Uric acid deposits and estivation in the invasive apple-snail, Pomacea canaliculata

Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Uric acid deposits and estivation in the invasive apple-snail, Pomacea canaliculata Maximiliano Giraud-Billoud a, María A. Abud a, Juan A. Cueto a, Israel A. Vega a, Alfredo Castro-Vazquez a,b,⁎ a b

Laboratory of Physiology (IHEM-CONICET), Department of Morphology and Physiology (FCM-UNCuyo), Casilla de correo 33, 5500 Mendoza, Argentina Centro Nacional Patagónico (CENPAT-CONICET). Blvd. Brown 2915 (U9120ACV) Puerto Madryn, Chubut, Argentina

a r t i c l e

i n f o

Article history: Received 2 July 2010 Received in revised form 6 December 2010 Accepted 10 December 2010 Available online 21 December 2010 Keywords: Reoxygenation Oxidative stress Oxyradicals scavengers Antioxidants Nitrogen recycling Urate oxidase Gastropods

a b s t r a c t The physiological ability to estivate is relevant for the maintenance of population size in the invasive Pomacea canaliculata. However, tissue reoxygenation during arousal from estivation poses the problem of acute oxidative stress. Uric acid is a potent antioxidant in several systems and it is stored in specialized tissues of P. canaliculata. Changes in tissue concentration of thiobarbituric acid reactive substances (TBARS), uric acid and allantoin were measured during estivation and arousal in P. canaliculata. Both TBARS and uric acid increased two-fold during 45 days estivation, probably as a consequence of concomitant oxyradical production during uric acid synthesis by xanthine oxidase. However, after arousal was induced, uric acid and TBARS dropped to or near baseline levels within 20 min and remained low up to 24 h after arousal induction, while the urate oxidation product allantoin continuously rose to a maximum at 24 h after induction, indicating the participation of uric acid as an antioxidant during reoxygenation. Neither uric acid nor allantoin was detected in the excreta during this 24 h period. Urate oxidase activity was also found in organs of active snails, but activity shut down during estivation and only a partial and sustained recovery was observed in the midgut gland. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Estivation is a state of behavioral quiescence and metabolic arrest which occurs in response to drought and/or high ambient temperature in several vertebrate and invertebrate taxa and may be associated with a profound metabolic depression (Guppy and Withers, 1999). The physiological mechanisms of estivation have been mostly studied in anurans (Amphibia) and land-living pulmonates (Gastropoda) (HermesLima and Zenteno-Savin, 2002; Storey, 2002), where a critical aspect is the defense against the excess production of free oxygen radicals at the time of arousal from estivation. Hermes-Lima et al. (1998) pointed to the overall similarities between tissue hypoxia/re-oxygenation occurring during the estivation/arousal sequence of events in land-living gastropods, and those occurring after ischemic injury in man (as in myocardial infarction and stroke), in which reperfusion with oxygenated blood does not simply reverse the stress but instead triggers a series of post-ischemic oxidative injuries caused by oxyradicals (McCord, 2000; Young and Woodside, 2001; Rahman, 2007; Halliwell, 2009). Several authors (Hermes-Lima et al., 1998; Hermes-Lima and Zenteno-Savin, 2002; Ramos-Vasconcelos et al., 2005; Nowakowska et al., 2009) have shown that different enzymatic mechanisms and glutathione, protect pulmonate gastropods from the damaging effects of re-oxygenation during post-estivation arousal. In contrast, uric acid has been shown to act as a non-enzymatic antioxidant (Ames et al.,

1981; Becker, 1993) and it has been suggested that it may play such role in post-estivation arousal (Hermes-Lima and Storey, 1995b; Vega et al., 2007; Giraud-Billoud et al., 2008). The apple-snail Pomacea canaliculata (Lamarck, 1822) (Architaenioglossa, Ampullariidae) shows an elaborate array of urate tissues distributed in lung, gill, coiled gut, midgut gland, testis, ampullary heart cavity and anterior kidney containing intracellular urate crystalloids (Vega et al., 2007; Giraud-Billoud et al., 2008). The latter study showed a sequential process of crystalloid formation and lysis, which occurs asynchronously in cells within the same tissue, suggesting an active turnover of uric acid in these cells. Pomacea canaliculata is able to estivate in the field (d'Orbigny, 1847; Cowie, 2002), and the current study has dealt with changes occurring during experimental estivation and during post-estivation arousal in both uric acid and allantoin concentration in soft tissues of this snail, to test the hypothesis that uric acid and allantoin are involved in antioxidant mechanisms of the arousing snails. Allantoin is an oxidation product of uric acid that may be produced either spontaneously (when uric acid acts as an electron acceptor) or by urate oxidase (EC 1.7.3.3) catalysis.

