumina (Figs. 1c, 1d).

 Class 3 – Spawning: Predominance of spermatozoa in lobules and ducts; little or no occurrence of spermatogenesis (Figs. 1e, 1f).

 Class 4 – Spent: No spermatogenesis; some residual spermatozoa in shrunken lobules and ducts (Figs. 2a, 2b).

 Class 5 – Regressed: Large transverse section compared to an immature male; little or no spermatocyte development; empty lobules and ducts; some recrudescence (spermatogonia through primary spermatocytes) possible at end of class (Figs. 2c, 2d).

Literature Cited

Grier, H.J. 1993. Comparative organization of Sertoli cells including the Sertoli cell barrier. Pp. 704-739, In L.D. Russell and M.D. Griswold (Eds.). The Sertoli Cell. Cache River Press, Clearwater, FL.

Nagahama, Y. 1983. The Functional Morphology of Teleost Gonads. pp. 223-275, In W.S. Hoar, D.J. Randall, and L.M. Donaldson (Eds.). Fish Physiology, Vol. IXA, Part A, Endocrine Tissues and Hormones. Academic Press, New York.

Takashima, F. and T. Hibiya (Eds.). 1995. An Atlas of Fish Histology. Normal and Pathological Features. Second Edition. pp. 129-153, Kodansha Ltd, Tokyo.195 pp.

Wenner, C.A., Roumillat, W.A., and C.W. Waltz. 1986. Contributions to the Life History of Black Sea Bass, Centropristis striata, off the Southeastern United States. Fishery Bulletin 84(3): 723-741.

47

a) b)

sg MD Class Class 1 1

sg

c) d)

st Class sc TD 2 sc

st

e) f) sz TD sc

Class TD 3 sc sz

Figure 1. a and b) Immature male specimen (ca. 177 mm TL) of Rhomboplites aurorubens in which lobules are not yet clearly defined; c and d) Developing male specimen (334 mm TL) of R. aurorubens; e and f) Spawning male specimen (353 mm TL) of R. aurorubens. MD = main testicular duct, TD = testis duct system, sc = spermatocytes, sg = spermatogonia, st = spermatids, sz = spermatozoa.

48 a) b)

sz sg Class 4

TD sz

c) d)

Class sg 5 TD

e) f)

TD sg Class 0

Figure 2. Transverse sections of testicular tissue from Rhomboplites aurorubens. a and b) Spent male specimen (321 mm TL); c and d) Regressed male specimen (320 mm TL) showing lobular structure of testis; e and f) Inactive male specimen (ca. 201 mm TL) of uncertain maturity (immature or regressing). TD = testis duct system, sg = spermatogonia, sz = spermatozoa.

49

HOW USEFUL HISTOLOGY CAN BE? The European Hake (Merluccius merluccius) case. Cristina Morgado ([email protected]) & Patrícia Gonçalves ([email protected]) DRM – IPIMAR, Av. Brasília, 1449-006 Lisbon, Portugal INTRODUCTION One of the main criteria for judging the status of an exploited fish population is the size of the spawning stock (Bromley, 2003). In Southern stock (ICES div. VIIIc and IXa) of hake (Merluccius merluccius L.), the spawning stock biomass (SSB) is currently assessed analytically on an annual basis, using an annual sex combined maturity ogive based on macroscopic gonad identification (ICES, 2006). Macroscopically, both the virgin ovaries (immature) and resting ovaries (mature) are similar. Misclassification of resting mature fish leads to an underestimation of SSB conducting to inadequate management options. Misclassification depends on several parameters, namely fish length/age and spawning season period. The older the fish, the higher the probability of being mature and the more distant to the spawning peak, the more probable to find mature fish in resting stages (Birkeland & Dayton, 2005). This work highlights the macroscopic similarity and the microscopic differences of immature and resting mature hake gonads. Material and Methods 20 female hake’s fresh gonads were analysed. Samples were collected at the end of the spawning period (April) according to Piñeiro & Sainza (2003). Macroscopic maturity stages were attributed according to an international 4 stages maturity scale (I: immature; II: developing; III: spawning; IV: post-spawning) (BIOSDEF, 1998). The gonads collected after sampling procedure were transferred to 70% alcohol and fixed 2 days later in 10% formaldehyde solution. Ovaries samples were dehydrated and embedded in paraffin using standard routine histology procedures. From each gonad, sections of 5 μm were cut in a rotating microtome and stained with Harri's haematoxylin and eosin. The slides were mounted using Entellan® with a coverslip on surface. The slides were observed by means of a binocular microscope under transmitted light at different magnifications. In the ovaries, the reproductive status was based only on the larger and more developed oocytes. Macroscopic immature female hake gonads are small, cylindrical, transparent or pinky with no opaque and hyaline oocytes. The main microscopic characteristic of immature female gonads is the presence only of unyolked oocytes and similar diameter of oocytes among them, while mature ones present signs of vitellogenic development, atretic reabsorption and the presence of residual oocytes. The annual sex combined maturity ogives used for Southern hake stock assessment were fitted in accordance to the detected errors based on the histological results. Assumptions include a correct male maturity stage attribution and sex proportion of the maturity samples to estimate maturity ogives of 1:1, and similar misclassification of immature female errors for all length distribution and spawning season. Results 6 of the 20 females were macroscopically classified as immature (inactive). However, the histological analysis shows that 3 were immature and the remaining 3 resting mature. The total length of those specimens is between 30.5 and 46.1 cm, which correspond to 2004 maturity ogive (ICES, 2006) to around 0.32 to 0.75 proportion of 50 mature. In both cases, macroscopically, gonads are small, cylindrical, pinky and without visible opaque and hyaline oocytes (Figures 1.A2-B2 and 2.C2-D2). The slides observation of immature females revealed that all oocytes present in the gonad had no signs of vitellogenesis (Figures 1.A3 and 1.B3). In the histological preparation of Figures 2.C3 and 2.D3 atretic vitellogenic oocytes and oocytes are visible in perinucleolar or circumnucleolar stage, which indicates signs of maturation. The following picture shows the mean mature proportion for the time series 1982–2004 (dashed line: mean maturity ogive used by the ICES WGHMM for the 2004 assessment; solid line: the same with the resting mature correction; grey line: correspondent age for L50% for each case). The resting mature females’ correction reveals that fish began to mature younger, since the first maturity age is lower. This means that, assuming the correction as an approach to the reality, in the ICES WGHMM the SSB is underestimated. The 2004 SSB estimated by the WGHMM is 10,900 tones; while with the resting mature correction is around 13,100 tones. Discussion Macroscopically, both ovaries (virgin/resting) show no signs of development, the distinction is only possible after histological analysis. The main microscopic difference between virgin and resting gonads is the presence in the latter of residual eggs, or atretic vittelogenic oocytes and oocytes in perinucleolar or circumnucleolar stage. In order to get an accurate estimation of the number of individuals with reproductive contribution for the stock maintenance it is imperative to estimate maturity ogive based on histological criteria. In the future, we expect to be able to validate our data to estimate maturity ogive only using samples which their maturity stage was histological identified, to improve stock assessment estimative for European hake. Further work should be aimed to this subject, namely an error estimation of the resting mature misidentification according to the fish length/age and the spawning season period.

