Reference: Biol. Bull. 185: 174-185. (October, 1993) Spermatophores and Plug Substance of the Marine Shrimp Trachypenaeus similis (Crustacea: Decapoda: Penaeidae): Formation in the Male Reproductive Tract and Disposition in the Inseminated Female RAYMOND T. BAUER AND LIN JUN MIN Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana, 70504 Abstract. Sperm are packaged into many small sper- matophores of variable size in the median vas deferens (MVD) of the male. A substance is intermixed with sperm in the proximal coils of the MVD, separating groups of sperm that will be ejaculated as spermatophores. Most of the ejaculatory duct is occupied by a chamber filled with a transparent, viscous fluid termed the "plug substance." When males are artificially ejaculated, spermatophores are emitted, followed by plug substance that quickly so- lidifies. This latter material fills and stoppers a space on the female, the median pocket, which serves as an ante- chamber to the apertures of the internalized seminal re- ceptacles, where sperm from ruptured spermatophores are stored. The slit-like openings to the receptacles are func- tionally divided into a posterior aperture, stoppered by plug substance after insemination, a closed mid-section, and an anterior exit for sperm release during spawning. Direct insemination by an everted male gonopore is considered more likely than transmission of spermato- phores and plug substance via the male gonopod (pe- tasma). In addition to its hypothesized roles during in- semination and sperm release, the mass of plug substance (mating plug) may act as a paternity assurance device that prevents subsequent inseminations by other males. Introduction There is considerable variation in the form and com- plexity of materials transferred from the male to the female during insemination in penaeoid shrimps (Decapoda: Penaeoidea) (Bauer, 1991). Sperm may be packaged in Received 18 December 1992; accepted 14 July 1993. Contribution No. 40 of the USL Center for Crustacean Research. structurally complicated spermatophores composed of an assortment of accessory substances secreted in the male reproductive tract. The most complex spermatophores are those attached externally to the genital area, or thelycum, of the female, as in the white shrimp, Penaeus setiferus, and other species of the subgenus Litopenaeus (Perez Far- fante, 1975; Bauer and Cash. 1991; Chow et a/.. 1991). In contrast, in the rock shrimps, Sicyonia spp., sperm in a seminal fluid are produced by the male and transferred to, and stored in, internal seminal receptacles of the female (Clark et al, 1984; Perez Farfante, 1985; Bauer, 1991, 1992). Other penaeoid shrimps, such as Trachypenaeus spp.. show intermediate degrees of spermatophore com- plexity and of internalization of sperm storage (Burken- road, 1934: Heldt. 1938a, b; Hudinaga, 1941; Malek and Bawab, 1 9 74a, b; Perez Farfante, 1971. 1982; Champion, 1987; Bauer and Cash, 1991). Detailed knowledge of the nature and formation of spermatophores and associated substances and of their disposition in the inseminated female is essential to an understanding of the mechanics of insemination, sperm storage, and sperm release during fertilization. In addition, characters associated with spermatophores and the in- semination morphology of the male and female are im- portant in analyses of the evolutionary relationships among taxa of penaeoid shrimps, as well as to an evolu- tionary interpretation of their mating systems (Bauer, 1991). Observations on the spermatophores, seminal recep- tacles, and thelyca of various species of Trachypenaeus (Penaeidae) have been made by Andrews ( 1 9 1 1), Burken- road (1934), Kubo (1949), Perez Farfante (1971), and Bauer (1991). The structure of the Trachypenaeus sper- 174 INSEMINATION IN TRACHYPENAEUS 175 matophore is somewhat unusual among penaeoids in that sperm are packaged into numerous small spherieal bun- dles (Burkenroad, 1934; Bauer. 1991), superficially very similar to spermatophores produced by male brachyuran crabs (Spalding, 1942;Cronin, 1947; Beninger et al.. 1988; Hinsch. 1988). However, little detailed information on Trachypenaeus spp. is available on the formation and structure of spermatophores, on a male accessory sub- stance that plugs or seals the female seminal receptacles, or on the placement of sperm and "plug substance" in the thelyca and seminal receptacles of females. In this report, we describe in Trachypenaeus similis (Smith) ( 1 ) the structure and formation of spermatophores and the plug substance in the male reproduction tract, and (2) the thelycum/seminal receptacle system of the female, especially the placement of sperm and plug sub- stance therein. We discuss alternative hypotheses on the mechanics of insemination, based both on these mor- phological observations and on observations from artificial ejaculation of living males. We make comparisons of spermatophores and insemination morphology of T. sim- ilis with that of other penaeoid shrimps and other decapod taxa. Materials and Methods Specimens of Trachypenaeus similis were obtained by bottom trawling at night in various locations in the north- ern Gulf of Mexico. Some material used for dissection and histology was obtained on cruises in 1987 and 1990 within an area at 28-29N latitude and 88-94W longi- tude. From 1989 to 1992, other specimens were trawled from the Mississippi Sound, just off the northwest end of Horn Island, Mississippi. Observations on living shrimps were taken on specimens collected from the Horn Island location and transported in oxygen-saturated water within sealed plastic bags to recirculating seawater facilities at the University of Southwestern Louisiana. Segments of or entire reproductive tracts of 33 males and the thelycum/seminal receptacle area of 30 females were prepared for histology or paraffin carving. Live spec- imens, anesthetized with chilling when possible, were in- jected with and initially preserved in Davidson's solution (Shaw and Battle. 