Available online at www.sciencedirect.com Comparative Biochemistry and Physiology, Part B 149 (2008) 202 – 208 www.elsevier.com/locate/cbpb Epitoky in Nereis (Neanthes) virens (Polychaeta: Nereididae): A story about sex and death Étienne Hébert Chatelain ⁎, Sophie Breton, Hélène Lemieux, Pierre U. Blier Département de Biologie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1 Received 11 July 2007; received in revised form 11 September 2007; accepted 11 September 2007 Available online 16 September 2007 Abstract The nereidid Nereis (Neanthes) virens undergoes drastic behavioural, morphological and physiological changes during its sexual maturation (epitoky). This metamorphosis prepares benthic worms for a brief pelagic existence devoted to mating although in N. virens only mature males leave their burrows to swarm. After spawning, individuals of both sexes die. Specific adjustments of energy metabolism pathway allowing higher muscular activity and swimming capacity remain to be eluded. This study compared atokous worms (immature) and epitokous (mature) swimming males and benthic females of N. virens to detect metabolic changes that could occur during epitoky. Epitokous males showed significantly higher electron transport system, citrate synthase and aspartate aminotransferase activities (p b 0.01) and significantly lower lactate dehydrogenase activity (p b 0.01) compared to atokous worms and epitokous females. There was no difference in antioxidant enzyme capacities between epitokes and atokes. Lipase and trypsin activities were significantly lower (p b 0.01) in epitokous males. The enzymatic changes observed are likely related to the metabolic adjustments required to support higher swimming abilities. Maintenance of antioxidant capacities could be related to protection of germinal tissues more than long term survival, since N. virens die after spawning. © 2007 Elsevier Inc. All rights reserved. Keywords: Polychaete; Nereis virens; Epitoky; Metabolism; Oxidative stress 1. Introduction Polychaete worms of the family Nereididae exhibit a wide range of reproductive modes, including external brooding, viviparity and hermaphrodism. Most species undergo morphological and physiological modifications when they become sexually mature, suiting many of them for a brief pelagic existence and improving the chances that sexual partners will find each other (Clark, 1961; Schöttler, 1989; Fischer, 1999). This metamorphosis of the immature worm into a special reproductive form is known as epitoky, a process that is particularly well described in the sandworm Nereis (Neanthes) virens, which is found in shallow marine soft-bottoms of the Northern hemisphere (Bass and Brafield, 1972; Dean, 1978; Wissocq, 1978; Kristensen, 1984; Hoeger, 1991; Fischer and Hoeger, 1993; Franke, 1999; Breton et al., 2003). In this species, the gravid males leave their burrows and swarm before spawning and ⁎ Corresponding author. Tel.: +1 418 7231986 #1262; fax: +1 418 7241849. E-mail address: etienne.hebertchatelain@uqar.qc.ca (É. Hébert Chatelain). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2007.09.006 releasing gametes in the sea while the females apparently spawn in their burrows, minimizing predation and avoiding adverse environmental conditions that males encounter (Bartels-Hardege and Zeeck, 1990). This sexual difference is reflected in a range of epitokous modifications that occur in males but are lacking or less pronounced in females. Morphological changes accompanying the swimming phase of the reproductive males include enlarged parapodia, formation of natatory chaetae, atrophy of the gut, histolysis of body wall and reorganization of body musculature (Bass and Brafield, 1972; Wissocq, 1978; Hoeger, 1991; Fischer and Hoeger, 1993). In contrast to iteroparous polychaete species which breed several times in their life, N. virens is a strictly semelparous species breeding only once during its lifetime. A reversion back to the atokous form (i.e. not metamorphosed or strictly benthic) is impossible because neither of the sexes appear to survive the process of spawning (Schroeder and Hermans, 1975; Fischer et al., 1996). Epitoky also involves considerable changes in the metabolism of the worms. Mature nereidid polychaetes rely