References BIOSDEF (1998). Biological Studies of Demersal Fish to European Commission, Final Report to the Commission of European Communities (BIOSDEF Study Contract 95/038). 522 pp. Birkeland, C. & P. K. Dayton (2005). The importance in fisheries management of leading the big ones. Trends in Ecology and Evolution, 20: 7. Bromley, P.J. (2003). The used of market sampling to generate maturity ogives and to investigate growth, sexual dimorphism and reproductive strategy in central and south-western North Sea sole (Solea solea L.), ICES J. Marine Science, 60: 52-65. ICES (2006). Report of the Working Group on the Assessment of Hake, Monk and Megrim (WGHMM), ICES CM 2006/ACFM:01. Piñeiro, C. & M. Sainza (2003). Age estimation, growth and maturity of the European hake (Merluccius merluccius) (Linnaeus, 1758) from Iberian Atlantic waters, ICES J. Marine Science, 60: 1086–1102.

51

A1 B1

A2 B2

A3 B3

Figure 1 – Immature hake female gonads A: fish with 30.2cm total length; B: fish with 36.9cm total length A1-2 and B1-2: macroscopic view (gonads ca. 4cm length) A3 and B3: microscopic view after histological preparation (U. – unyolked oocyte) 52

D1 C1

C2 D2

C3 D3

Figure 2 – Immature hake female gonads

C: fish with 46.2cm total length; D: fish with 32.1cm total length C1-2 and D1-2: macroscopic view (gonads ca. 4cm length) C3 and D3: microscopic view after histological preparation (U. - unyolked oocytes; P. - perinucleolar oocytes) 53

Gonad histology of male round stingrays (Urobatis halleri): seasonal patterns in testes morphology and spermatogenesis Christopher G. Mull, Jennifer J. Granneman and Kelly A. Young, Department of Biology, California State University, Long Beach, CA [email protected]

Round stingrays breed annually during the late spring- early summer, with parturition occurring in autumn. While breeding behavior, gonadosomatic index (GSI), and hormone levels correlate in some species, this is not true of all elasmobranchs. We hypothesized that annual cycles of spermatogenesis, GSI and mating behavior would be correlated in male round stingrays. Round rays were collected monthly for 12 months in Seal Beach, CA. Gonadal tissue was collected for each ray and fixed in 10% buffered formalin for 9 days and transferred to 70% ethanol for nine days prior to paraffin embedding. Tissue was sectioned at 6 µm and stained using hematoxylin and eosin. Based on seasonal changes in GSI the annual reproductive cycle categorized into three phases: inactive (May-July), recrudescent (August-October), and degenerative (November-April). Round stingrays have cystic testes and sperm production occurs within membrane bound spermatocysts in discrete testicular lobes, composed of a primary and secondary lobe. Testisticular lobes, which are not apparent during the quiescent phase, appear on the dorsal surface of the testis during the recrudescent phase in close association with the epigonal organ. During recrudescence testicular lobes increase in size as spermatocysts mature. A spermatogenic index was used to assess spermatogenesis throughout an annual reproductive cycle. The adapted spermatogenic index described seven distinct stages of spermatocyst development (SI-SVII) (Figure 1). Stage I (SI) consists of mainly the primary or germinal zone and is characterized by loosely organized germ cells and primary spermatagonial cells. In this stage, clusters of spermatagonia are associated with Sertoli cell nuclei but no membrane bound structure resembling a spermatocyst has yet developed. Stage II (SII) consists of early spermatocysts containing a peripheral layer of spermatagonia and an inner layer of Sertoli cells surrounding a hollow lumen. In Stage III (SIII), Sertoli cells migrate to the surface of the spermatocyst and spermatagonia begin to undergo meiosis to produce primary and secondary spermatocytes. Stage IV (SIV) is characterized by spermatids that have resulted from the second meiotic division of secondary spermatocytes. These spermatids are smaller than spermatocytes and completely fill the lumen. SIV spermatocysts are seen to have migrated radially toward the periphery of the testicular lobe and collecting ducts. Stage V (SV) spermatocysts are characterized by the immature sperm that have developed from spermatids. This spermiogenesis involves differentiation of the sperm head, midpeice and tail. Individual sperm nuclei are associated with the peripheral Sertoli cells but there is little organization. Stage VI (SVI) spermatocysts consist of mature sperm organized into tight packets. Each sperm pack is associated with a single Sertoli cell and sperm heads are oriented outwards. SVI spermatocysts are only found at the lobe periphery in close proximity to the collecting ducts. Stage VII (SVII) is described as the degenerate zone, and results from the 54 breakdown of spermatagonia once development has ceased. Spermatocysts appear flattened and empty. The primary lobe, on the dorsal surface, contains early stage spermatocysts (SI- SII). As spermatocysts mature they migrate radially through the secondary lobe to the efferent collecting ducts on the ventral side of the testis. Each stage was measured as the relative abundance of each stage in each testicular lobe. During the inactive phase testes were composed primarily of stage I spermatocysts and GSI was reduced as compared to other stages (p<0.05). The recrudescent phase was characterized by an increase in GSI which peaked in October (p<0.001). Maturation of spermatocysts and spermatocytes from early stage I and II spermatocysts to later stages (SIII-SV) was noted during the recrudescent stage. Finally, the degeneration phase was characterized by a decrease in GSI, following the peak in October, (p<0.001) and a final maturation of sperm and degeneration of spermatocysts (SVI and SVII). Interestingly, GSI peaked in October, 5 months before the established mating season, and peak sperm production occurred in December, two months after peak GSI and 3 months prior to mating.

55

Figure 1

A B

C D

E F

Figure 1. Spermatogenic Index: A) Stage II spermatocyst: an outer layer of spermatagonia surrounds and inner layer of Sertoli cells, the lumen appears hollow (40x). Bar = 25 µm. B) Stage III spermatocyst: Meiosis I complete, lumen completely filled with primary spermatocytes (40x). Bar = 25 µm. C) Stage IV spermatocyst: Meiosis II complete, spermatids formed which can be identified by elliptical nuclei (20x). Bar = 50 µm. D) Stage V spermatocyst: spermiogenesis occurring. Sperm tails may be visible but mature sperm are disorganized (20X). Bar = 50 µm. E) Stage VI spermatocyst: sperm mature, organized into tight packets at the periphery of spermatocyst (20x). Bar = 50 µm. F) Stage VII spermatocyst: sperm has been released, may appear flatten or deflated. Some unreleased sperm may be present. Bar = 50 µm.

56

Figure 2

A B

PL

SL

C D

Figure 2. Hematoxylin and Eosin staining of representative tissue from A) February, B) April, C) October and D) December. A) Photo of entire testes cross section from February (degenerative phase). The primary lobe (PL) is visible on the dorsal surface of the testes . Bar = 5 mm. B) April is representative of the quiescent gonadal state observed throughout the summer months when testes are composed solely of SI tissue, with few or no early stage spermatocysts apparent. Bar = 0.5 mm. C) October is the time of peak GSI and is characterized by early-mid spermatocyst stages (SII-SIV). Bar = 0.5 mm. D) December is the time of peak sperm production and is composed of predominantly later stage spermatocysts (SV- SVII). Bar = 0.5 mm.