1957). Within a few days of fixation, specimens were washed in running water for 1-2 h and were then taken through a graduated series of alcohol changes (25%, 35%, 50%, 70%) with final storage in 70% ethyl alcohol. Standard alcohol dehydration and toluene infiltration were used to prepare dissected material for embedding in a compound of paraffin and plastic poly- mers (Galigher and Kozloff, 1971 ). Sections were stained with Mallory's Triple Stain, using the variation in which sections are rinsed with 1% phosophotungstic acid after staining in acid fuchsin and prior to staining in aniline blue-orange G mixture (Galigher and Kozloff, 1971 ). In paraffin carving, material was sectioned to a desired lo- cation, deparaffined in toluene, gradually re-infiltrated with 100%. ethyl alcohol, and prepared for scanning elec- tron microscopy (SEM) by critical-point drying with car- bon dioxide and sputter coating with a 10-20 nm thick- ness of gold/palladium. Methods for SEM of external morphology are described in Bauer ( 1987). Living shrimps were maintained on water tables with recirculating seawater. Adult females were isolated indi- vidually and checked daily for molting. Upon molting, females cast off stored sperm and other male products from previous inseminations, and several such females were preserved for SEM 2-3 days after molting. Exuviae of many females (>50) were collected and preserved for later examination. Living males, held ventral side up im- mersed in seawater in a dish under a stereomicroscope, were artificially ejaculated by squeezing both sides of the posterior cephalothorax at the level of the gonopores with a pair of fine forceps. Ejaculated spermatophores or fresh dissections of ejac- ulatory ducts were deposited in a drop of seawater on plastic coverslips coated with polylysine (Mazia et al., 1975). After 5-10 minutes to allow spermatophores and sperm from spontaneously ruptured spermatophores to adhere to coverslip surfaces, coverslips were gently im- mersed in 4%. seawater formalin in petri dishes for fixation of material. Washes with distilled water and a standard alcohol series were made by gently pipetting solutions out of and into the petri dishes. The coverslips with adhering material were then prepared for SEM as described above. Results Gross morphology of the male reproductive tract The male reproductive system, situated above the gut and hepatopancreas in the posterior half of the cephalo- thorax, consists of paired testes, vasa deferentia, and ejac- ulatory ducts (Fig. 1 ). The testis on each side is composed of 5-6 lobes, the first with an anterior elongation, and a posterior extension inserted among the coils of the median vas deferens. In freshly sacrificed specimens, a testicular lobe can be teased apart, revealing that each lobe is com- posed of a single, highly convoluted seminiferous tubule. The vas deferens can be divided on the basis of gross morphology into proximal, median, and distal portions (Fig. 1). The structure we identify as the proximal vas deferens (PVD) is a very fine duct, difficult to identify in, and illustrate from, gross dissections but apparent in serial sections (Fig. 2). A PVD runs along the mesial edges of the testicular lobes, merging into the proximal part of the median vas deferens at the posterior end of the testis (Fig. 1 ). The connections between the seminiferous tubules and the PVD were not clearly observed. We refer to the highly 176 R. T. BAUER AND L. J. MIN dvd Figure 1. Diagram of male reproductive tract of Trachypenaeus similis, dorsal view. Testicular tissue covering coils of right median vas deferens(MVD)isnot illustrated. In most preserved specimens, the coils of the MVD are usually bunched into a more compact, tangled mass than illustrated in this diagram, with the distal (anterior) coils somewhat below the more proximal (posterior) ones, dvd, distal vas deferens; mvd, coils of median vas deferens: psc. plug substance chamber of ejaculatory duct; pvd, posterior end of proximal vas deferens merging into the median vas deferens; sc, spermatophore chamber of ejaculatory duct; sf, semi- niferous tubule; t, testicular lobes. Scale bar = 1 mm coiled portion of the vas as the median vas deferens (MVD), which lies under the posterior part of the testis (Fig. 1 ). Each MVD begins posteriorly, gradually increas- ing in diameter distally (anteriorly). The distal vas deferens (DVD) is defined as the long, curved, uncoiled portion of the vas that lies outside of the