Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682004 Blackwell Science Asia Pty Ltd 704August 2004 841 Unidentified tissue of P. clarkii T Murakami et al. 10.1111/j.1444-2906.2004.00841.x Original Article561568BEES SGML

FISHERIES SCIENCE 2004; 70: 561–568

An unidentified gland-like tissue near the androgenic gland of red swamp crayfish, clarkii

Tomoya MURAKAMI,1* Tai-Hung LEE,2 Kunihiro SUZUKI3 AND Fumio YAMAZAKI2

1Hiroshima Prefectural Fisheries Experimental Station, Aki, Hiroshima 737-1207, 2Graduate School of Fisheries Science, Hokkaido University, Hakodate 041-8611 and 3Shizuoka Prefectural Fisheries Resources Office, Shizuoka 420-8601, Japan

ABSTRACT: Among all of the decapods that have been studied, only one kind of gland, the androgenic gland, has been found attached to the subterminal ejaculatory region of the vas deferens. In the present study of the red swamp crayfish, it was found that two kinds of gland-like tissues were attached to the subterminal ejaculatory region of the vas deferens. One of the tissues, which is referred to as TIB, is located inside the body cavity, whereas the other tissue, which is referred to as TIC, is inside the coxa. The characteristics of TIB resemble those of the androgenic gland of other malacostracans. The TIC cells, however, are smaller and more basophilic due to the small size and high density of their cytoplasm and nuclei. Moreover, black fibers, similar to the nerve fibers of the ganglion, were detected among the TIC cells after Bodian’s stain. The conclusion is that TIC may be a newly found male sexual characteristic and further research is needed to confirm its functions.

KEY WORDS: androgenic gland, crayfish, decapod, sexual characteristic.

INTRODUCTION The same mechanism, however, does not seem to be fully applicable to the decapods for two main In aquaculture, sex control is an extremely impor- reasons. First, it is reported that the AG in the deca- tant technique for mono-sex culture and breeding pods does not appear prior to the sex differentia- research. To establish this technique, the study of tion as in the amphipods or isopods but posterior sex differentiation is necessary. to the beginning of sex differentiation.18–20 Second, The androgenic gland (AG) has been detected in although it is suggested in two studies that com- the males of most orders of .1–7 It is plete sex reversal in the gonochoristic freshwater attached to the ejaculatory region of the vas defer- Macrobrachium rosenbergii can be achieved ens, except for isopods where the AG is located at by implanting21 and ablating the AG,22 other studies the end of each testicular utricle, or the external show that ablation and implantation of the AG in side of the seminal vesicle or on the surface of the the decapods only result in partial feminization or vas deferens.8–10 masculinization.23–28 The role of the AG has been well-studied in the The red swamp crayfish Procambarus clarkii, Amphipod Orchestia gammarella2,11,12 and the Iso- one of the best-known decapod , has been pod Armadillidium vulgare.13–17 Research on the studied for a wide variety of purposes by research- ablation and implantation of the AG in these orders ers, yet little is known about the location and struc- indicates that the AG is related to the inducement ture of its AG. Unlike what is reported on the other and development of male sexual characteristics decapods, all of which possess only one kind of and the suppression of female sexual characteris- gland, that is, the androgenic gland, at the subter- tics. The mechanism of sex differentiation in these minal ejaculatory region of the vas deferens,29–33 we orders is thus explained. found in a preliminary study of the red swamp crayfish that two kinds of AG-like tissues are attached to the subterminal ejaculatory region of the vas deferens. Moreover, the tissues are mor- *Corresponding author: Tel: 81-823-51-2171. phologically different and located in separate Fax: 81-823-52-2683. Email: [email protected]. places. In this study, we describe in detail the loca- jp tion and structure of the two AG-like tissues by Received 7 July 2003. Accepted 5 March 2004. histological and histochemical methods. We also 562 FISHERIES SCIENCE T Murakami et al.