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Female Reproductive Strategies of Marine Fish Species of the North Atlantic H. Murua1, F. Saborido-Rey2, J. Tomkiewicz3, P. King4 and R. Rideout5 1 AZTI Foundation, Herrera Kaia–Portualde, 20110 Pasaia, Basque Country (Spain). E-mail: [email protected] 2 Institute of Marine Research, Eduardo Cabello, 6. 36208 Vigo (Spain). E-mail:[email protected] 3 Danish Institute for Fisheries Research, DK-2920 Charlottenlund (Denmark). E-mail: [email protected] 4 Mayo Institute of Technology, Galway, Dublin Road, Galway (Ireland). E-mail [email protected] 5 DFO Newfoundland and Labrador Region, P. O. Box 5667, St. John’s (Canada). E-mail: rideoutr@dfo- mpo.gc.ca

Fishes all over the world exhibit great diversity in reproductive strategies and associated traits (Helfman et al., 1997). This includes breeding mode, maturation and spawning pattern, fecundity, number of spawning occasions and partners, gender role, spawning habitat and season, etc. Many common marine, anadromous and catadromous fish species of commercial importance in the North Atlantic and North Pacific are iteroparous, i.e., can breed more than once; they are gonochoristic, i.e., separate sexes without distinctive sexual dimorphism, exhibit external fertilization and provide no parental care, e.g. gadoids, clupeids and flatfishes. However, there are many exceptions e.g., Pacific salmonids (Oncorhynchus spp.) and Lampreys (Petromyzontiformes) are semelparous, i.e., they spawn once and die. Fishes of the genus Sebastes (Atlantic redfishes and Pacific rockfishes) and some elasmobranchs are viviparous, i.e., embryos develop inside the ovary with internal fertilization of eggs. Viviparity in Sebastes is lecithotrophic, which means that larvae absorb nutrients from the accumulated yolk in the egg (formerly known as ovoviviparity). However, two Pacific Sebastes species (S. melanops and S. schlegeli), females seem at least partially to provide food for the embryos during their develoment, i.e., their viviparity is matrotrophic. Also a few hermaproditic species both sequential and synchronous are fished, but mainly in the recreational fishery, such as grouper, seabass and wrass. Their reproductive pattern includes sex-change and sex-reversion. In fisheries biology, the reproductive potential of a fish stock, i.e. its capacity to produce viable eggs and larvae, is used in stock-recruitment relationships to define biological reference points and evaluate stock status. However, the true stock reproductive potential is normally not determined and the total biomass of spawners is used as an index. However, this index rarely reflects the true variability of the stock reproductive capacity and substantial improvements of stock-recruitment relationships can be made by considering the reproductive strategy of the species. In this context, our aim is to classify the reproductive strategy of common commercially exploited fish species of the North Atlantic updating the contribution to the NAFO Working Group on Reproductive Potential (Murua and Saborido-Rey, 2003). In the classification system presented, we included the mode of reproduction as the opportunity to breed once or more times, the pattern of oocyte development and organization in the ovary, oocyte recruitment and fecundity type as well as spawning pattern as these reproductive traits were those that differed most among the exploited species in the region studied (Murua and Saborido- Rey, 2003). The breeding opportunities distinguished between semelparous (species that reproduce only once in their lifetime) and iteroparaous (species that are capable of producing offspring in successive annual or seasonal cycles). Three types of ovarian organization 58 were applied on the basis of Wallace and Selman (1981): Synchronous (all oocytes develop and ovulate at unison); Group-synchronous (at least two populations of oocytes can be recognized in the maturing or ripe ovary i.e. a synchronous population of larger oocytes to be spawned in the coming season and a heterogeneous population of small residual oocytes for future spawning seasons; and Asynchronous (oocytes in various stages of development occur at the same time in the ovaries of ripening and spawning females). Two types of fecundity were applied to describe the strategy by which oocytes are recruited to the advanced stock of yolked oocytes to be shed (Hunter et al., 1992). Determinate fecundity i.e. all oocytes to be spawned in the coming spawning season are recruited at an early stage of the ovarian ripening process. The total number of vitellogenic oocytes prior to the onset of spawning thus is equivalent to the total potential fecundity of the individual female within the spawning season. Indeterminate fecundity describes that first growth phase oocytes continue to be recruited, matured and spawned for a protracted period of time during the spawning season with successive spawning events. Consequently, estimation of the potential annual fecundity requires information about the number of batches spawned per season and batch sizes. The spawning pattern is based on Tyler and Sumpter (1996), who distinguish between: “Synchronous ovulators” or total spawners, i.e. the whole clutch of yolked oocytes are hydrated and ovulated at the same time and the eggs are spawned in a unique event or over a shorter period but as part of a single event, and “Asynchronous ovulators” or batch spawners, i.e. yolked oocytes are recruited in batches for final maturation and successive batches of hydrated eggs are spawned with several days intervals over a longer period. Iteroparous species and with group-synchronous oocyte development, determinate fecundity and batch spawning are frequent among North Atlantic fishes (e.g., gadoids, pleuronectoids). Synchronous oocyte developement, determinate fecundity, and total spawning is a common female reproductive strategy which occurred in a number of semelparous (capelin, Mallotus villosus) and iteroparous species (e.g., redfishes, Sebastes spp., monkfishes, Lophius spp., herring, Clupea harengus, and elasmobranchs). Asynchronous development, indeterminate fecundity, and batch spawning species include e.g. anchovies (Engraulis spp.), sprat (Sprattus sprattus), European hake (Merluccius merluccius), mackerels (Scomber spp. and Trachurus spp.), swordfish (Xiphias gladius). A rare spawning strategy is observed in European eel (Anguilla anguilla) that seems to be an asynchronous, determinate, batch spawner.

HELFMAN, G. S, B. B. COLLETTE, and D. E. FACEY. 1997. The diversity of fishes. Chapter 20. USA: Blackwell Science. HUNTER, J.R., B. J. MACIEWICZ, N. C. H. LO, and C. A. KIMBRELL. 1992. Fecundity, spawning, and maturity of female Dover Sole, Microstomus pacificus, with an evaluation of assumptions and precision. Fishery Bulletin, U.S. 90: 101-128. MURUA, H. and SABORIDO-REY, F. 2003. Female reproductive strategies of commercially important fish species in the North Atlantic. Journal of Northwest Atlantic Fishery Science. Vol. 33, 23-32. TYLER, C. R. and SUMPTER, J. P. 1996. Oocyte growth and development in teleosts. Review in Fish Biology and Fisheries. 6: 287-318. WALLACE, R. and K. SELMAN. 1981. Cellular and dynamic aspects of oocyte growth in teleosts. American Zoologist 21:325-43.

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1 2

3 4

Figure.- (1) and (2) Synchronous oocyte development in late ripening stage of herring and cod. The ovaries show oocytes in first growth phase stage (perinuclear stage and circumnuclear stage) and a homogenous group of vitellogenic oocytes from which batches will be selected for final maturation. (3) Asynchronous oocyte development of European eel in late ripening stage with different cohorts of maturing oocytes, from early lipid vesicle stage to late vitellogenic stage. No first growth phase oocytes are present. (4) Asynchronous oocyte development of European hake in late ripening stage with different cohorts of immature and maturing oocytes, from first growth phase oocytes to lipid vesicle and late vitellogenic stage oocytes.