testicular-M VD mass. Each DVD extends to the body wall of the last cephalothoracic segment, descending down ventrally to join the ejaculatory duct in the floor of the cephalothorax. The ejaculatory duct has two distinct but confluent chambers (Fig. 1 ). The spermatophore chamber (SC), an extension of the DVD, is located anteriorly, opening me- sially to the outside through the gonopore. situated ex- ternally between the mesial edge of the coxa of the fifth (most posterior) pereiopod and the sternum of the male. A large chamber, the "plug substance chamber" (PSC), lies posterior to the SC. The PSC is partially divided into two regions, which are continuous mesially. The two chambers of the ejaculatory duct are quite distinct in color and transparency, with the SC bright white or opaque, and the PSC clear or transparent with a yellow tint in living and preserved specimens, respectively. Spermatophore structure and formation When males are artificially ejaculated, a short cord composed of many spermatophores is emitted from the gonopores. The spherical spermatophores, which separate from each other in seawater within seconds to minutes, are quite variable in size (Fig. 6). Each spermatophore is composed of a group of sperm cells surrounded by a thin film or pellicle (Figs. 3, 4). Undisturbed spermatophores remain intact in seawater for at least an hour, but rupture easily when moved about or handled, releasing the sperm cells (Figs. 3, 4, 7). Many sperm cells were examined with SEM and light microscopy, and all showed the same ex- ternal structure of a main body, cap, spike, and a long, delicate filament extending away from the main body (Fig. 7). A striking decrease in the thickness of the epithelium lining the MVD from the proximal to distal coils (Fig. 5) coincides with spermatophore formation. In the most proximal coils of the MVD, the epithelium is composed of large cuboidal to columnar cells with distinct nuclei (Fig. 2), and the lumens of these coils are filled with a solid mass of sperm (Figs. 2, 5, 8). In this region, a material, termed here MVD substance, which apparently forms the spermatophore pellicle, first appears (Fig. 8). In sections stained with Mallory's Triple Stain, the MVD substance appears a pale blue when viewed with light microscopy. The MVD substance increases in volume more distally in the MVD, where it intermingles with sperm, surround- ing and separating out groups or packets, which are in- cipient spermatophores (Fig. 9). In the DVD, the epithelium is quite flattened and thin, as in the distal-most part of the MVD (Fig. 5), and the lumen is filled with sperm packets surrounded by MVD substance (Fig. 12). When the wall of the DVD is stripped away to expose the underlying contents, the separate, variably sized groups of sperm are exposed. INSEMINATION IN TRACHYPENAEUS 177 P V dvd Figure 2. Cross section through proximal (posterior) coils of median vas deferens of Trachypenaeus simi/is, showing left and right proximal vasa deferentia (pvd) dorsal to coils, g, gut; t, tissue of posterior extension of testes. Figure 3. Single spermatophore showing pellicle (p) surrounding group of sperm, s, sperm free in water from ruptured spermatophores. Figure 4. Group of spermatophores including one (unlabeled arrow) in which most sperm have escaped, leaving behind a largely unbroken pellicle, s. sperm from ruptured spermatophores. Figure 5. Cross section through coils of median vas deferens (bordered by arrows labeled mvd) of one side approximately halfway along its length, showing proximal-to-distal decrease in duct wall thickness. Note thin-walled distal vas deferens (dvd) lateral to median vas deferens. d, dorsal side of section; g, gut. Scale bar in Figure 2 represents 800 /^m in Figure 2, 300 ^m in Figures 3, 4, and 2 mm in Figure 5. coated by MVD substance (Fig. 10). The MVD sub- stance surrounding the sperm packets is completely continuous with that of adjacent groups of sperm (Figs. 10, 12). Internal anatomy of and materials in the ejaculatory duel Sections through the ejaculatory duct show that the spermatophore chamber (SC) is a continuation of the thin- wailed DVD. filled with sperm packets separated by MVD substance (Figs. 11, 1 3). A large duct, the "plug substance" chamber (PSC), occupies the bulk of the ejaculatory duct (ED) (Figs. 1,11) and is lined with a well-developed ep- ithelium (Figs. 11, 14). The PSC is partially subdivided with a posterior chamber (Figs. 1, 14), which is confluent mesially with the main PSC chamber. The PSC and the SC are separated on the mesial side of the ED (Fig. 1 1 ), but laterally, in the area of the gonopore, the two chambers are continuous (Fig. 15). Plug substance intrudes into the mass of sperm packets above the posterior gonopore area (Fig. 15), but more anteriorly sperm packets fill the space over the gonopore (Fig. 1 3). The interior of the PSC is filled with a material (Figs. 11, 14), appearing a reddish purple when stained with Mallory's Triple Stain and viewed with light microscopy, which is very hard and difficult to section in paraffin-embedded material. When living males are artificially ejaculated, the contents of the PSC flow out of the gonopore after emission of a cord of sper- matophores. The PSC material