Fish Sci (2010) 76:281286 DOI 10.

1007/s12562-009-0208-8

ORIGINAL ARTICLE

Aquaculture

Detection of prostaglandin E2 in polychaete Perinereis sp. and its effect on Penaeus monodon oocyte development in vitro
O. Meunpol E. Duangjai R. Yoonpun S. Piyatiratitivorakul

Received: 5 February 2009 / Accepted: 16 December 2009 / Published online: 4 February 2010 The Japanese Society of Fisheries Science 2010

Abstract Prostaglandins are involved in the reproductive processes in a variety of animals, including crustaceans. It was found that polychaetes, the best maturation diet for shrimp broodstock, possessed the greatest variation of prostaglandin E2 (PGE2) when compared with other live feeds. The level of PGE2 varied according to sizes, feed intake, sources and type of polychaete. The matured and also larger sand polychaete Perinereis sp. contained higher PGE2 levels than younger and smaller sand polychaetes (18.16 5.82 ng PGE2 mg-1 protein for polychaetes at an average length of 10 cm up to 160.8 37.09 ng PGE2 mg-1 protein for polychaetes at an average length of 17 cm). The PGE2 levels in ovaries and haemolymph of female shrimp uctuated with the developmental stage of the ovaries. The highest concentration of PGE2 in haemolymph was at stage 3 of ovarian development, whereas the highest concentration of PGE2 in shrimp ovaries was at stage 4. In vitro incubation of Penaeus monodon pre-vitellogenic oocytes with
O. Meunpol (&) R. Yoonpun Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand e-mail: omeunpol@hotmail.com E. Duangjai Department of Animal Science and Fisheries, Faculty of Science and Agricultural Technology, Rajmangala University of Technology Lanna, Nan Campus, Phupiang, Nan 55000, Thailand S. Piyatiratitivorakul Center of Excellence in Marine Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand S. Piyatiratitivorakul Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

polychaete extract and synthetic PGE2 demonstrated that both PGE2s enhanced oocyte development, especially during late development and ovulation. The putative role of PGE2 from polychaetes or the presence of PGE2 in polychaetes may be a factor in their role as a dietary constituent required for shrimp oocyte development. Keywords Polychaete Prostaglandin E2 Shrimp oocyte In vitro incubation

Introduction Prostaglandins (PGs) have a regulatory role in crustacean reproduction in addition to well-known peptide hormones such as gonad-inhibiting hormone and gonad-stimulating hormone [1]. In crustacean aquaculture, polychaetes are accepted as the best shrimp broodstock diet due to their abilities to improve the reproductive performance of shrimp [2, 3]. Aside from their high nutritional value, it is also possible that polychaetes contain some reproductive hormones that are similar to those found in shrimp, since annelids and arthropods may have evolved from the same ancestors [4]. To date, a number of hormones have been identied in polychaetes, for example, osmoregulatory hormones [5], oxytocin/vasopressin hormones [6, 7] reproductive hormones [8], sex hormones [9], sex pheromones [10], immunity and inammation hormones [5, 11]. Some hormones of polychaetes, such as progesterone and 17ahydroxyprogesterone, have been reported to be capable of inducing shrimp oocyte development [12]. Based on our previous nding on high contents of arachidonic acid in whole polychaetes [13], since arachidonic acid is a potent precursor for prostaglandin E2, we were interested in

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investigating whether PGE2 was also present in these animals and if this hormone from polychaetes can affect shrimp oocyte development in vitro. The physiological levels of PGE2 in the female reproductive system of shrimp broodstock were also measured in order to establish the concentration of PGE2 in the trials.