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PIGMENTED MACROPHAGE AGGREGATIONS AND OVARIAN HAEMOSIDEROSIS IN SILVER CATFISH Chrysichthys nigrodigitatus FROM LOCATIONS ON THE LAGOS LAGOON COMPLEX Oluwatosin M. Olarinmoye. Department of Fisheries, the Lagos State University, Ojo, Lagos, Nigeria. P.M.B. 1087, Apapa, Lagos, Nigeria. [email protected]

Introduction As part of the results of a continuing series of experiments to investigate the effects of aquatic pollutants on fish from estuaries in Lagos state, Nigeria, histological investigations of the gonads of a local species were carried out. Pigmented macrophage aggregates with heavy deposition of Haemosiderin in the ovaries of the silver catfish C. nigrodigitatus from the Lagos lagoon complex are reported. Taking into consideration the polluted nature of the Lagos lagoon complex, it is suggested by the authors that the presence of pigmented macrophage aggregations and hemosiderosis are consequent on exposure of resident fish to a cocktail of aquatic pollutants, and that these features as reported are indicators of pathology occasioned by such exposure. No disease agent (s) has been implicated as the cause of hemosiderosis in the lagoon complex. Toxic and pollution related causes are thus the most probable. Materials and methods Adult C. nigrodigitatus were purchased from fishermen at designated landing sites on the lagoon on consecutive days of the month, spanning a period of six months (December 2004-June 2005). Necropsy and collection of organs were then done in the laboratory. Sectioning, staining and microscopic procedures were carried out according to established, standard histologic methods. Results Hyperplasia of the ovarian capsule was a consistent finding in ovarian sections from all the test locations. This hyperplasia was accompanied by mononuclear cell infiltration, involving macrophages and lymphocytes generally. Melanomacrophage aggregates were common, and ovarian capsular and stromal hemosiderosis were also recorded in specimens. Discussion Results generated in the course of this research (Melanomacrophage aggregates, lymphocytic infiltration and hemosiderosis) were consistent with reports in literature. Macrophage aggregates are known to be putative tissue biomarkers of contaminant exposure in fish, and increase in occurrence with decreasing water quality (Patino et. al, 2003; Facey et. al., 2005), cachectic disease and stress. They have been employed in the monitoring of aquatic pollution ( Couillard and Hodson ,1996). This study focused on the gonadal histology of female C. nigrodigitatus, and the observed features of increased pigmented macrophage aggregates, and ovarian hemosiderosis are indicative and pathognomonic of chronic exposure of fish to chemical pollutants and heavy metals, particularly iron. No chemical analysis of the identity and levels of pollutants in test waters were attempted, but this is intended to be done in later trials, at which time observed symptoms could then be associated to specific individual chemicals and chemical cocktails. This study has however established through histologic evidence, potentially disruptive and pathologic potentials of polluted Nigerian estuaries on resident aquatic fauna and the continuing utility of histology in the biomonitoring of water bodies. 61

The use of histology in refuting claims of sexual lability: an example of a cichlid fish Ronald G. Oldfield University of Michigan Museum of Zoology, 1109 Geddes Ave., Ann Arbor, MI, 48109, U.S.A. [email protected]

The Midas cichlid, Amphilophus citrinellus, has been reported to have sex determined by social conditions (Francis and Barlow 1993). The current report relates a series of investigations that show that social conditions do not play a role in sex determination in this species. The accompanying presentation in the ASIH Neotropical Ichthyology session will provide an overview of these investigations. The presentation described here will focus on the role of gonad histology in these investigations, with emphasis on the developmental pattern observed in this species, the gonad structure of an individual suspected of changing sex, and the presence of ambiguous, unidentified structures.

The first use of histology was to sex wild groups of juvenile fish. According to Francis and Barlow (1993), relatively large juveniles differentiate as males and smaller fish differentiate as females, and this results in the observed size difference in adults. This pattern of development means that any group of Midas cichlids that has differentiated sexually should possess males that are larger than all of the group’s females. Two groups of juveniles were captured in Lake Apoyo, Nicaragua. Their gonads were removed and analyzed histologically. Males were not larger than females in either group. No bisexual gonads were observed: all were undifferentiated or contained exclusively either female or male tissue (Fig. 1a-1c). This indicated that sex is not controlled socially in Midas cichlids in Lake Apoyo.

Several lab experiments were conducted to determine if social control of sex determination might occur in other lineages of Midas cichlids. Captive groups of individually marked Midas cichlids were established from fish originating from different localities. These included fish from the type locality Lake Nicaragua, fish from the pet trade, fish crossed between parents obtained from Lake Nicaragua and from the pet trade, and individuals of the closely related Arrow cichlid, Amphilophus zaliosus, from Lake Apoyo. Sex was not associated with size, except in groups that exhibited high levels of growth. In these groups, males grew faster than females, indicating that the large size of adult males is due to faster post-pubescent growth rates in males rather than large individuals differentiating as males because they are large. Gonads of several individuals were sectioned histologically for verification of sex, comparison of structure to Midas cichlids from the complementary investigations, and to identify the presence of bisexual gonads. Gonad morphology was consistent with the other investigations and no bisexual gonads were observed.

The evidence presented above strongly indicated that sex is not determined by social factors in Midas cichlids. However, the possibility remained that sex is socially controlled in Midas cichlids from Lake Masaya, the source of the fish studied by Francis and Barlow (1993). Twenty five Lake Masaya specimens from the 166 individuals in Lot #76050 at the California Academy of Sciences were dissected and their gonads 62 analyzed histologically. The smallest and largest individuals were intentionally sampled from the lot (20.5-169 mm SL). There seemed to be no association between body size and sex except in adults: the largest males were much larger than the largest females. Gonad structures were consistent with complementary investigations. Despite the presence of atretic oocytes in the testes of many cichlid species, at no point in development were bisexual individuals observed in Midas cichlids: individuals differentiated directly as either female (Fig. 1d, e) or male (Fig. 2a, b). Some ambiguous structures were observed. In some of the females, small, darkly staining structures were observed in a region separate from the ovarian tissue (Figs. 1d, 2c). These structures resembled spermatozoa. However, no spermatogenic tissue was observed so they were interpreted not to be spermatozoa, although their identity remains unknown. In two of the females (82.5 and 99 mm SL) and two of the males (79 and 86 mm SL) amoeba- like cells were present in undeveloped regions of the gonads (Fig. 2d). Midas cichlids from Lake Masaya were concluded not to have sex influenced by social conditions.

Finally, anecdotal reports exist of Midas cichlids changing sex as adults, although such events are thought to be atypical. One Midas cichlid was obtained from a trusted dealer as a previously spawned female. This individual was housed as a member of a group of other adult Midas cichlids. It paired with another female and over several months they attempted to reproduce. After several infertile spawns, one resulted in living offspring. To confirm that this individual changed sex, its gonads were analyzed histologically. No structures that typically indicate previous protogynous sex change were present. Testicular tissue was well developed but no ovarian tissue was present (Fig. 2e). There were no sperm sinuses in the wall of the gonad. A lumen was present in the gonad. At first examination, it was thought that this might be the remnant of an ovarian lumen. However, comparison with ovarian lumena from other specimens revealed a very different structure. This lumen appeared to have formed by overgrowth of the gonadal tissue. The position of the blood vessel indicated that in the posterior region of the gonad, where the mesentary no longer occurred, the gonad tissue wrapped upon itself to form the lumen (Fig. 2f). The lumen was bounded by a membrane indistinguishable from the membrane surrounding the outside of the gonad. The lumen was round, and revealed no indication of dividing the gonad tissue into lamella. I concluded that this individual did not change sex, and that the original supposition was based on misinformation.