entirely on their body reserves during the metamorphosis and the histolysis of the É. Hébert Chatelain et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 202–208 tissues provides material for the formation and growth of the gametes (Clark, 1961; Golding and Yuwono, 1994). Coupled with these changes there is usually a growth of the vascular system and respiratory surface, suggesting a respiratory adaptation as a mean of improving the swimming ability of the worms (Clark, 1961). Nereidids that are usually active and frequent swimmers, such as Nereis succinea, have a more efficient and larger capillary circulation in the parapodia than infrequent swimmers like N. virens, even in the immature phase (Clark, 1961). In this latter species, muscular activities are generally restricted to ventilation and feeding movements (Evans et al., 1974). The observation that epitokous N. virens show significantly higher O2 consumption compared to atokous worms is consistent with the hypothesis of increased respiratory capacity during the period of increased muscular activity when the worms are swarming (Schöttler, 1989). Although little is known about biochemical changes occurring during nereidids' metamorphosis (Schöttler, 1989), it appears reasonable to predict that the development of swimming ability would also be fostered by metabolic adjustments to support higher muscular activity. According to the duration of swarming of epitokous N. virens males (1–3 days, Kristensen, 1984), we suspect that major proportion of activity during this period relies on aerobic metabolism. Since N. virens do not survive reproduction, it is possible that these animals could reach levels of aerobic capacity that otherwise would be limited by induced redox stress. We have carried out a comparative study on atokous (immature worms) and epitokous (mature swimming males and benthic females) Nereis (Neanthes) virens to detect metabolic changes that could occur during sexual maturation. We have analyzed enzymes involved in different energy producing pathways: (i) mitochondrial aerobic metabolism was measured by the activities of electron transport system (ETS), which comprises the activity of two enzymes of the respiratory chain, NADH dehydrogenase and cytochrome c reductase (complexes I and III, respectively), cytochrome c oxydase (CCO or complex IV), and citrate synthase (CS) and (ii) anaerobic metabolism by the activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH). Amino acid metabolism was measured with aspartate aminotransferase (AAT). Defense capacities against oxidative stress have been estimated by the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Finally, as N. virens reproduction mode is associated with food deprivation; digestive capacity has been determined by the measure of trypsin and lipase activities. 2. Materials and methods 2.1. Sampling Epitokous N. virens individuals (dark green colored swimming males and brown-red benthic females) were collected in an intertidal sandflat in Ste-Luce, Québec, Canada, in May 2003 and 2006. Atokous individuals were collected in the same area in September 2003 and 2006. These sampling periods were chosen because atokous and epitokous worms were at equivalent sizes and because a hypothetic higher metabolism in 203 epitokes could not be related to better conditions since they were ending a low food supply season compared to atokes (Desrosiers et al., 1994). After capture, each individual was stored at − 80 °C until used for enzymatic analyses (less than 2 months). 2.2. Enzymatic assays Each individual was weighed and homogenized three times for 10 s in 5 volumes of ice cold buffer (50 mM imidazole, 2 mM MgCl2 and 5 mM EDTA, pH 7.5) with a Tekmar homogenizer. Homogenates were centrifuged at 500 g for 5 min at 4 °C and the supernatant was used for enzymatic analyses. All the manipulations were performed on ice. Individuals sampled in 2003 were used to determine AAT, CS, LDH, PK, trypsin and lipase activities while ETS, CCO, SOD, CAT and GPX activities were measured with worms collected in 2006. Enzyme activities were measured at 25 °C using a UV/VIS spectrophotometer (Perkin Elmer, Lambda 11) equipped with a thermostated cell holder and a circulating refrigerated water bath. All