is a viscous, clear, ad- hesive fluid when first leaving the gonopore. Within Figure 6. Spermatophores of Trachypcnacur, unit/is, showing variation in size. Figure 7. Sperm cell of T similis. c, cap; f. filament; mh, main body; sk, spike. Figure 8. Section through median vas deferens near proximal end. showing appearance of M VD substance (ms) just inside epithelium and inside solid mass of sperm, s, sperm mass. Figure 9. Section through four adjoining sperm packets separated hy MVD substance (ms) in distal (anterior) end of median vas deferens. s. sperm mass. Figure 10. View of outer surface of spermatophores exposed when wall of distal vas deferens was dissected away. Note continuity of MVD substance among spermatophores. Figure II. Sagittal section taken on mesial side of right ejaculatory duct, dvd, distal vas deferens; ps, plug substance in plug substance chamber; sp, mass of spermatophores in spermatophore chamber. Scale bar in Figure 6 represents 33 ^m in Figure 6, 1.4 fjm in Figure 7, 35 /jm in Figure 8, 18 /jm in Figures 9 and 10, and 216 /urn in Figure 1 I. 178 INSEMINATION IN TR.lCHYPENAEi'S 179 14 Figure 12. Longitudinal section through segment of distal vas deferens. Section was stretched by heating during preparation, separating groups of sperm (spermatophores) from surrounding MVD substance, ms. MVD substance: sp. spermatophores. Figure 13. Cross section through spermatophore chamber (sc) of ejaculatory duct, left side, at level of gonopore (gp). dvd. distal vas deferens. Figure 14. Cross section through plug substance chamber, posterior end. of right ejaculatory duct, e; epithelium; ps. plug substance. Figure 15. Cross section through right ejaculatory duct near posterior end of gonopore. Note plug substance (ps) surrounded by spermatophores (sp). gp, gonopore. Scale bar in Figure 12 represents 300 urn in Figure 12 and 2 mm in Figures 13-15. seconds of emission, it becomes only weakly adhesive, and it changes from a viscous fluid to a soft, malleable, solid mass. It begins a change in transparency and color within seconds, becoming opaque and white within a few minutes. The gross appearance of the PSC material is. after some minutes of exposure to seawater, identical to the material, termed here "plug substance," which plugs and seals the median pocket and seminal receptacles of the female (see below). Disposition of sperm and plux substance in the inseminated female Modifications of the sternum of the penultimate (XIII) and ultimate (XIV) segments of the cephalothorax com- prise the genital area, or thelycum. of the female. The external morphology of the thelycum can be best observed in recently molted, uninseminated females (Fig. 16). On sternite XIV, a pair of flaps (Fig. 16), separated by a lon- gitudinal gap, form the floor of a space, the median pocket (Figs. 18-20). On sternite XIII is the undivided median protuberance, part of which extends posteriorly and dorsal to the sternite XIV flaps. In most inseminated females, the plug substance (PS) from the male can be seen pro- truding from the median pocket in the transverse gap be- tween the median protuberance and the flaps, and from between the mesial margins of the latter (Figs. 17. 18- 20). The PS is quite hard in living females and firmly seals the thelycum flaps together. In contrast, in living or pre- served uninseminated females, the flaps can be pulled back and moved quite easily. The PS in the median pocket of the female thelycum has similar staining properties as that 180 R. T. BAUER AND L. J. MIN Figure 16. Ventral surface of posterior cephalothorax of an uninseminated female of Trachypenaeus .V/HH//V. showing thelycum. ax, apex of median protuberance; cx3, cx4, cx5, coxae of third, four, and fifth pereiopods. respectively, fl. flap; gp. gonopores; mp, median protuberance. Figure 17. Thelycum of inseminated female, showing plug substance (ps) protruding from median pocket, en. endite of coxa of pereiopod 4. Scale bar in Figure 16 represents 590 nm in Figure 16 and 421 urn in Figure 17. in the PSC of the male, as well as comparable physical properties such as hardness and homogeneity of texture in paraffin-carved material viewed with SEM (Figs. 1 1, 18-20). Examination of exuviae of inseminated females shows that PS remains in the median pocket from the time of insemination until the next molt, at which time it is cast off with the exuviae. Inside the cephalothorax, dorsal to the thelycum, lie a pair of cuticular invaginations, the seminal receptacles, each with large posterior and smaller anteromesial and antero- lateral lobes (Figs. 22-23). The posterior lobes are dorsal to sternite 14, while the anterior lobes lie dorsal and lateral to the median protuberance. Sections through the seminal re- ceptacles showed no spermatophores but rather sperm in a solid mass. Within the sperm mass, small, irregular patches of material, identical in appearance and staining properties to that of the MVD substance of the male, were usually observed in sectioned material. Examination of exuviae show that sperm remaining in the receptacles, often in considerable quantity, are cast off with the cuticular lining of the recep- tacles when an inseminated female molts. The opening into each seminal receptacle is a