discuss whether those tissues are in fact andro- After the specimens were fixed in Bouin’s solu- genic glands and the significance of those uniden- tion for 24 h, they were preserved in 80% ethanol. tified glands in the study of the AG. Sections (8-mm thick) were made by the usual paraffin embedding methods and cut vertically to the body axis. After the paraffin of the sections MATERIALS AND METHODS were removed with xylen and ethanol, the sections were washed with distilled water (DW; 5 min) and Adult P. clarkii were purchased and kept in a stained with 1% silver protein solution containing concrete tank (3 ¥ 3 ¥ 0.5 m) filled with freshwater 5 g of copper per 100 mL, as a catalyst (24 h). The at the Faculty of Fisheries, Hokkaido University, sections were then washed with DW (90 s ¥ three Japan. They were reared in an aerated, closed cir- times) and soaked in a reducing solution contain- culating system at 24∞C, and were fed frozen vege- ing 4 g hydroxinon per 100 mL of 4% Na2SO4 tables (corn, carrots and green peas) and every (10 min). This procedure was followed by washing day until mating and egg-laying. Juveniles hatched with DW (60 s ¥ three times) and soaking in 2% from the eggs of one female, were kept individually C2H2O4 (30 min). After that, the sections were in plastic pots (5 ¥ 5 ¥ 5 cm). For water circulation, washed with DW (5 min ¥ three times), soaked in five holes of 2 mm in diameter were made on each 5% Na2S2O3 (5 min), and washed with DW (10 min). side of the pots which were placed in large plastic For comparison, sections of the abdominal gan- containers (25 ¥ 35 ¥ 7 cm) with a closed water cir- glion of P. clarkii and E. japonicus and the AG of culating system. The juveniles were reared at 24∞C E. japonicus as specified by AG implanting experi- and fed frozen vegetables and krill every day until ment,28 were prepared and stained in the same their carapace length (CL) reached about 10 mm. way. Sections stained only with HE were also pre- After that, they were kept in a large concrete tank pared to compare with those after treatment with until they became adults. Bodian’s stain. For comparison with the AG of P. clarkii and that of the Eriocheir japonicus, adult males of E. japonicus were collected from the estuary of the RESULTS Shiodomari River in Hakodate, Hokkaido, Japan. They were kept in the same way as the adults of Location and morphology of androgenic P. clarkii at 20∞C. gland-like tissues In order to determine the timing of the appear- ance of the gland-like tissues near the ejaculatory Two kinds of gland-like tissues attached to differ- region of the vas deferens, samples of P. clarkii ent regions of the subterminal ejaculatory duct of (entire animals) were collected at the second, third, the vas deferens are observed near the boundary fourth and seventh intermolt (CL 5 mm), and between the body cavity and the coxa of the fifth between CL 7.5 mm and CL 10 mm after the sev- walking leg. One of the gland-like tissues is located enth intermolt. Five specimens were taken at each inside the body cavity. We call this tissue TIB. TIB stage and were fixed in Bouin’s solution for 24 h. is cord-like in shape and firmly attached to the After that, the exoskeleton of the specimens were ejaculatory duct. The other gland-like tissue is decalcified by decalcification solution (decalcifica- located inside the coxa of the fifth walking leg. We tion solution B, Wako Pure Chemical Industries call this tissue TIC. TIC is also cord-like in shape Ltd.) and then the whole specimens were pre- but only one of its ends is attached to the ejacula- served in 80% ethanol. Sections (8-mm thick) were tory duct while the rest of it is separate from the made by the usual paraffin embedding methods, ejaculatory duct and protrudes posteriorly (Fig. 1) cut vertically to the body axis, and stained with Delafield’s hematoxylin and eosin (HE). For histological and histochemical observations Timing of the appearance of androgenic of the gland-like tissues, samples of P. clarkii adult gland-like tissues males (entire animals, CL 20–35 mm) were used. Sections of the specimens, which included the Up to the seventh intermolt (CL 5 mm), no TIB is coxa of the left fifth walking leg, were made and detected along the vas deferens of the males. At CL stained with Bodian’s stain (i.e. a method that turns 7.5 mm, primordial TIB appears on the surface of cytoplasm of nerve cells, nerve fibers and axon the ejaculatory duct (Fig. 2). At CL 10 mm, TIB is processes into black)34 following the procedures highly visible (Fig. 3). described below. After Bodian’s stain, the sections In contrast, no TIC is detected along the vas were stained with HE and observed with a light deferens of the males at the second intermolt. At microscope. the third intermolt, primordial TIC appears on the Unidentified tissue of P. clarkii FISHERIES SCIENCE 563