assays were run in duplicate and specific activities were expressed as U/g protein where U represents μmol of substrate transformed per minute. Conditions for enzymatic assays were as follows: ETS: 0.1 M sodium phosphate (pH 8.5), 0.85 mM NADH, 2 mM iodonitrotetrazolium chloride and 0.03% Triton X-100. Activity was recorded at 25 °C by following the reduction of INT, which yields an increase in absorbance at 490 nm (ε490 = 15.9 mL cm– 1 μmol– 1) (Lannig et al., 2003). CCO (EC 1.9.3.1): 100 mM potassium phosphate and 0.05 mM cytochrome c (pH 7.4). Cytochrome c was reduced by the addition of dithionite (4 mM) and the solution was bubbled with air to eliminate the excess of reducing agent (Hodges and Leonard, 1974). Control samples consisted of 50 mM cytochrome c oxidized with potassium ferricyanide (0.05%). Activity was measured at 25 °C by following the oxidation of cytochrome c at 550 nm and calculated using an extinction coefficient (ε550) of 19.1 mL cm– 1 μmol– 1. CS (EC 4.1.3.7): 100 mM imidazole–HCl (pH 8.0), 0.1 mM 5,5′-Dithiobis(2-nitrobenzoic acid), 0.1 mM acetyl CoA and 0.15 mM oxaloacetate. Activity was measured at 25 °C by following the increase in absorbance due to the oxidation of DTNB at 412 nm (ε412 = 13.6 mL cm– 1 μmol– 1) (Thibeault et al., 1997). PK (EC 2.7.1.40): 50 mM imidazole–HCl (pH 7.0), 10 mM MgCl2, 100 mM KCl, 5 mM ADP, 0.15 mM NADH, 5.0 mM phosphoenolpyruvate and 0.6 U mL– 1 lactate dehydrogenase. PK was measured at 25 °C by following the oxidation of NADH at 340 nm (ε340 = 6.22 mL cm– 1 μmol– 1) (Pelletier et al., 1994). LDH (EC 1.1.1.27): 100 mM potassium-phosphate (pH 7.0), 0.16 mM NADH and 0.4 mM pyruvate. LDH was measured at 25 °C by following the oxidation of NADH at 340 nm (ε340 = 6.22 mL cm– 1 μmol– 1) (Thibeault et al., 1997). AAT (EC 2.6.1.1): 100 mM potassium-phosphate (pH 7.4), 0.025 mM pyridoxal phosphate, 0.32 mM NADH, 10 mM αketoglutarate, 22 mM aspartate and 0.6 U mL– 1 malate dehydrogenase. AAT was measured at 25 °C by following the 204 É. Hébert Chatelain et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 202–208 oxidation of NADH at 340 nm (ε340 = 6.22 mL cm– 1 μmol– 1) (Pelletier et al., 1994). SOD (EC 1.15.1.1): 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 0.01 mM cytochrome c, 0.05 mM xanthine, 0.005 unit of xanthine oxidase and 1 unit of superoxide dismutase. Superoxide anion was generated by a xanthine/ xanthine oxidase system and the reduction of cytochrome c was monitored at 25 °C and 550 nm (pH 7.8). Enzyme activity was expressed as SOD units, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per minute and per g of protein (Moraes et al., 2006). CAT (EC 1.11.1.6): 100 mM sodium phosphate (pH 7.5) and 60 mM H2O2. CAT activity was determined at 25 °C by following the decomposition rate of H2O2 at 340 nm (ε340 = 6.22 mL cm– 1 μmol– 1) (Nusetti et al., 2005). GPX (EC 1.11.1.9): 100 mM potassium phosphate (pH 7.5), 0.06 mM NADPH, 2 mM reduced glutathione, 40 mM sodium azide, 1 U ml– 1 glutathione reductase and 60 mM H2O2. Activity of GPX was recorded at 25 °C by following the oxidation of NADPH at 340 nm (ε340 = 6.22 mL cm– 1 μmol– 1) (Nusetti et al., 2005). Lipase (EC 3.1.1.3): 0.8 mM 1,2-diglyceride, 1.5 mM 3-(NEthyl-3-methylanilino)-2-hydroxypropanesulfonic, 0.5 mM ATP, 650 U L– 1 monoglyceride lipase, 990 U L– 1 glycerol kinase, 29500 U L– 1 glycerol-3-phosphate oxidase, 990 U L– 1 peroxidase, 29500 U L– 1 co-lipase, 3.9 mM cholic acid, 0.04% sodium azide. This measure was realized with the Lipase-PS kit (procedure no. 805) from Sigma Diagnostics (St. Louis, Mo., USA). Lipase activity was recorded at 25 °C and 550 nm and estimated following the manufacturer’s protocol. Trypsin (EC 3.4.21.4): 0.2 M Tris–HCl, 0.04 M CaCl2 (pH 8.0), 1 mg ml– 1 N α-Benzoyl-DL-arginine 4-nitroanilide and 5% (v/v) dimethylformamide. Trypsin activity was determined at 25 °C by following p-nitro-alanine formation at 410 nm (ε410 = 8.8 mL cm– 1 μmol– 1) (Erlanger et al., 1961). 3. Results The average mass was not significantly different among epitokous females, epitokous males and atokous worms, whereas epitokous females had significantly higher protein content (p b 0.01) than both epitokous males and atokous worms (Table 1). 