Literature Cited

Francis, R. C. and Barlow, G. W. (1993) Social control of primary sex differentiation in the Midas cichlid. Proceedings of the National Academy of Science USA 90, 10673 – 10675. 63

a

c b

Figure 1. Ovary typical of females (a), testiculard structure typical of males (b), and an undifferentiated gonade (c, longitudinal section) in a group of young juvenile Midas cichlids caught in Lake Apoyo, Nicaragua. Typical ovary (d) and cortical alveolar oocyte (e) in Lake Masaya Midas cichlids. AC = amoeboid cells, BM = basement membrane, BV = Blood vessel, CA = cortical alveoli, CH = chromosome, CM = cell membrane, CNO = chromatin nucleolus phase oocyte, G = granulosa, LA = lamella, LU = lumen, M = mesentary, OL = ovarian lumen, PO = perinucleolar phase oocyte, SC = spermatocyte, 1ºSC = primary spermatocyte, 2ºSC = secondary spermatocyte, SD = sperm duct, SGA = spermatogonia A, SGB = spermatogonia B, SLC = sperm-like cells, SZ = spermatozoa, YB = yellow body, ZS = zygotene spermatocyte.

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a b

d c

e f

Figure 2. Testicular structure of male Midas cichlids from Lake Masaya showing (a) lobules composed of spermatocysts and sperm ducts containing sperm of various stages and (b) zygotene spermatocytes. Ambiguous structures in Lake Masaya specimens include (c) sperm-like cells, shown in this enlarged portion of Figure 1d, and (d) amoeboid cells. A gonad from an individual suspected of changing sex is shown in e (anterior) and f (posterior). See Fig. 1 for abbreviations. 65

Oogenesis and maturation of European eel induced by hormonal treatment J. Tomkiewicz1, P. Lauesen2, C. Graver3 and H. Jarlbaek4. 1 Danish Institute for Fisheries Research, DK-2920 Charlottenlund (Denmark). E-mail: [email protected] 2 Billund Aquaculture Service, DK-7190 Billund (Denmark). E-mail: [email protected] 3Danish Eel Farmers Association, DK-6760 Ribe (Denmark). E-mail: [email protected] 4 Danish Institute for Fisheries Research, DK-2920 Charlottenlund (Denmark). E-mail: [email protected] European eel, Anguilla anguilla, is a catadromous species living the continental part of its life cycle in European rivers, lakes and estuaries. During a prepuberty stage, their appearance changes from yellow to silver, they stop feeding and start migrating towards the ocean and the assumed spawning areas in the Sargasso Sea region. Hardly anything is known about the ripening process and spawning of the European eel in nature, but they are assumed to spawn once and die shortly afterwards. Existing knowledge about the gametogenesis and maturation originate from eels in the silvering process and experimental work applying hormonal treatments to ripen the gonads. Silver eels possess oocytes in the lipid vesicle stage, but true vitellogenesis is not observed in wild European eels probably due to the presence of a dopamine inhibitor. Treatment with CPE or SPE (carp or salmon pituitary extract) over some months induces vitellogenesis and maturation. Ovulation normally does not occur spontaneously, but can be induced by e.g. DHP (17α,20β-dihydroxy-4-pregnen-3-one) to obtain hydrated eggs for fertilization. Spermatozoa are obtained from males treated with HCG (human chorion gonadotropin). Embryos rarely develop and mass hatching of larvae has only been reported once by Russian researchers in the 1980’s with larvae living up to 3.5 days. Another mass hatching occurred in spring this year during a series of artificial maturation experiments applying the protocol of hormonal treatment developed for Japanese eel (Tanaka et al., 2003) and it is the results of this study that are reported here.

Farmed female eels in the length range 60 to 90 cm and body weight ranging between 800 and 1200 g were selected for the experiments. The females were transferred to experimental tanks with a water temperature of 20-21˚C and a salinity that gradually was increased from 9 ‰ to 35 ‰ over a period of 7 days. The female eels were treated weekly with SPE injected into the dorsal muscle. Final maturation was induced in the ripe females by injecting DHP in the abdomen, when biopsies taken through the abdominal wall revealed ripe eggs and ovulation took place around 12 hours after treatment. After stripping the eggs, the females were allowed to recover with no further SPE treatment. Some females that ripened again after some weeks were similarly treated with DHP to induce ovulation. The oocyte and ovarian development was followed by sacrificing females randomly chosen from the treated groups each week before the injections. Body and organ weights were recorded and ovary samples were preserved in buffered formalin for histological analysis. In the laboratory, the ovarian tissue was dehydrated and embedded in paraffin using standard procedures, sectioned at 4 μm, stained with haematoxylin and counterstained with eosin, and examined at x 40-100 under the microscope.

The ovaries of female eels sacrificed prior to the start the treatment possessed oocytes ranging from perinuclear stage up to late lipid vesicle stage surrounded by numerous, large adipose cells. This corresponds to the pre-puberty stage observed the onset of the 66 silver eel migration. The degree of development differed among oocytes, but in the most advanced ovaries all oocytes seemed to develop lipid vesicles. The SPE treatments induced true vitellogenesis in the oocytes with yolk grains appearing in the periphery of the cytoplasm among the lipid vesicles (and cortical alveoli which are however indistinct in the H-E stained tissue). As treatment proceeds, the yolk grains and lipid vesicles increase in numbers and gradually fills and expands the oocytes. The adipose cells diminish in size and their contours fade and they are hardly visible in the ripe ovary. During final maturation, the yolk grains coalesce and the lipid vesicles merge into a few large or a single droplet that also is prominent in the hydrated egg. Postovulatory follicles were visible among maturing oocytes in different development stages in stripped females.

The oocyte development was asynchronous mostly with 4-5 cohortes of oocytes at different stages being present in ovaries in late vitellogenesis. No residual oocytes in perionuclear stage were visible in the ripe ovaries. Some females naturally ripened again and a second or third ovulation could be induced. The eggs from these batches were viable and could be fertilized similarly to the first batch, and embryos and single larvae were also obtained. These findings suggest that oocyte development in European eel is asynchronous with several batches of eggs being spawned with a few weeks interval. This contradicts the often stated reproductive status of eels as being total spawners with synchronous oocyte development, but agrees with similar experiments reporting that the eels spawned more than once, and the asynchronous development is unlikely to an artifact caused by the treatment as inferred by Palstra et al. (2003). It is, however, likely that fecundity is determinate, as all oocytes to ripen seems to be recruited at an early stage. Some regulation may, however, take place in form of atresia, as atretic lipid vesicle oocytes were observed early in the treatment as well as atretic vitellogenic oocytes later in the process, but neither the intensity of atresia nor the fecundity has been quantified at present.

The lack of residual oocytes in the ripe eels indicates that the European eel is semelparous, i,e. spawn once and die soon afterwards, but the species seems not to be genetically primed to die after spawning like lampreys (Petromyzontiformes) as females that did not ripen again tended to recover. In an earlier experiment, artificially matured males recovered, resumed eating and were brought to ripen and spawn again (Dollerup and Graver, 1985). The reproductive investment seems, however, to be extreme particularly with respect to the females. In the ripe females, the ovary constituted up to 50 % of the total body weight! Explicitly, the muscle tissue diminished as the ovary grew indicating a successful transfer of lipids and proteins to the oocytes, and in the ripe and spawning specimens, the dorsal and ventral muscles was reminiscent. It is thus probable that eels in nature die after spawning, because they exhaust their physical resources. During the present series of experiments, females that have spawned and recover will be followed for a period of time to investigate their gonadal status and their ability to resume feeding.