concentration was quantied or the fraction was used in the shrimp oocyte in vitro incubation assay. PGE2 quantication The concentration of PGE2 in each sample was analyzed by high performance liquid chromatography (HPLC) analysis as described above [16]. For samples with a low PGE2 concentration, the Prostaglandin E2 Biotrak Enzyme immunoassay (EIA) kit (RPN222) from Amersham Biosciences was used [17]. The dried sample was re-dissolved in methanol and pipetted into 96 pre-treated wells, in duplicate. The colorimetric change due to the binding reaction was monitored by spectrophotometry (Microplate reader, Biorad 550, USA) at a wavelength of 450 nm. The minimum quantity of PGE2 that could be detected with our assay was 40 pg/ml, with a within-assay CV of less than 10.9%. In vitro incubation of shrimp oocytes with PGE2 One female black tiger shrimp with developing stage ovaries was chosen for the in vitro incubation experiments. The in vitro assays were carried out either with or without PGE2 extracted from polychaetes, or with synthetic PGE2 (Sigma-Aldrich, USA). A 5 9 5-mm2 piece of ovary fragment from the middle portion of the ovary lobe was dissected and washed in sterilized phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4) and then placed into a sterilized 24-well cell culture plate. Three different concentrations of PGE2 (1, 3 and 5 ng/ml) were prepared in M199 plus salt (NaCl 26.7; KCl 1.11; MgCl2 0.36; MgSO4 0.62 and CaCl22H2O 3.4 g/l) [18], and this PGE2 solution was then added into the designated well. The culture plate was gently shaken on an orbital shaker at 25C for 24 h [19]. At 1-h intervals during the incubation, the culture medium was replaced with new media plus hormone PGE2 in order to maintain optimal culture conditions of the culture. Histological examination Ovarian fragments were removed from the wells at the end of the experiment and then xed in Davidsons xative for histological examination [20]. The tissues were dehydrated with serially diluted methanol and replaced with parafn. The parafn block was cut into 5-lm sections and stained with haematoxylin and eosin. Under a light microscope (Nikon, Japan), the diameters of oocytes in the hormonaltreated ovary were compared to the control (without hormones). The percentage of oocytes present at each stage of development (pre-vitellogenic oocytes, vitellogenic oocytes

Materials and methods Sample collection The natural sand polychaete Perinereis sp. (1018 cm body length) were purchased from polychaete collectors in Chonburi Province, Thailand. The animals were transported to the Center of Excellence in Marine Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Other live feeds at their marketable sizes for shrimp broodstock, such as mud polychaete Marphysa sp., squid Loligo chinessis, horse mussel Perna viridis and blood clam Anadara granosa, were purchased from local shermen to be quantied for their PGE2 contents. Samples of female black tiger shrimp Penaeus monodon weighing between 160 and 180 g with different ovarian developmental stages (from 0 to 5) [14] were kindly donated by First Farm, Phuket, Thailand. The shrimp ovaries were dissected on ice and immediately extracted for analysis of PGE2 contents. The haemolymph of the same shrimp was drawn from the base of the rst segment of the abdomen by a 26-G needle that had been pre-treated with EDTA. The haemolymph was then stored at -80C before PGE2 analysis. Each sample was aliquoted prior to protein determination by the Bradford method [15]. PGE2 extraction Whole polychaete and shrimp tissues (ovaries, muscle and haemolymph) were homogenized separately in a small volume of 0.4% NaCl and extracted with water:ethanol (1:4), followed by acetic acid. The homogenate was mixed and centrifuged at 5739g for 5 min, and the supernatant was eluted with hexane and ethyl acetate onto a Sep Pak (C18) column (Alltech, MD, USA). The eluant was evaporated and re-dissolved in 20 ll of methanol. The supernatant was then injected onto a Prevail C18 column (4.6 mm 9 15 cm I.D.) (Alltech, MD, USA) and run on an isocratic solvent of 17 mM phosphoric acid/acetonitrile (7:3, v/v) (pH 3.0) at a ow rate of 1.5 ml/min. The PGE2 peak of the sample was measured at 195 nm [16]. The PGE2 HPLC fraction was collected and evaporated under nitrogen gas, and stored at -80C before the PGE2

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283
** 114.73

and matured oocytes with cortical rods [21]) from three replications were then calculated. Statistical analyses The experiment was composed of three different concentrations of polychaete extract PGE2 and three concentrations of synthetic PGE2 with three replicates for each treatment. The statistical signicance of the mean values was evaluated by a completely randomized design (CRD) at a 95% degree of condence. The differences among the treatment means were tested using Duncans multiple range test [22].