narrow slit between the anteromesial and posterior lobes that ex- tends up to the anterolateral lobe as well. Each seminal receptacle slit (SRS) begins in the roof of the median pocket just posterior to the transverse gap between flaps and median protuberance (Fig. 20), extending anteriorly along the sides of the median protuberance up to about the level of the coxal endite (Fig. 17) of pereiopod 4. Sec- tions through the posterior end of the SRS of inseminated females show that the borders of the SRS are separated but that the opening is stoppered with plug substance (Figs. 20, 2 1 ). More anteriorly, in the region just anterior to the transverse gap between flaps and median protuberance, the SRS appears functionally closed because its borders, the median protuberance on one side and a fold of the adjacent sternal wall on the other, are tightly pressed to- gether (Figs. 24, 25). The borders of the SRS separate again near its anterior end so that the anterolateral lobe of the seminal receptacle is open to the exterior near the coxal endite of pereiopod 4 (Fig. 26). In serial sections through the genital area of several females, sperm were found outside Figure 18. Cross section through thelycum ol inseminated female of Trachypenaeus similis at approx- imately halfway between anterior and posterior ends of transverse slit between flaps (see Figures 16-17 for orientation). Section is viewed from posterior side of animal, ventral (external) surface up. Median pocket is filled with plug substance (ps) that protrudes from between flaps (fl) to exterior. Posterior lobes of seminal receptacles (sr, lateral extent indicated by arrows) inside the cephalothoracic cavity are partially filled with sperm (s). mp. median protuberance. Figure 19. Higher magnification of median pocket filled with plug substance (ps) which protrudes to exterior between flaps (fl) of thelycum. Figure 20. Cross section through thelycum of inseminated female just posterior to transverse gap between flaps (fl) and median protuberance (mp). Orientation same as that for Figure 1 8. Note plug substance (ps) intruding into each seminal receptacle (sr) from median pocket, plugging each receptacle's slit or opening (srs). 1X1 182 R. T. BAUER AND L. J. MIN of the seminal receptacles, anterior to the SRS, in the channel or groove (Fig. 27) on each side of the median protuberance up to its apex near the female gonopores (Fig. 16). Discussion The packaging of sperm into numerous small sper- matophores, as described here in Trachypenaeus similis, is unusual for a penaeoid shrimp. In most species of pen- aeoids, a single large mass of sperm, surrounded by a va- riety of accessory materials, is emitted from each male gonopore during ejaculation (Heldt, 1938a, b; Malek and Bawab, 1 974a, b; Perez Farfante, 1975; Champion, 1987; Orsi Relini and Tunesi, 1987; Ro et ai. 1990; Bauer and Cash, 1991; Bauer, 1991, Chow el al, 1991). In species with such large, complex spermatophores, the vas deferens is subdivided into two ducts, one containing sperm mass and surrounding capsular substance, the other with one or more accessory materials that serve to attach or seal the sperm mass on or in the thelycum of the female. In T. similis, as in other Trachypenaeus spp., the vas deferens is a simple undivided tube containing a single material (MVD substance) that participates in the formation of many small spermatophores. The "plug substance" of T. similis, appearing only in the ejaculatory duct, is not ho- mologous to accessory materials produced in the median vasa deferentia of other penaeoid shrimps. Observations by Burkenroad ( 1934) indicate that males of another pen- aeid, Xiphocaris kroyeri (Heller), may have a similar sys- tem of many small spermatophores with a plug substance present in the ejaculatory duct, but the details of this sys- tem have not yet been described. Only males of the pen- aeoid genus Sicyonia have simpler reproductive tracts in which a simple mass of sperm in a fluid matrix is found in the vas deferens, is emitted from the gonopores, and is stored in the female seminal receptacles without any male accessory substances (Burkenroad, 1934; Bauer, 1991). Ultrastructural studies on the vasa deferentia of a va- riety of male decapods have demonstrated the secretion of spermatophore and accessory substances by the epi- thelium of the vas deferens [majid crab Libinia emargin- ata Leach, Hinsch and Walker, 1974; lobster Homarus americanus H. Milne Edwards, Kooda-Cisco and Talbot, 1986; majid crab Cliionoecetes opilio(O. Fabricius). Ben- inger et al.. 1988; crayfish Cherax albidus. Talbot and Beach, 1989; Penaeus shrimps, Ro et al.. 1990; Chow el al., 1991]. We did not study possible secretion of the ma- terial (MVD substance) that appears to form the pellicles of spermatophores in Trachypenaeus similis. Secretion of MVD substance might be revealed by ultrastructural studies on the epithelium of the proximal coils of the me- dian vas deferens where this substance first appears. Males of brachyuran crabs such as Carcinus maenas (Linnaeus), Callinectes sapidus Rathbun, Port units san- guinolentus (Herbst), Chionoecetes opilio, and Geryon spp. produce many spermatophores of small size, as in Tra- chypenaeus similis. The epithelium of the vas deferens of these brachyurans secretes a substance or substances that, after intermixing into the sperm mass and surrounding groups of sperm, condenses into the spermatophore pel- licle (Spalding, 1947;Cronin, 1947; Ryan, 1967; Beninger etal.. 1988;Hinsch, 1988, 1991). Our observations in this study on isolation of packets of sperm by MVD substance suggest a comparable mechanism of spermatophore for- mation in T. similis. We observed peristaltic contractions in the median and distal portions of vasa deferentia excised from freshly sacrificed males, similar to observations made in other decapod species (Ryan, 1967; Ro et al.. 1990; suggested from vas deferens ultrastructure by Kooda-Cisco and Talbot. 