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References: Tanaka, H., Kagawa, H., Ohta, H., Unuma, T. and Nomura, K., 2003. The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture. Fish Physiology and Biochemistry 28: 493–497. Palstra, A.P., et al., 2005: Artificial maturation and reproduction of European silver eel: Development of oocytes during final maturation. Aquaculture, 249, 533 - 547. Dollerup, J. and Graver, C.M., 1985. Repeated induction of testicular maturation and spermiation, alternating with periods of feeding and growth in silver eels, Anguilla anguilla (L). Dana, 4, 19 – 39

Figure 1. Ovary of European eel (Anguilla anguilla) early in the pre-puberty stage. The majority of oocytes are in the first growth phase, but single vesicles are seen in the most developed oocytes. Apipose cells constitute a large proportion of the ovary. This specimen is a wild ell. Scale bar: 50 μm. Figure 2. Ovary of European eel by the end of the pre-puberty stage. Oocytes of different sizes are present with the majority being in the lipid vesicle stage. The apipose cells are large and many small blood vessels are visible. Scale bar: 200 μm.

Figure 3. Maturing ovary of European eel: Early vitellogenesis has started and yolk grains and numerous small lipid vesicles accumulate in the periphery of the largest oocytes with a circle of larger vesicles towards the nucleus. The cytoplasm is still visible and the nucleus appears large. Apipocytes have become irregular in shape. Scale bar: 500 μm. Figure 4. Maturing ovary of European eel: The most advanced oocytes have reached mid vitellogenesis. These oocytes have expanded significantly and are filled with numerous small lipid vesicles and yolk grains that give the cytoplasm a red appearance. Less developed oocytes can be grouped as early vitellogenic, and late and and early lipid vesicle stage. Scale bar: 500 μm.

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Figure 4. Ovary of European eel in late ripening stage. The most advanced ooctes are in late vitellogenesis, their cytoplasm is filled with yolk grains and uniformly sized lipid vesicles surround the nucleus. Four less developed groups or cohorts of oocytes are present; one in early vitellogenesis and one approaching mid vitellogenesis as well as late and early lipid vesicle stage. The adipose cells have vanished and no perinuclear oocytes are visible. Scale bar: 500 μm.

Figure 5. Ripe ovary of European eel: Oocyte in final maturation showing coalescence of yolk grains, a few large lipid droplets and nuclear migration, surrounded by oocytes representing three different vitellogenic stages. Scale bar 500 μm.

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OVARIAN STRUCTURE AND OOGENESIS IN VIVIPAROUS FISHES: GOODEIDS and POECILIIDS. Mari Carmen Uribe. Laboratorio de Biología de la Reproducción, Facultad de Ciencias, Universidad Nacional Autónoma de México, México, DF. [email protected]

The gestation in viviparous teleosts occurs in reproductive structures non-homologous to those seen in other vertebrates. These structures are determinated during the ontogenia of female reproductive system, and define the particular type of gestation in viviparous teleosts: the intraovarian gestation. This type of gestation is a consequence of the confluence of three main reproductive characteristics of teleosts: a) the internal position of the germinal epithelium which lines the ovarian lumen, consequently, the ovulation occurs into the lumen, instead of into the coelom, as it is described in the rest of vertebrates; b) the lack of oviducts derived of Müllerian ducts; and, c) the development of internal fertilization. Therefore, the ovary of viviparous teleosts differs from those of other vertebrates because it is the site, not only for oogenesis and endocrine secretion, but also for the entrance and storage of spermatozoa, internal fertilization and gestation. Thus, ovarian structure of viviparous teleosts becomes clearly modified, because the viviparity, and is unique among vertebrates, according to these ontogenetic, morphological and physiological aspects. The features of the ovarian morphology of viviparous teleosts shows similarities and differences between them. The analysis of its similarities and differences in compared studies, have special importance in the understanding of this successful mode of teleost reproduction. This is clear comparing the ovarian morphology of species of two families of viviparous teleosts: Goodeidae and Poeciliidae. Based on these aspects, the aim of this contribution is illustrates the morphological features of reproductive structures in three species of goodeids (Goodea atripinnis, Ilyodon whitei, and Xenotoca eiseni) (Figs. 1A-F), and in three species of poeciliids (Poeciliopsis gracilis, Poecilia latipinna, and Heternadria formosa) ) (Figs. 2A-E), such as, type of ovary, presence of sperms in the ovary, intraluminal gestation (goodeids), intrafollicular gestation (poeciliids), and developing embryos into the ovary. Type of ovary. During the early development of the female embryos, the right and left ovaries fuse forming a single, medial ovary. The ovary is a sacular structure (Figs. 1A-B,2C,D). The wall of the ovary contains germinal and somatic histological components. The germinal components are oogonia and oocytes. The somatic components are: 1) epithelial cells lining the surface of the internal lumen (Fig. 1C); 2) vascularized connective tissue or stroma, subyacent to the epithelium (Figs. 1C,F), and separated by a basement membrane; 3) muscle fibers, in circular and longitudinal disposition; and, 4) serosa surrounding externally the ovary. The internal epithelium is the germinal epithelium, formed by somatic cells and germinal cells. The germinal cells are oogonia and early primary oocytes, at the beginning of the folliculogenesis (Fig. 1C), irregularly situated between the somatic cells. The oocytes in different stages of development and atretic oocytes are surrounded by follicular cells, theca layers and stroma. Because teleosts have not oviducts, the comunication of the ovary to the exterior occurs by the caudal region, where the wall of the ovary forms the gonoduct. The gonoduct opens to the exterior at the genital pore. The ovary of goodeids presents 70 a longitudinal septum in the middle of the ovary (Figs. 1A,B,F). The germinal tissue could be seen in the septum, and all around the wall of the ovary (Goodea atripinnis and Xenotoca eiseni) (Fig. 1A), or it could be situated only in two folds of the wall (Ilyodon whitei) (Fig. 1B). The ovary of poeciliids doesn’t present septum, and the germinal tissue is observed all around the wall of the ovary (Fig. 2D). Presence of sperms in the ovary. The ovary of goodeids presents sperms in the ovarian lumen and in the germinal epithelium around the time of fertilization. No sperms were seen in previtellogenic ovaries, or during middle and late gestation stages. The ovary of poeciliids presents sperms during all the stages of the reproductive cycle. The sperms were seen in the lumen of the ovary and in folds of the wall, irregularly distributed since the anterior to the posterior parts of the ovary. The presence of spermatozoa inside the ovary of viviparous teleosts shows another special characteristic, unique among vertebrates. The germinal epithelium assumes a nurse- cell function, and the spermatozoa are seen embedded, in some of the cells of the germinal epithelium. In other vertebrates, it is the oviduct which assumes the sperm- storage, never the ovary. Oogenesis. The process of oogenesis presents five stages of development: 1) early primary oocytes during folliculogenesis (Fig. 1C); 2) primary oocyte growth during the increase of basophilic ooplasm (Figs. 1C,2A,B); 3) secondary oocyte growth during the formation of lipid vesicles (Fig. 2A); 4) vitellogenesis during the deposit of yolk platelets (Fig. 2B); and, 5) maturation when the yolk is fluid and homogeneous (Figs. 1D,E,2C), and the continuation of meiosis occurs. Different size of oocytes because the reduction of vitellogenesis were seen. The most intense reduction of yolk was seen in the poeciliid Heterandria formosa. The different number of eggs produced at a single cycle, has an evident incidence in ovarian morphology. Viviparous teleosts have a much lower fecundity, than oviparous teleosts. The number of eggs produced in viviparous teleosts, is much lower compared with those in oviparous teleosts Gestation. Goodeid embryos in the lumen of the ovary, since cleavage stage (Fig. 1E), until the latest stage of development (Figs. 1D,F), before birth, were observed. The embryos develop the trophotaeniae, extensions of the hindgut to the ovarian lumen for the absorption of maternally-supplied nutrients, the histotrophe, secreted to the lumen. The competency for secretion of the oviduct, where occurs the gestation in other viviparous vertebrates, in viviparous teleosts is provided by the germinal epithelium which secrets the histotrophe. Poeciliid embryos are situated inside the follicle (Figs. 2C,D), developing a clear vascularized region surrounding the embryos (Fig. 2E). Even there is little information on the regulation of embryonic metabolic wastes in viviparous teleosts, it is assumed that waste products are transported across the germinal epithelium or follicular cells and, subsequently, removed via the maternal vascular system. Superfoetation. The development of embryos in different stages of development, at the same time, was evident in Heterandria formosa (Fig. 2D).