a
ng PGE2 / mg Protein

120

PGE2 in Live feed

80

* 42.09

40
17.65 3.21 5.73 2.77

Loligo chinensis

Anadara granosa

Perna viridis

Results The results showed that polychaetes contained PGE2 at higher levels than any other live feeds (160.80 37.09 ng PGE2 mg-1 protein for sand polychaetes, 58.99 11.11 ng PGE2 mg-1 protein for mud or mud polychaetes, 3.09 0.71 ng PGE2 mg-1 protein for horse mussels, 8.03 2.42 ng PGE2 mg-1 protein for squid and 4.50 1.37 ng PGE2 mg-1 protein for clams (Fig. 1a). The concentrations of PGE2 varied according to the sizes and sources of the polychaetes. Smaller and also younger polychaetes (polychaetes at an average length of 10.70 0.06 cm) displayed the lowest concentration of PGE2 (18.16 5.82 ng PGE2 mg-1 protein) when compared to larger polychaetes (89.52 9.61, 114.05 10.58 and 160.80 37.09 ng PGE2 mg-1 protein for polychaetes at an average length of 11.30 0.80, 11.62 0.08 and 17.80 0.40 cm, respectively) (Fig. 1b). The ovaries and haemolymph of female shrimp possessed signicant amounts of PGE2 with varying levels related to the degree of ovarian development (Fig. 2a, b). Matured ovaries at stage 3 and 4 displayed the highest amounts of PGE2 (from 25 to 30 ng PGE2 mg-1 protein) compared to any other ovarian stages. The PGE2 concentrations in shrimp rapidly declined after spawning (ovary stage 5) (Fig. 2a). The level of PGE2 in shrimp haemolymph was much lower than that of PGE2 in shrimp ovary. Interestingly, the PGE2 level in shrimp haemolymph was at its highest (ovary stage 3; 4.60 ng PGE2 ml-1 haemolymph) before the highest peak of PGE2 in ovary occurred (ovary stage 4; 30.30 ng PGE2 mg-1 protein). A surge of PGE2 levels in the haemolymph might be essential as a lead to the nal stages of development of oocytes. A trace of PGE2 was determined in shrimp muscle and also with varying levels of PGE2 in this tissue according to the ovarian developmental stage (1.321.94 ng PGE2 mg-1 protein).
b
mg PGE2 / mg Protein
180

Ovary of Macrobrachium

PGE2 in natural Perinereis sp.

a
160.80

120

114.05

b
89.52

60
c
18.16

10

11

12

17

Length (cm)

Fig. 1 Levels of PGE2 measured in various samples: a live feeds of Penaeus monodon broodstock, b natural sand polychaetes Perinereis sp. at size differences. The data represent the mean SD of ve replicates (letters above each bar indicate a signicant difference from the mean, P \ 0.05)

Incubation of shrimp ovarian fraction with various concentrations of PGE2, either extracted from polychaetes or synthetic PGE2, for a period of 24 h can comparably stimulate in vitro oocyte development of P. monodon. At the beginning of the experiment, the shrimp ovary was composed of 62.0 (5.7)% pre-vitellogenic oocytes, 32.0 (5.7)% vitellogenic oocytes and 6.0 (0)% oocytes with cortical rods. After a 24-h incubation with either PGE2 extracted from polychaetes or synthetic PGE2, the percentage of pre-vitellogenic oocytes was decreased, and there was a concomitant giving way for the increase in the proportioning of vitellogenic oocytes and oocytes with cortical rods (Fig. 3). The highest percentage of oocytes with cortical rods was received from oocytes incubated with 5 ng ml-1 of polychaete PGE2 extract (22.7 1.2%). This concentration of PGE2 was also found to be equal to the physiological level of PGE2 concentration in shrimp

Perinereis sp.

123

Marphysa sp.

