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, Procambarus 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 Malacostraca.1–7 It is plete sex reversal in the gonochoristic freshwater attached to the ejaculatory region of the vas defer- prawn 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 animals, 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 krill 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 crab 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.