Fig. 1 The coxa of the fifth walking leg of a male Pro- cambarus clarkii showing the location of TIB and TIC. ed, ejaculatory duct; ps, penis. Fig. 3 TIB (arrow) on the surface of the ejaculatory duct of a male Procambarus clarkii at CL 10.0 mm. ed, ejaculatory duct.

Fig. 2 Primordial TIB (arrow) on the surface of the ejac- ulatory duct of a male Procambarus clarkii at CL 7.5 mm. ed, ejaculatory duct. Fig. 4 Primordial TIC (arrow) on the surface of the ejac- ulatory duct of a male Procambarus clarkii at the third intermolt. ed, ejaculatory duct. surface of the ejaculatory duct (Fig. 4). TIC is highly visible at the fourth intermolt (Fig. 5). Fibrous tissues are found among the TIC cells (Fig. 6). Histological and histochemical observations of Unlike the TIC cells, the nucleus of the TIB cells androgenic gland-like tissues stained with HE in adult P. clarkii is nearly spheri- cal and about 7.5 mm in diameter with a distinct The nuclei of the TIC cells stained with HE in adult nucleolus. The cytoplasm of the TIB cells is abun- P. clarkii are oval in shape. Their sizes are irregular, dant but not as basophilic as that of the TIC cells. with a long axis of about 6.5 mm. Compared with No fibrous tissue is found in TIB (Fig. 7). the TIB cells, the volumes of the cytoplasm and The cells of the AG in E. japonicus has similar nuclei of the TIC cells are smaller. Both the nuclei features as the TIB cells: the nuclei are spherical, and cytoplasm of the TIC cells are more basophilic. the nucleolus are distinct, the cytoplasm is abun- 564 FISHERIES SCIENCE T Murakami et al.

dant but not as basophilic as that of the TIC cells, and no fibrous tissue is found among the cells (Fig. 8). Fibers in TIC, the ganglion of P. clarkii and that of E. japonicus turn black after being stained with HE and Bodian’s stain (Figs 9,10a,11a) but not after they are stained with HE only (Figs 10b,11b). The black fibers in these three kinds of tissues are all similar in terms of thickness. No such black fiber is found in TIB and the AG of E. japonicus (Figs 12,13). The nuclei of the TIC cells appear to be darker than those of the TIB cells and the AG of E. japonicus (Fig. 9).

DISCUSSION

Among all of the malacostracans that have been Fig. 5 TIC (arrow) on the surface of the ejaculatory duct studied, only one kind of gland, AG, has been of a male Procambarus clarkii at the fourth intermolt. ed, found attached to the ejaculatory region of the vas ejaculatory duct. deferens (e.g. decapods), or the top of the testis or

Fig. 6 TIC on the surface of the ejaculatory duct in an adult male Procambarus clarkii stained with Delafield’s hematoxylin and eosin. Arrows indicate fibrous tissues. (a) Low magnification, (b) enlargement. ed, ejaculatory duct; n, nucleus.

Fig. 7 TIB on the surface of the ejaculatory duct in the same adult male Procambarus clarkii as that of Fig. 6 stained with Delafield’s hematoxylin and eosin. (a) Low magnification, (b) enlargement. ed, ejaculatory duct; n, nucleus. Unidentified tissue of P. clarkii FISHERIES SCIENCE 565

Fig. 8 Androgenic gland on the surface of the ejaculatory duct in an adult male Eriocheir japonicus stained with Delafield’s hemat- oxylin and eosin. (a) Low mag- nification, (b) enlargement. ed, ejaculatory duct; n, nucleus.

Fig. 9 TIC on the surface of the ejaculatory duct in an adult male Procambarus clarkii stained with Delafield’s hema- toxylin and eosin after Bodian’s stain. Arrows indicate black stained fiber. (a) Low magnification, (b,c) enlargement. ed, ejaculatory duct; n, nucleus.