284 Fig. 2 Data are average values (SD) of PGE2 levels measured in female shrimp broodstock Penaeus monodon (n = 5) at different ovarian developmental stages: a ovaries, b haemolymph and c muscle. Bars with the same letter did not differ signicantly from each other (P \ 0.05)

Fish Sci (2010) 76:281286

a
ng PGE 2 / mg Protei n

35 30 25 20 15 10 5 0

ng PGE 2 /ml haemolymp

PGE2 in ovary of female P. monodon broodstock a 30.3 a 25.0 b b b b 16.2 13.7 14.7 13.6

b
5 4 3 2 1 0

PGE2 in haemolymph of female P. monodon broodstock a a 4.60 ab 3.84 3.30 c c 2.33 2.47 d 0.33
0 1 2 3 4 5

Ovarian stages

Ovarian stages

c
ng PGE2 / mg Protein

1.5

PGE2 in muscle of female P. monodon broodstock a a a 1.9 1.8 1.7 b b 1.4 1.3

a 1.7

0.5

Ovarian stages

haemolymph. The effect of synthetic PGE2 on oocyte development was signicantly lower than those of the polychaete extracts (P \ 0.05). At a concentration of 3 ng ml-1 of synthetic PGE2, 18 (2)% of oocytes with cortical rods was reached. However, increasing dosages of synthetic PGE2 (5 ng ml-1) decreased the percentage of oocytes with cortical rods (13 2.3%) instead of enhancing the process.

Discussion Although a wide range of aquatic organisms have been used as feeds for shrimp broodstocks [2], farmers prefer polychaetes over other live feeds [3]. Previous research stated that one factor that differentiated polychaetes from other live feeds (squid, mussels and oysters) was a significantly higher ratio of arachidonic acid compared to docosahexaenoic acid and eicosapentaenoic acid [13]. From this, we discovered that polychaetes not only possess a specic pattern of fatty acid prole, but they also displayed a very high content of the crustacean reproductive hormone, i.e., PGE2. Therefore, the presence of PGE2 in

polychaetes might be another explanation for its ability to induce shrimp broodstock development. Levels of PGE2 present in polychaetes varied according to the sizes, sources and types of polychaete. Sand polychaetes contained higher amounts of PGE2 than mud polychaetes. In normal shrimp farming practice, without recognizing their contents of PGE2, Thai shrimp farmers have traditionally preferred using sand polychaetes as feed for shrimp broodstocks to mud polychaetes because of the higher percentage of ovarian development of sand polychaete-fed shrimp when compared to mud polychaete-fed shrimp. The PGE2 contents increased as the polychaetes matured. It might be indicated that PGE2 also acts as a reproductive hormone in the polychaetes themselves in addition to the gonad-inhibiting hormone from brain [23]. Ovarian development in the penaeid shrimp is composed of primary and secondary stages of vitellogenesis. Yolk protein deposition in oocytes is dened as the initial stage of secondary vitellogenesis [24, 25]. After completion of yolk accumulation, the oocytes form cortical rods around the periphery of the oocyte plasma membrane and undergo nal development and ovulation, followed by oviposition

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Previtellogenic oocytes 70 Vitellogenic oocytes Matured oocytes with cortical rod

285

a 62 59 53 b b 40 46 b 45 42 39 A B C D D b 35 37 45 58 b 47 b 39

60

Percentage of oocytes

50

40

b 32

30

23
18 D

20

17

18 7

12

13

10

Fixed

media

Ex1

Ex3 Ex5 Treatment

Syn1

Syn3

Syn5

Fig. 3 Percentage of different Penaeus monodon oocyte stages after incubation with PGE2 for 24 h. The letter above a bar indicates a signicant difference between treatment means (P \ 0.05). Fixed previtellogenic oocytes prior to incubation, Media previtellogenic oocytes incubated with non-hormone media, EX1 previtellogenic oocytes incubated with polychaete extract at 1 ng ml-1, EX3

previtellogenic oocytes incubated with polychaete extract at 3 ng ml-1, EX5 previtellogenic oocytes incubated with polychaete extract at 5 ng ml-1, Syn1 previtellogenic oocytes incubated with synthetic PGE2 at 1 ng ml-1, Syn3 previtellogenic oocytes incubated with synthetic PGE2 at 3 ng ml-1 and Syn5 previtellogenic oocytes incubated with synthetic PGE2 at 5 ng ml-1