3.1. Metabolic capacities Significant differences among groups were observed for ETS (ETS and CCO activities were not measured for epitokous females), CS, LDH and AAT. Epitokous males had significantly higher ETS activity than atokous worms (p b 0.01; Fig. 1a), whereas no significant difference was found for CCO between these groups (p = 0.41; Fig. 1b). Significantly higher activity was also observed in epitokous males compared to both epitokous females and atokous worms for CS (p b 0.01; Fig. 1c). There was no significant difference among groups for PK activity (p = 0.29; Fig. 2a), whereas LDH was significantly lower in epitokous males compared to other groups (p b 0.01; Fig. 2b). AAT activity was significantly higher in epitokous males compared to both epitokous females and atokous worms (p b 0.01; Fig. 3). 3.2. Antioxidant capacities No significant difference in antioxidant enzymes activities (SOD, p = 0.33; CAT, p = 0.67; GPX, p = 0.83) was observed between epitokous males and atokous worms (Table 2). These enzymes were not measured in epitokous females because no significant difference in aerobic enzyme activities (and likely oxidative stress level) was observed between females and atokous worms. 3.3. Digestive capacities 2.3. Protein assays Total protein content of each sample was determined by the bicinchoninic acid method (Smith et al., 1985). Lipase and trypsin activities were significantly lower in epitokous males than in epitokous females and atokous worms (p b 0.01; Fig. 4a, b). 2.4. Chemicals 4. Discussion All chemicals used for enzymatic and protein assays were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Our results show a clear parallel between morphological transformation and metabolic modifications to afford swimming and spawning in epitokous N. virens males. The reengineered 2.5. Statistical analyses SAS software (9.1.3) was used for all statistical analyses. Student t-test for independent samples was used to compare SOD activities between atokous and epitokous worms. Normality assumption was verified with Shapiro–Wilk test and variance equality was confirmed with Levene test. Non parametric Kruskal–Wallis test was used to compare all other parameters, as these parameters violated normality assumptions according to Shapiro–Wilk test. Significance was assessed at the 0.05 (or lower) level for all tests. Table 1 Mean (± SD) body mass and mean (±SD) protein content in atokes, epitokous females and epitokous males of Nereis virens Atokes (n = 34) Epitokous females (n = 32) Epitokous males (n = 18) p-value a Statistically different. Body mass Protein content (g) (mg g− 1) 3.92 ± 1.28 3.87 ± 1.69 3.75 ± 1.28 0.80 36.74 ± 7.65 42.25 ± 7.37 a 38.05 ± 8.75 b0.01 É. Hébert Chatelain et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 202–208 205 Fig. 3. Mean (± SD) aspartate aminotransferase (AAT) activity in atokes (n = 10), females (n = 29) and males (n = 25) of Nereis virens. ⁎ Indicates statistical difference; p b 0.01 according to Kruskal–Wallis test. Fig. 1. Mean (± SD) enzymatic activities of aerobic metabolism (a: ETS, electron transport system; b: CCO, cytochrome c oxidase; c: CS, citrate synthase) in atokes (n = 10), females (n = 29) and males (n = 26) of Nereis virens (ETS and CCO were not measured for epitokous females). ⁎ Indicates statistical differences; ETS and CS; p b 0.01 according to Kruskal–Wallis test. body musculature, which support high speed contractions required by sustained swimming (Schroeder and Hermans, 1975), is associated with an increase of CS and ETS activities and a decrease of LDH activity, indicating a higher aerobic activity and a lower anaerobic capacity in epitokous swimming males. Previous data presented by Schöttler (1989) supported a similar reorganization of metabolism by showing a 3-fold in- crease in VO2 max and glutamate dehydrogenase (a mitochondrial enzyme) and a 7-fold decrease in lactate dehydrogenase during epitoky in N. virens. These biochemical findings also corroborate previous observations of increment in size and number of mitochondria in epitokous worms (Clark, 1961; Wissocq, 1978), revealing that swarming males are adapted to aerobic activity. Indeed, since the major end product of polychaetes in early anaerobiosis is lactate (Scott, 1976; Schöttler, 1979), a