Fig. 10 Abdominal ganglion in an adult male Procambarus clar- kii. (a) Specimen stained with Delafield’s hematoxylin and eosin after Bodian’s stain. Arrows indi- cate fiber stained in black color. (b) Specimen stained with Delafield’s hematoxylin and eosin. 566 FISHERIES SCIENCE T Murakami et al.

Fig. 11 Thoracic ganglion in an adult male Eriocheir japonicus. (a) Specimen stained with Delafield’s hematoxylin and eosin after Bodian’s stain. Arrows indi- cate fiber stained in black color. (b) Specimen stained with Delafield’s hematoxylin and eosin.

Fig. 12 TIB on the surface of the ejaculatory duct in the same adult male Procambarus clarkii as that of Fig. 9 stained with Delafield’s hematoxylin and eosin after Bodian’s stain. (a) Low mag- nification, (b) enlargement. ed, ejaculatory duct; n, nucleus.

Fig. 13 Androgenic gland on the surface of the ejaculatory duct in an adult male Eriocheir japonicus stained with Delafield’s hema- toxylin and eosin after Bodian’s stain. (a) Low magnification, (b) enlargement. ed, ejaculatory duct; n, nucleus. external side of the seminal vesicle, or on the sur- to as TIB, is located inside the body cavity, whereas, face of the vas deferens (e.g. isopods). In the the other tissue, which we refer to as TIC, is inside present study, however, we observed two kinds of the coxa. gland-like tissues, both of which are attached to The TIB cells have large spherical nuclei, abun- the subterminal ejaculatory duct near the bound- dant cytoplasm, distinct nucleolus, and are slightly ary between the body cavity and the coxa of the basophilic. No fibrous tissue is found in TIB after fifth walking leg. One of the tissues, which we refer HE and Bodian’s stain. These characteristics Unidentified tissue of P. clarkii FISHERIES SCIENCE 567

resemble those of the AG of E. japonicus and In contrast, more research is needed to confirm the AG of other malacostracans that have been whether tissues other than the AG at the ejacula- studied,6,35 suggesting that TIB may be the AG tory duct exist in other decapods. In a preliminary of P. clarkii. study of the crayfish Pacifastacus trowbridgii, for Unlike the TIB cells, the TIC cells are smaller and example, we observed two gland-like tissues that more basophilic due to the small size and high resemble those reported in this study (Lee TH, density of their cytoplasm and nuclei. Moreover, unpubl. data, 2003). black fibers were detected among the TIC cells The existence of gland-like tissues may help to after Bodian’s stain. The black fibers are similar to explain why previous researchers failed to achieve the nerve fibers of the ganglion of P. clarkii and that complete sex reversal in their studies of AG of E. japonicus. implantation24,28,37,38 and why the rate of achieve- These morphological and histochemical obser- ment of complete sex reversal was so low in the vations clearly indicate that the two gland-like tis- study of Malecha et al.21 In other words, the sues are always different from each other, researchers might have mistaken other gland-like regardless of the size of the (which ranged tissues for AG or neglected the existence of other from CL 20 mm to CL 35 mm). In addition, the two gland-like tissues which may be important in gland-like tissues appear at different times, with inducing sex reversal. TIC appearing first. These observations clearly show that the two tissues are not of the same kind, and that the differences between them are inher- ACKNOWLEDGMENTS ent and permanent, and not due to the seasonal or physiological changes. Therefore, we conclude that The authors would like to thank Dr Akira Goto of TIC may be a newly found male sex characteristic. Aquatic Breeding Science Laboratory, Graduate According to Taketomi,30 the AG of P. clarkii is School of Fisheries Science, Hokkaido University, located inside the coxa of the fifth leg. In addition, for his helpful suggestions. We also wish to express the cord-like tissue, as Taketomi et al.36 described our hearty thanks to Dr Enid Lee, Mrs Yumi the AG in juveniles of P. clarkii, resembles TIC in Murakami and Ms. Makoto Sakurada for their this study. This suggests that Taketomi et al. might continuous assistance. have mistaken the TIC for the real AG. 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