[26]. Each step of ovarian development is responsible for a number of hormones. As observed from this study, PGE2 was found to act as a vitellogenesis-inducing factor and nal development step in penaeid shrimp since it not only inuences the development of yolk globules in oocytes, but also increases the percentage of oocytes with cortical rods compared with controls. The presence of cortical rods is a good indicator of successful treatment because cortical rods are synthesized only at the nal stages of oocyte development and are required for protective coats for free-spawning shrimp [27, 28]. Our ndings that PGE2 acts as a stimulator of vitellogenesis in P. monodon are in agreement with results obtained from a study on craysh Procambarus paeninsulanus [29] and crab Oziotelphusa senex senex [1]. Enhancing effects of polychaete PGE2 were found on P. monodon oocyte development in vitro. An effective dosage of PGE2 from polychaetes coincided with the concentration of PGE2 in the haemolymph of matured female shrimp broodstock. When replacing the polychaete PGE2 with synthetic PGE2 at the same concentration, a negative outcome was achieved. The results demonstrated in this experiment were in agreement with some works [16, 30] that have stated that prostaglandins and related compounds from the polychaetes were possible inducers of ovarian development in Penaeid shrimp. The proportion of three stages of oocyte development cultured with polychaete extract was not identical to that cultured with authentic

PGE2. Especially polychaete extract corresponding to 5 ng/ ml PGE2 induced oocyte development, whereas the same dose of authentic PGE2 did not. This can be explained that whole polychaetes might contain some other unknown substances apart from PGE2 that can also induce shrimp oocyte development. This speculation could only be conrmed by full structural analysis using mass spectrometry and NMR following capillary gas-liquid chromatography. The synthesis rate of PGE2 and PGF2a in craysh ovarian tissue increased prior to ovulation [29], whereas some research found increased activity of prostaglandin H synthase in the ovary in later stages of reproduction (vitellogenesis II) [1]. Both groups reported that PGs controlled ovarian growth of the crab, craysh and the freshwater shrimp Macrobrachium rosenbergii. On the contrary, there was no correlation of GSI with PGE2 and PGF2a in Marsupenaeus japonicus [16, 30, 31]. The concentration of PGF2a and PGE2 in haemolymph was high in previtellogenic stage ovaries and gradually decreased toward maturation. This was an indicator that PGF2a and PGE2 may stimulate previtellogenesis and vitellogenesis of Marsupenaeus japonicus [16, 30]. Our ndings in P. monodon, however, showed that PGE2 was not only an inducer of vitellogenesis, but also of nal maturation and spawning, as can be detected from the presence of cortical rods in the oocytes. The concentration of PGE2 in haemolymphs of P. monodon was different from those reported for Marsupenaeus japonicus, where the level of PGE2