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Female reproductive classification in marine teleost fishes David M. Wyanski and Oleg Pashuk

Marine Resources Research Institute, S. Carolina Dept. of Natural Resources P.O. Box 12559, Charleston, SC 29422 E-mail: [email protected]

Reproductive stage in gonochoristic females is categorized using the following criteria based on Wallace and Selman (1981), Hunter and Macewicz (1985), Hunter et al. (1986), Wenner et al. (1986), and West (1990).  Stage 0 – Uncertain maturity: Inactive ovaries, primary growth oocytes only. Unable to assess maturity (i.e., specimen is immature or resting).  Stage 1 – Immature (virgin): Oogonia and primary growth oocytes only, no evidence of atresia. In comparison to resting female, most primary growth oocytes <80 um (size is species dependent), area of transverse section of ovary is smaller, cords in lamellae lack muscle and connective tissue bundles and are not as elongate, oogonia abundant along margin of lamellae, ovarian wall is thinner (Figs. 1a, 1b).  Stage E – Early developing: Most-advanced oocytes in cortical-alveoli stage (Fig. 1c).  Stage F – Developing: Most-advanced oocytes in yolk-granule or yolk-globule stage (Fig. 1d).  Stage G – Late Developing: Most-advanced oocytes in migratory-nucleus stage; partial coalescence of yolk globules possible (Fig. 1e).  Stage 3 – Ripe: Completion of yolk coalescence and hydration in most- advanced oocytes; zona radiata becomes thinner (Fig. 1f).  Stage B – Developing, recent spawn: Vitellogenic oocytes and postovulatory follicles <12 h old, sensu Hunter et al. (1986), (Fig. 2a).  Stage C – Developing, recent spawn: Vitellogenic oocytes and postovulatory follicles 12-24 h old, sensu Hunter et al. (1986), (Fig. 2b).  Stage D – Developing, recent spawn: Vitellogenic oocytes and postovulatory follicles >24 h old, sensu Hunter et al. (1986), (Fig. 2c).  Stage 4 – Spent: More than 50% of vitellogenic oocytes in alpha or beta stage of atresia, sensu Hunter and Macewicz (1985), (Fig. 2d).  Stage 5 – Regressed: Oogonia and primary growth oocytes only, with traces of atresia possible. In comparison to immature female, most primary growth oocytes >80 um (size is species dependent), area of transverse section of ovary is larger, cords in lamellae have muscle and connective tissue bundles, lamellae are more elongate and convoluted, oogonia are less abundant along margin of lamellae, ovarian wall is thicker and exhibits varying degrees of expansion due to previous spawning (Figs. 2e, 2f).  Stage 8 – Mature, stage unknown: Mature, but inadequate quantity of tissue or postmortem histolysis prevent further assessment of reproductive stage.  Stage 9 – Unknown: Postmortem histolysis or inadequate quantity of tissue prevent assessment of reproductive stage.

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Clarifying comments  Stage 1 vs. stage 5 (immature vs. resting) – Distinguishing these two stages is often the most difficult interpretive exercise, especially if the specimen is in the size range at which sexual maturity is attained. Don’t rely on one or two criteria - attempt to assess all criteria.

The presence of traces of atresia in resting specimens does not refer to melano- macrophage centers (MMC), also known as “gold masses” or “brown bodies”. These macrophage centers, called gamma and delta atresia by Hunter and Macewicz (1985), are not part of oocyte atresia (resorption of yolked and unyolked oocytes), but rather a stage of the immune response by white blood cells to tissue damage caused by spawning or environmental stress/disease (Zapata et al. 1996). Oocyte atresia is accomplished by the granulosa cell layer of the follicle, whereas the immune response is usually initiated by neutrophils that leave blood vessels and move toward the damaged tissue. Monocytes then arrive and immediately become phagocytic macrophages that consume damaged and foreign bodies, including pigments that give them their gold to dark brown color. These macrophages may aggregate to form various sizes of MMC. The presence of MMC in ovarian tissue is highly correlated with sexual maturity given the damage to structural tissue in the ovary during spawning.

 Stages B, C, and D – The rate of postovulatory follicle (POF) degradation is a function of water temperature. POFs were assigned approximate ages based on criteria developed by Hunter et al. (1986) for skipjack tuna Katsuwonus pelamis, a species that spawns at temperatures similar (23-24oC) to those encountered by vermilion snapper spawning on the outer shelf and shelf-edge during summer months. Literature Cited Hunter, J.R., and B.J. Macewicz. 1985. Rates of atresia in the ovary of captive and wild northern anchovy, Engraulis mordax. Fish. Bull. 83:119-16. Hunter, J.R., B.J. Macewicz, and J.R. Sibert. 1986. The spawning frequency of skipjack tuna, Katsuwonus pelamis, from the South Pacific. Fish. Bull. 84:895-903. Wallace, R.A., and K. Selman. 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Am. Zool. 21:325-343. Wenner, C.A., W.A. Roumillat, and C.W. Waltz. 1986. Contributions to the life history of black sea bass, Centropristis striata, off the southeastern United States. Fish. Bull. 84:723-741. West, G. 1990. Methods of assessing ovarian development in fishes: a review. Aust. J. Mar. Freshwater Res. 41:199-222. Zapata, A.G., A. Chiba, and A. Varas. 1996. Cells and tissues of the immune system of fish. Pp. 1-62 In: Iwama, G., and T. Nakanishi (eds.), The fish immune system, Academic Press, Boston. 380 p.