1986; Talbot and Beach, 1989). Such con- tractions of the vas deferens wall might serve to intermix MVD substance with the sperm mass in spermatophore formation as well as move all duct contents more distally. The sperm cells of Trachypenaeus similis are similar to those described for other penaeoid shrimps (Clark et al.. 1984; Griffin et al.. 1988; Felgenhauer and Abele, 1991) except for a novel structure, a long delicate but conspicuous filament on the end of the cell opposite the spike. The filaments were not only visible in sperm re- leased from spermatophores but also apparent within sec- tioned vasa deferentia, indicating that the filaments were not artifacts nor results of any type of reaction of sperm to seawater. The distribution of plug substance in the median pocket of the female thelycum, and its emission after the sper- matophores in artificially ejaculated males, suggest alter- native hypotheses on the mechanics of insemination in Trachypenaeus similis. One alternative we propose is that Figure 21. Close-up of cross section through one seminal receptacle from Figure 20. Slit or opening (srs) to receptacle from median pocket is filled with plug substance (ps). Note that cuticle (ct) of median pocket (arrow towards upper nght) is continuous with that (arrow towards lower left) of seminal receptacle (sr. empty part of lumen of receptacle), s, sperm mass with bits of darker MVD substance inside receptacle. Figure 22. Dorsal view of left and nght seminal receptacles inside cephalothorax. Internal organs and soft tissues were removed by treatment with KOH, leaving only cuticular structures, a, anterior direction. Figure 23. Close-up of left seminal receptacle from Figure 22, rotated 90. al. anterolateral lobe; am, anteromesial lobe; pi, posterior lobe. Scale bar in Figure 18 represents 492 ^m in Figure 18. 230 ^m in Figure 19, 402 ^m in Figure 20, 126 ^m in Figure 21, 466 ^m in Figure 22. and 300 ^m in Figure 23. INSEMINATION IN TRAClIYI'KMtLS 183 Figure 24. Cross section through median protuberance (mp) just anterior to thelvcum flaps and median pocket (same orientation as Figures 18 and 20). Unmarked black arrowheads point to locations on external surface which would be open to passageway leading to the slits or openings (unmarked white arrowheads) of seminal receptacles (sr| if passageway were not blocked by abutting cuticles of median protuberance and adjoining sternal wall, mpc, cuticle of median protuberance; s, sperm mass: sr. lumen of seminal receptacle: swc, cuticle of sternal wall. Figure 25. Close-up of blocked passageway between opening to seminal receptacle and external surface. Rotated 90 clockwise from Figure 24. left side (right side of animal). Note bit of sperm mass trapped about midway (middle arrows of mpc and swc) along the closed passageway. Labels same as Figure 24. Scale bar in Figure 24 represents 385 ^m in Figure 24 and 179 ^m in Figure 25. the spermatophores are introduced into the median pocket, followed by an injection of plug substance, directly from the male gonopore that is perhaps everted into a papilla during ejaculation. In this scenario, the plug sub- stance would displace the spermatophores from the me- dian pocket (rupturing them in the process), would force the sperm mass into the posterior ends of the slits that open into the seminal receptacles, and would stopper these openings, preventing backflow and loss of sperm from the receptacles. Alternately, Burkenroad (1934) suggested that Figure 26. Cross section through median protuberance (mp) and area just lateral on right side (section viewed from posterior, dorsal is up in micrograph). Note that opening (small arrowhead) from anterolateral lobe of right seminal receptacle is open to the exterior, en. section through coxal endite of pereiopod 4: sal. sperm mass inside anterolateral lobe; sam, sperm mass inside anteromesial lobe of right seminal receptacle. Figure 27. Cross section through the median protuberance (mp) near its apex. Same orientation as Figure 26. Note sperm (s) outside of seminal receptacles in channel between median protuberance and sternum lateral to it. sr. very anterior ends of anteromesial lobes of seminal receptacles, partially filled with sperm. Scale bar in Figure 26 represents 2 mm in Figures 26 and 27. 