slightly toxic waste, the use of anaerobic metabolism should reduce fitness of epitokous males by limiting their capacity to swarm and spawn. Inversely, aerobic pathway would likely allow for longer effort without the noxious side effects of anaerobic metabolism. Although our results indicate that high aerobic capacity is likely essential to enable mature males to swim before releasing their gametes, we found no simultaneous change in the specific activity of the cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain. These findings suggest that an increase in CCO activity is not required to improve aerobic aptitude. This enzyme is known to present an excess capacity in mammal tissues (Gnaiger et al., 1998; Rossignol et al., 2003) and in ectotherm species (Blier and Lemieux, 2001), suggesting that small changes in its activity barely affect the overall mitochondrial respiration. In the absence of CCO responsiveness, however, an increase in ETS activity in N. virens males could increase the reduction state of the entire respiratory chain, which could in turn stimulate the production of reactive oxygen species (ROS) and generate oxidative damage (see below) (Chen et al., 2003). Alternatively, within the context of retrograde signaling, ROS could have regulatory (as opposed to cytotoxic) effects, and upregulate gene expression to Table 2 Mean (±SD) activities of antioxidant enzymes (SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase) in U/g protein in atokous and epitokous males of Nereis virens Fig. 2. Mean (±SD) enzymatic activities of anaerobic metabolism (a: PK, pyruvate kinase; b: LDH, lactate dehydrogenase) in atokes (n = 10), females (n = 32) and males (n = 26) of Nereis virens. ⁎ Indicates statistical difference; p b 0.01 according to Kruskal–Wallis test. Atokes (n = 8) Epitokous males (n = 8) SOD CAT GPX 137.32 ± 29.46 141.57 ± 30.60 1.33 ± 0.59 1.42 ± 0.64 1.93 ± 0.83 1.96 ± 0.67 No significant difference (p N 0.05) detected. 206 É. Hébert Chatelain et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 202–208 Fig. 4. Mean (±SD) digestive capacities (a: lipase; b: trypsin) in atokes (n = 10), females (n = 32) and males (n = 26) of Nereis virens. ⁎ Indicates statistical differences; lipase and trypsin, p b 0.01 according to Kruskal–Wallis test. compensate for an energetic deficit (Chen et al., 2003). The alternative terminal oxidase (AOX) could also represent a means to maintain respiration when the cytochrome pathway is impaired (McDonald and Vanlerberghe, 2004). This enzyme, which has been noted in several polychaete species such as Nereis pelagica (Tschischka et al., 2000), provides an alternative route for electrons by transferring them directly from reduced ubiquinol to oxygen. In other words, even when CCO does not follow the general increase in ETS activity, electrons can be transferred to oxygen via AOX. As several proton-pumping steps are bypassed in this alternative pathway, activation of the oxidase should however reduce ATP generation (McDonald and Vanlerberghe, 2004). Further studies will be needed in order to evaluate the importance of changes in the specific activity and kinetic regulation of mitochondrial respiratory chain complexes in determining oxidative energy metabolism and ATP production during the epitokous phase of polychaete worms. The elevated activity of AAT in epitokous males may bear evidence for high catabolism and anabolism of amino acids derived from the extensive histolysis of the body wall and a reorganization of body musculature. It may also indicate an increase in energy metabolism due to the production of oxaloacetate by the malate–aspartate shuttle and the related increase production of reducing equivalents to supply the electron transport system in mitochondria (Scaraffia et al., 1997). The higher mitochondrial metabolism observed in epitokous males could lead to an overall reduction of the mitochondrial respiratory system and to an increase of ROS production, which could damage DNA, proteins and lipids and accelerate senescence (Beckman and Ames, 1998; Barja and Herrero, 2000; Das et al., 2001; Bokov et al., 2004). However, one might also expect that oxidative damage would hardly have a significant impact on long term survival of N. virens