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Stage 1 Stage 1

a) W b)

pgo

c

Stage E Stage F

c) o d)

n

z ca z

100 u y 100 u

Stage G Stage 3 e) f)

ym n o z 100 u ym 100 u

Figure 1. a and b) Immature specimen of Rhomboplites aurorubens (214 mm TL) with thin ovary wall, short lamellae with thin fibromuscular cord, and small primary growth oocytes; c) Early developing specimen of Lutjanus campechanus (417 mm TL) with cortical alveoli formation in oocytes; d) Developing specimen of R. aurorubens (367 mm TL) with vitellogenic oocytes; e) Late developing specimen of R. aurorubens (242 mm TL) with oocytes undergoing final oocyte maturation, which includes migration of nucleus to animal pole and initial coalescence of yolk globules; f) Ripe specimen of R. aurorubens (256 mm TL) with oocytes undergoing completion of yolk coalescence and hydration. c = cord, ca = cortical alveoli, n = nucleus, o = oil droplet, pgo = primary growth oocyte, w = ovary wall, y = yolk globule, ym = yolk mass, z = zona radiata. 76

Stage B Stage C a) b) pof

g t

100 u 100 u

Stage D Stage 4 c) pof d)

ao-  vo

100 u 100 u pof

Stage 5 Stage 5 e) m f) w

mb

pgo 100 u 100 u

Figure 2. a) Developing specimen of Mycteroperca microlepis (840 mm TL) with <12 h old postovulatory follicle; b) Developing specimen of Rhomboplites aurorubens (258 mm TL) with 12-24 h old POF; c) Developing specimen of R. aurorubens (274 mm TL) with >24 h old POF; d) Spent specimen of R. aurorubens (420 mm TL) with oocytes undergoing alpha () stage of atresia; e and f) Resting specimen of R. aurorubens (351 mm TL). In comparison to immature specimen, ovary has thicker wall with well-developed muscle layer, longer lamellae with noticeably- thickened fibromuscular cord, and larger primary growth oocytes. ao-  = alpha stage of atresia, g = granulosa epithelial cell layer, pgo = primary growth oocyte, pof = postovulatory follicle, m = muscle, mb = muscle bundle in cord, t = thecal cell layer, vo = vitellogenic oocyte, w = ovary wall.

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Reproductive classification of gray triggerfish (Balistes capriscus) and female blackbelly rosefish (Helicolenus dactylopterus) David M. Wyanski, H. Scott Meister, Oleg Pashuk, and Allison E. Williams Marine Resources Research Institute, S. Carolina Dept. of Natural Resources P.O. Box 12559, Charleston, South Carolina 29422-2559 E-mail: [email protected]

Gonad classification in male gray triggerfish and female blackbelly rosefish will be focused on because the gonad morphology and classification of these two species is atypical of marine teleosts off the Atlantic coast of the southeastern United States. Gray triggerfish The gray triggerfish (Balistidae) is a gonochoristic species with females exhibiting group-synchronous oocyte development and carrying no more than three to four batches of oocytes in the ovary at one time. The male of the species has testes that are separate, small, oval-shaped structures which lie close together along the ventral side of the swimbladder. The testes of the gray triggerfish do not join at the posterior end, but remain separate throughout their length. Spermatogenesis is atypical, in that most of the spermatocysts in lobules are filled with spermatocytes, whereas a small number of the spermatocysts contain spermatozoa. The lobules of the testes are drained via efferent ducts into the main testicular duct, which is positioned along the dorsal medial surface of each testis. The system of efferent ducts and the main testicular duct fuse outside of the testes to become the long common spermatic duct. Unlike most teleosts, the common spermatic duct in gray triggerfish is lined with secretory epithelial cells. This duct is surrounded by a bilateral outgrowth with tubules and secretory ducts for storage of spermatozoa, a structure that has been called an accessory gland in other fishes such as gobies and blennies. This accessory gland in gray triggerfish functionally resembles both a seminal vesicle and an epididymus, structures common to higher vertebrates, and it is connected to the ventro-posterior surface of the urinary bladder duct via connective mesenteries along the outgrowth. In addition, the epithelial cells in ducts of the accessory gland appear to be secreting fluids to nourish spermatozoa prior to spawning.

This atypical morphology and physiology of the male reproductive system in gray triggerfish necessitates the addition of two new reproductive classes, Storage and Recent Spawn. Histological criteria to assign reproductive class in male gray triggerfish were developed by Moore (2001), based on a modification of criteria presented by Wenner et al. (1986) for black sea bass (Centropristis striata). Ripe males with testes and ducts filled with spermatozoa were not observed among the 1167 specimens examined. Developing (Class 2): Limited spermatogenesis; elongation of lobules and some accumulation of spermatozoa in lobules and ducts.

Storage (Class H): Spermatic ducts are densely packed with spermatozoa that are oriented perpendicular to duct walls; little or no spermatogenesis in testes.

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Recent spawn (Class 7): Residual spermatozoa in extremely enlarged ducts lined with columnar secretory cells, spermatozoa oriented parallel to duct walls; little or no spermatogenesis in testes.

Spent (Class 4): Compressed and collapsing ducts with little or no residual sperm; no spermatogenesis evident in testes.

Regressed (Class 5): Little or no residual sperm in lobules, efferent ducts and main spermatic duct; some early spermatogenesis may be present in testes.

Blackbelly rosefish

Blackbelly rosefish (Scorpaenidae) exhibit an atypical reproductive mode for teleosts, as they have internal fertilization and intraovarian gestation (White et al. 1998). Free spermatozoa are found primarily in resting ovaries (Fig. 2f) from July through early December with peak occurrence in September through November. There is a delay of 1–3 months before fertilization, as oocyte development does not begin until December. Developing oocytes are suspended on stalks radiating from lamellae that are attached to the central stroma, not to the ovary wall (Fig. 2e). The stroma extends longitudinally through the ovary and is connected to the ovarian wall only at the anterior end of each lobe. The stalk functions to place the oocyte into the ovarian cavity and near the ovary wall, where a clear gelatinous matrix envelops the eggs after ovulation and fertilization. Eggs develop within the gelatinous matrix into early-celled embryos composed of an undifferentiated mass of cells approaching the blastula stage and a large yolk mass. The life history class at parturition is not yet known.

Literature Cited

Moore, J.L. 2001. Age, growth and reproductive biology of the gray triggerfish (Balistes capriscus) from the southeastern United States, 1992-97. M.S. Thesis, College of Charleston, 69 p.

Wenner, C. A., W.A. Roumillat, and C.W. Waltz. 1986. Contributions to the life history of black sea bass, Centropristis striata, off the southeastern United States. Fish. Bull. 84:723–741.

White, D.B., D.M. Wyanski, and G.R. Sedberry. 1998. Age, growth, and reproductive biology of the blackbelly rosefish, Helicolenus dactylopterus (Teleostei:Scorpaenidae), from the Carolinas, USA. J. Fish Biol. 53:1274-1291.

79 a) b) sz Class sz 2

sc

c) d)

sz sc Class H

sz

e) f) sc

Class 7

sz sz

Figure 1. Transverse sections of testis and common spermatic duct/accessory gland in male Balistes capriscus. a and b) 429 mm TL specimen in Developing class; c and d) 475 mm TL specimen in the Storage class; e and f) 553 mm TL specimen in the Recent Spawn class. sc = spermatocytes, sz = spermatozoa.

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a) b)

sz Class 4 sz

sz

c) d)

Class 5

sc

e) f)

Class sz w F

vo Class 5

Figure 2. Transverse sections of gonadal tissue from Balistes capriscus and Helicolenus dactylopterus. a and b) Testis and common spermatic duct/accessory gland from 439 mm TL specimen of B. capriscus in Spent class; c and d) Testis and common spermatic duct/accessory gland from 310 mm TL specimen of B.

capriscus in Regressed class; e) Vitellogenic oocytes radiate from lamellae attached to the central stroma in a 286 mm TL female specimen of H. dactylopterus; and f) Free spermatozoa in a 242 mm TL female specimen of H. dactylopterus in the Regressing class. sc = spermatocytes, sz = spermatozoa, vo = vitellogenic oocyte, w = ovary wall.

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