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Fish Sci (2010) 76:281286 10. Zeeck E, Harder T, Beckmann M (1998) Uric acid: the spermrelease pheromone of the marine polychaete Platynereis dumerilii. J Chem Ecol 24(1):1322 11. Salzet M (2001) The neuroendocrinology systems of annelids. Can J Zool 79:175191 12. Meunpol O, Iam-Pai S, Suthikrai W, Piyatiratitivorakul S (2007) Identication of progesterone and 17a-hydroxyprogesterone in polychaetes (Perinereis sp.) and the effects of hormone extracts on penaeid oocyte development in vitro. Aquaculture 270:485492 13. Meunpol O, Meejing P, Piyatiratitivorakul S (2005) Maturation diet based on fatty acid content for male Penaeus monodon (Fabricius) broodstock. Aquac Res 36:12161225 14. Primavera JH (1988) Biology and culture of Penaeus monodon. Brackishwater aquaculture information system. Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan 15. Bradford M (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 28:350356 16. Tahara D, Yano I (2003) Development of hemolymph prostaglandins assay systems and their concentration variations during ovarian development in the kuruma shrimp, Penaeus japonicus. Aquaculture 220:791800 17. Curtis GH, Macnaughton WK, Wallace JL (1995) Intraluminal pH modulates gastric prostaglandin synthesis. Can J Physiol Pharmacol 73:130134 18. Webster SG (1986) Neurohormonal control of ecdysteroid biosynthesis by Carcinus maenas Y-organ in vitro and preliminary characterization of the putative moult-inhibiting hormone. Gen Comp Endocrinol 61:237247 19. Tsukimura B, Kamemoto FI (1991) In vitro stimulation of oocytes by presumptive mandibular secretions in the shrimp Penaeus vannamei. Aquaculture 92:5966 20. Bell TA, Lightner DV (1988) A handbook of normal penaeid shrimp histology. The World Aquaculture Society, Baton Rouge 21. Tan-Fermin JD, Pudadera RA (1989) Ovarian development stages of the wild Giant tiger shrimp, Penaeus monodon Fabricius. Aquaculture 77:229242 22. Zar JH (1996) Biostatistical analysis, 3rd edn. Prentice-Hall, New Jersey 23. Golding DW (1967) The diversity of secretory neurons in the brain of Nereis. Z Zellforsch 82:321344 24. Yano I (1988) Oocyte development in the kuruma shrimp Penaeus japonica. Mar Biol 99(4):547553 25. Quackenbush LS (1992) Yolk synthesis in the marine shrimp, Penaeus vannamei. Comp Biochem Physiol 103A:711714 26. Clark WH Jr, Yudin AI, Lynn JW, Grifn FJ, Pillai MC (1990) Jelly layer formation in penaeoidean shrimp eggs. Biol Bull 178:295299 27. Anderson SL, Chang ES, Clark WH Jr (1984) Timing of postvitellogenic ovarian change in the ridgeback shrimp Sicyonia ingentis determined by ovarian biopsy. Aquaculture 42:257271 28. Clark WH Jr, Lynn JW (1977) A Mg2? dependent cortical reaction in the eggs of penaeid shrimp. J Exp Zool 200:177183 29. Spaziani EP, Hinsch GW, Edwards SC (1993) Changes in prostaglandin E(2) and F2-alpha during vitellogenesis in the Florida craysh Procambarus paeninsulanus. J Comp Physiol 163:541 545 30. DCroz L, Wong LV, Justine G, Gupta M (1988) Prostaglandins and related compounds from the polychaete worm Americonuphis reesi Fauchald (Onuphidae) as possible inducers of gonad development in penaeid shrimps. Rev Biol Trop 36:331332 31. Tahara D, Yano I (2004) Development-related variations in prostaglandin and fatty acid content of ovary in the kuruma shrimp (Marsupenaeus japonicus). Comp Biochem Phys 137A:631637

was low at the earliest stage of ovarian development, but was higher at the maturing stage ovary. Furthermore, our previous research investigating fatty acid contents of ovaries in P. monodon showed that there were no signicant differences in the contents of arachidonic acid during gonad ovarian development (Leelahanon P, unpublished data, 2003). Although these two species are closely related, the role of each hormone might be more species-specic; therefore, more information may be required in order to make any exclusive conclusions. Finally, a number of polychaete shrimp broodstockenhancing factors have been claried, partly from this study, as being both nutritional and/or hormonal. Our laboratory is currently in the process of making articial feed pellets with similar properties based on polychaetes, particularly with respect to the hormones and fatty acid content of polychaetes. The outcome of these studies is to help improve shrimp reproductive performances by dietary feeding factors and to provide alternative approaches to the enhancement of female shrimp development by eyestalk ablation.
Acknowledgments This work was funded by the National Research Council of Thailand to O. Meunpol. Many thanks to First Farm and Sam Dao Farm for providing P. monodon broodstock, the CP Group of Companies for some polychaete samples, CENTEX and Mahidol University, Bangkok, for histology facilities.

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