since both epitokous males and females die within 3 days after males have emerged from burrows (Kristensen, 1984; Fischer, 1999). This is supported by detecting no significant increase in superoxide dismutase, catalase and glutathione peroxidase (antioxidant enzymes) between epitokous males and atokous worms despite an increase in aerobic capacity in epitokous males. These results suggest that death following increased reproductive effort in N. virens males is at least partly mediated by oxidative stress, although future work is needed to determine the level of oxidative damage in both epitokous males and females. For example, antioxidant capacities could be more important in germinal tissues for the protection of the gametes until spawning. A comparative study of antioxidant capacities among different tissues would be needed to test this hypothesis. Further investigations into the role of non-enzymatic antioxidants in oxidative stress regulation would also help to evaluate their potential influence during epitokous transformation. For example, AbeleOeschger and Oeschger (1995) proposed that the presence of non-enzymatic antioxidants in the mucus layer of eggs cocoons of the polychaete Phyllodoce mucosa should compensate for the low enzymatic antioxidant defenses, which are acquired later during the ontogeny of the worm. Combined with reproductive stress, food deprivation (even for few days) could also be implied in the rapid senescence and natural death that occur after spawning in semelparous species. For example, semelparous pacific salmons do not feed during their extreme migration and rely on the catabolism of fat and muscle protein to fuel breeding activity: their investment in reproduction is associated with weight loss and atrophy of digestive organs (Mommsen et al., 1980; Morbey et al., 2005). In this case, it was proposed that the salmon rapid senescence is primarily caused by stress and starvation (Hochachka and Somero, 2002; Morbey et al., 2005). A similar phenomenon likely occurs in nereidid polychaetes as they are known to cease feeding at the end of maturation and rely solely on body reserves in order to maximize the growth of gametes (Hoeger, 1991). In N. virens epitokous males, the maintenance of gamete formation during food deprivation could be achieved by eleocytes, which are specialized coelomic cells that synthesize compounds such as lipids, supplying material for growth and development of spermatogonia cells (Hoeger and Kunz, 1993). In females, these cells assume a central role in the process of oogenesis by producing important quantity of vitellogenin, likely explaining the higher protein content in females. While our results of significantly reduced digestive capacities in epitokous swimming males appear to support Hoeger (1991), no significant difference in digestive capacities and AAT was obtained in epitokous females compared to atokous worms. This may be due to their feeding behaviour: as N. virens females do not appear to leave their burrows to spawn, they could continue to feed during the period when the eggs are maturing, and then conserve their digestive capacities. This feeding behaviour during egg maturation has already been observed for the female Hediste diversicolor (Clark, 1961). The importance of food deprivation in the death of N. virens is probably more important in males than in females. É. Hébert Chatelain et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 202–208 Indeed, by contrast with males that acquire a modification of the posterior end, the pygidial rosette, which serves for sperm emission, most female nereids spawn (and probably die) through ruptures in the body wall (Hofmann, 1964). In conclusion, epitokous swimming males have a different metabolic pattern compared to atokous worms and epitokous females, likely due to their requirement for an increase in swimming capacity for swarming. The higher aerobic capacity in epitokous swimming males is paralleled by a decrease in anaerobic metabolism, but is not correlated with an increase in antioxidant enzymes activities. To perform reproduction, males must swim vigorously for an important period, which may only be afforded through an aerobic burst. 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