184 R. T. BAUER AND L. J. MIN the petasma, a semi-closed tubular structure on the male's first pleopods, might function as the injection apparatus, with each of its grooved horns inserting into one of the seminal receptacles during copulation. He hypothesized that spermatophores would first be deposited, and then the petasma would withdraw slightly and deposit the "sperm free secretion" or plug substance into the median pocket. It seems unlikely to us that plug substance, only slightly fluid when first ejaculated, hardening to a soft mass almost immediately, could flow through the petasma and out its narrow terminal channels. In many vertebrates and invertebrates, hardened or co- agulated male products ("mating or copulatory plugs") block the openings to the female reproductive tract after mating (Mann, 1984). Parker (1970) and Thornhill and Alcock ( 1 983), citing examples of mating plugs in various insect species, suggested that one function of mating plugs is to prevent re-insemination of a female once it has mated. Mating plugs ("sperm plugs") have been described in a variety of brachyuran crabs by Hartnoll (1969), who sug- gested their major role was to prevent loss of sperm from receptacles after copulation. However, Diesel (1991) dis- cussed their possible function as a paternity assurance mechanism in certain brachyurans. In Trachypenaeus similis, the male plug substance that blocks access to the seminal receptacles of the females may serve, in addition to preventing loss of sperm from the receptacles after cop- ulation, as a mating plug precluding subsequent insemi- nations by other males. We have observed during artificial ejaculation of males that the amount of plug substance in and protruding from the median pocket of the female thelycum is approximately that emitted from one ejacu- latory duct of one male. Plug substance may also play an indirect but important role in sperm release from the seminal receptacles. During spawning and concomitant fertilization, which occur sep- arately from mating and insemination in most penaeoids (Anderson el a/., 1985; Pillai ct ai. 1988; Bauer, 1991), sperm being forced out of receptacles by whatever means would be prevented from flowing back out the wrong di- rection (into the median pocket) by the plug substance that stoppers the receptacle apertures. Sperm must leave the receptacles through what appears to be the only avail- able exits, the openings from the anterolateral lobes of the receptacles. From these exits, sperm would enter the grooves on each side of the median protuberance, stream- ing forward to just below the gonopores where eggs would be emerging during spawning. Acknowledgments The senior author (R.T.B.) wishes to thank Dr. Isabel Perez Farfante for the stimulating and useful discussions on the thelyca and seminal receptacles of Trachypenaeus spp. We wish to thank George Cantrell, captain of the R/V Bill Demoran out of the Gulf Coast Research Lab- oratory, for his skill and patience in trawling Trachypen- aeus on our collecting trips to Horn Island, Mississippi. Our participation in cruises on the NOAA vessel R/V Oregon II was quite useful in collecting material used in this study. We thank Dr. Bruce Felgenhauer for alerting us to the polylysine technique used in preparing sper- matophores for SEM. We also thank the anonymous re- viewers of this manuscript for their helpful suggestions and comments. This research was supported by grants from the National Oceanographic and Atmospheric Administration's Louisiana Sea Grant Program (No. NA89AA-D-SG226) and the Louisiana Educational Quality Support Fund (1989-92-RD-A-20). Literature Cited Anderson, S. L., \V. H. Clark, Jr., and E. S. Chang. 1985. Multiple spawning and molt synchrony in a free spawning shnmp (Sicyonia inxenlis: Penaeoidea). Bioi Bull 168: 377-394. Andrews, K. A. 1911. Sperm transfer in certain decapods. Proc. U.S. Null Mil* 39: 419-434. Bauer, R. T. 1987. Stomatopod grooming behavior: functional mor- phology and amputation experiments in Gonodactylus oerstedii. J. Crustacean Bio/ 7:414-432. Bauer, R. T. 1991. Sperm transfer and storage structures in penaeoid shrimps: a functional and phylogenetic perspective. Pp. 183-207 in Crustacean Se.\ual Biology. R. T. Bauer and J. W. Martin, eds. Co- lumbia University Press, New York. Bauer, R. I. 1992. Repetitive copulation and variable success of in- semination in the marine shrimp Sicyonia dorsa/is (Decapoda: Pen- aeoidea). J. Crustacean Biol. 12: 153-160. Bauer, R. I., and C. E. Cash. 1991. Spermatophore structure and anatomy of the ejaculatory duct in Penaeus setileriis. P. duorarum, and P. a:tecus (Crustacea: Decapoda): homologies and functional significance. Trail*. Am. Micrasc Soc 110: 144-162. Beninger, P. G., R. VV. Elner, T. P. Foyle, and P. H. Odense. 1988. Functional anatomy of the male reproductive system and the female spermatheca in the snow crab Chionoecetes opilio (O. Fabricius) (Decapoda: Majidae) and a hypothesis for fertilization. ./ Crustacean Bin/. 8: 322-331. Burkenruad, M. D. 193-4. The Penaeidea of Louisiana, with a discussion of their world relationships. Bull Am. Mils. Nat. Hist. 68: 61-143. Champion, II. F. B. 1987. The functional anatomy of the male repro- ductive system in Penaeus indicus. S. Afr. J Zool 22: 297-307. Chow, S., M. M. Dougherty, \V. J. Dougherty, and P. A. Sandifer. 1991. Spermatophore formation in the white shrimps Penaeus se- ll /erus and P vannamei. J Crustacean Bio/. 11: 201-216. Clark, W. H., Jr., A. I. Vudin, F. J. Griffin, and S. Shigekawa. 1984. The control of gamete activation and fertilization in the ma- rine Penaeidae, Sicyonia ingenlis. Pp. 459-472 in Advances in In- rertehrate Reproduction 3. W. Engels el ill., eds. Elsevier Science Publishers B. V., Amsterdam. Cronin, L. E. 1947. Anatomy and physiology of the male reproductive system of ( 'allincclcs sapidits Rathbun. ./. Miirplwl. 81: 209-239. Diesel, R. 1991. Sperm competition and the evolution of mating be- havior in Brachyura. with special reference to spider crabs ( Decapoda, Majidae). Pp. 145-163 in Crustacean Sexual Biology. R. T. Bauer and J. W. Martin, eds. Columbia University Press, New York. Felgenhauer, B. E., and L. G. Abele. 1991. Morphological diversity of decapod spermatozoa. Pp. 322-341 in Crustacean Se\uiil Biology. INSEMINATION IN TRACHYPENAEUS 185 R. T. Bauer and J. \V. Martin, eds. Columbia University Press, New York. Galigher, A. E., and E. N. Ko/loff. 1971. Essentials <>/ Practical Mi- crotechnique. Lea & Febiger, Philadelphia. 531 pp. Griffin, K. J., K. Shigekawa, and \V. H. Clark, Jr. 1988. Formation and structure of the acrosomal filament in the sperm of Sicyonia iii K cnlis J- Exp. Biol 246: 94- 102. llarlnull. R. G. 1969. Mating in the Brachyura. Cnistaceana 16: 161- 181. Heidi. J. II. I938a. La reproduction chez les crustaces decapodes de la famille de peneides. Ann. lust. Oceanogr. 18: 31-206. Heldt, J. H. I938b. De 1'appareil genital des Penaeidae. Relations morphologique entre spermatophore. thelycum et petasma. Travaux tic In Sim ion /.oo/ogique lie MY/mrnn 13: 349-358. Hinsch. G. \V. 1988. Ultrastructure of the sperm and spermatophores of the golden crab Geryiin fenneri and a closely related species, the red crab G' quinquedens. from the eastern Gulf of Mexico. J. Crus- tacean Biol. 8: 340-345. Hinsch, G. \V. 1991. Structure and chemical content of the spermato- phores and seminal fluid of reptantian decapods. Pp. 290-307 in Crustacean Sexual Biology. R. T. Bauer and J. W. Martin, eds. Co- lumbia University Press. New York. Hinsch, G. \\ '., and M. H. Walker. 1974. The vas deferens of the spider crab. Lihinia emarginala. J Morphol 143: 1-20. Hudinaga, M. 1941. Reproduction, development, and rearing of Pen- aeus iiiponicus Jpn. J Zool. 10: 305-393. Kooda-Cisco, M., and P. Talbot. 1986. Ultrastructure and role of the lobster vas deferens in spermatophore formation: the proximal seg- ment. ./. Morphol. 188: 91-104. Kubo, 1. 1949. Studies on penaeids of Japanese and its adjacent waters. ./ Tokyo Coll Fish 36: 1-467. Malek, S. R. A., and F. M. Baah. 1974a. The formation of the sper- matophore in Penaeus kerallmrus (Forskal. 1775) (Decapoda: Pen- aeidae). I. The initial formation of a sperm mass. Cntstaceana 26: 273-285. Malek, S. R. A., and F. M. Bawab. 1974b. The formation of the sper- matophore in Penaeus keralhuru.s (Forskal, 1775) (Decapoda: Pen- aeidae). II. The deposition of the main layers of the body and of the vsmg. Cruslaceana 27: 73-83. Mann, T. 1984. Spermatophores. Spring-Verlag, Berlin. 240 pp. Mazia, D., G. Schatten, and W. Sale. 1975. Adhesion of cells to surfaces coated with polylysine. J. Cell Biol 66: 198-200. Orsi Relini, L., and L. Tunesi. 1987. The structure of the spermalophore in Arisieus antenniilus (Risso, 1816). Invest. Pesq. 5l(Supl. I): 461- 470. Parker, G. A. 1970. Sperm competition and its evolutionary conse- quences in the insects. Biol. Rev Camb. Philos. Soc. 45: 525-567. Perez Farfante, I. 1971. A key to the American Pacific shrimps of the genus Trachypenaeus (Decapoda: Penaeidae), with the description of a new species. L'.S. Nail. Mar Fish. Sen: Fish. Bull 69: 635-646. Perez Farfante, I. 1975. Spermatophores and thelyca of the American white shrimps, genus Penaeus. subgenus Litopenaeits. U.S. Nail. Mar. Fish. Sen: Fish. Bull. 73: 463-486. Perez Farfante, I. 1982. The geminate shrimp species Parapenaeus longimstris and P. polilits (Crustacea: Decapoda: Penaeoidea). Quad. Lah. Teen. Pesca Ancona 3: 187-205. Perez Farfante, I. 1985. The rock shrimp genus Sicyonia (Crustacea: Decapoda: Penaeoidea) in the eastern Pacific. U.S. Natl. Mar. Fish. Sen: Fish Bull. 83: 1-78. Pillai, M. C, F. J. Griffin, and \V. H. Clark, Jr. 1988. Induced spawning of the decapod crustacean Sicyonia ingentis. Biol. Bull. 174: 181- 185. Ro, S., P. Talbot, J. Leung- 1 rujillo, and A. L. Lawrence. 1990. Structure and function of the vas deferens in the shrimp Penaeus setiferus: segments 1-3. J. Crustacean Biol. 10: 455-468. Ryan, E. P. 1967. Structure and function of the reproductive system of the crab Ponunus sanguino/entus (Herbst) (Brachyura: Portunidae). I. The male system. Proc. Symp. Crustacea. Mar. Biol. Assoc. India 2: 506-521. Shaw, B. L., and H. I. Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster Crassostrea virginica (Gmelin). Can J. Zool. 35: 325-347. Spalding, J. F. 1942. The nature and formation of the spermatophore and sperm plug in Carcinus nuienus. Q J Microsc. Set. 83: 399- 422. Talbot, P., and D. Beach. 1989. Role of the vas deferens in the formation of the spermatophore of the crayfish (Cherax). J Crustacean Biol. 9: 9-24. Ihornhill, R., and J. Alcock. 1983. The Evolution of Insect Mating Svstems. Harvard University Press. Cambridge. Massachusetts. 547 PP.