2. Materials and methods 2.1. Animals and culturing conditions

⁎ Corresponding author. Fisiología, Casilla de Correo 33, M5500 Mendoza, Argentina. Tel.: +54 261 413 5000x2715; fax: +54 261 449 4117. E-mail address: [email protected] (A. Castro-Vazquez). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.12.012

Adult males and females (30–40 mm shell length) obtained from a cultured strain of P. canaliculata were used. All groups in this study

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

507

were composed by an equal number of males and females. The original stock was collected at the Rosedal Lake (Palermo, Buenos Aires, Argentina) and voucher (ethanol preserved) specimens of the original population, were deposited at the collection of Museo Argentino de Ciencias Naturales (Buenos Aires, Argentina; lots MACN-In 35707 and MACN-In 36046, respectively). Temperature was regulated at 24–26 °C and artificial lighting was provided 14 h per day. Room relative humidity varied around 80%. Aquarium water was changed thrice weekly. Animals were fed ad libitum with a mixed diet made mostly of fresh lettuce, supplemented weekly with carp food pellets, desiccated and powdered eggs and toilet paper. 2.2. Mortality rate and body mass changes during experimental estivation and after arousal The animals were induced to estivate by leaving them on a dry surface in the culture room. Day 0 of estivation of each snail was defined as the day when the operculum appeared firmly attached to the shell aperture (see Results section). At that time each snail was put in an individual dry container in the culture room. In one set of observations, the mortality rate was recorded on days 2, 15, 30, 45, 60 and 75 of experimental estivation (independent groups of N = 24 for each day; this was made since in previous observations the snails appeared extremely sensitive to handling during estivation). Four of the dead snails showed fly puparia in their proximity, and these were collected and kept in close containers (to prevent the adults to escape after emergence) and they were later fixed in 70% ethanol for taxonomic identification (see Acknowledgements). In a second set, body mass changes during experimental estivation were recorded for 18 animals, by weighing them on day 0 (as defined above) and on days 2, 15, 30 and 45 thereafter. Body mass losses were expressed as percent of the initial value on Fig. 1B. In a third set of observations, arousal was induced in 30 animals after 45 days of estivation by transferring each snail to an individual vessel with 45 mL of tap water, so that the animals were only partly submersed. Time 0 of arousal was defined as the time of operculum detachment, and the animals were drained and weighed 20 min, and 24, 48, 72 and 96 h thereafter (N = 6 per group). A large piece of fresh lettuce was offered 90 min after water exposure. Body mass recovery was computed for each snail as the body mass difference between day 0 of estivation and the corresponding post-arousal value. These results were expressed as percent recovery on Fig. 1C.

Fig. 1. Mortality rate and body mass changes during experimental estivation and after arousal in P. canaliculata. A. Mortality rate during experimental estivation (N = 72). B. Body mass loss during experimental estivation (mean % change ± SE, N = 18). C. Body mass recovery after induction of arousal (mean % change ± SE, N = 30).

2.3. Behavioral observations Eighteen animals which were induced to estivate by leaving them on a dry surface were observed at 2-h intervals during the first 10 h, and at 24, and 48 h after induction. Also, 18 animals that were estivating for 45 days, were induced to arouse by transferring them to water containing vessels. A large piece of fresh lettuce was added to each vessel 24 h after induction. Also, behavioral observations of the arousing snails were made at 5, 10, 20, 30 and 50 min, and at 5 and 10 h after induction. 2.4. Experimental groups and sacrifices Four groups (six animals each) were set for exploring the changes in uric acid and allantoin concentration in the soft tissues of control, estivating and aroused animals. The control group was kept in the standard culturing conditions, while the other three groups were induced to estivate. One of the latter groups was sacrificed in the morning of day 45 of estivation, while the other two groups were aroused from estivation on that morning, and were sacrificed either 20 min or 24 h after the operculum was detached from the shell (see Results section).

Also, 24 h-excreta were collected from six active control snails and from six estivating snails that were exposed to water to induce arousal. For such purpose, they were weighed (the active snails were previously drained) and each one was placed in a vessel containing 45 mL of tap water with no food, and with the addition of penicillin-G 0.6 g/L and streptomycin sulfate 0.6 g/L, to prevent possible bacterial action on the excreted compounds, as described in Vega et al. (2007). Arousal of the estivating snails was induced by water exposure in these vessels, and 24 h later the animals were removed and water with the excreta was thoroughly mixed and then centrifuged at 3000 g for 20 min at 4 °C. Aliquots (2.5 mL) of the obtained supernatants (soluble excreta) were collected and frozen while the precipitates (particulate excreta) were homogenized for 10 min at 4 °C in 1.5 mL of 0.5% lithium carbonate (UltraTurrax® homogenizer; IKA® Werke GmbH & Co., Germany), and centrifuged at 3000 g for 20 min at 4 °C. The supernatants were aliquoted (2.5 mL) and kept frozen until uric acid and allantoin determinations were made.

508

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

2.5. Lipid peroxidation Thiobarbituric acid reactive substances (TBARS) were quantified as an index of lipid peroxidation. The visceral mass (including mantle skirt, gill and lung, and the copulatory apparatus in males) and the head-foot mass were separated in each snail, and were desiccated at 60 °C until constant weight, and they were mortar powdered separately because of their different toughness. One hundred mg of powder from each mass were homogenized (UltraTurrax® homogenizer) in 900 μL of 0.1 mol/L sodium phosphate buffer, pH 7.0, centrifuged at 10,500 g for 5 min at room temperature, and the supernatants were kept frozen until assaying with a colorimetric method described by Wasowicz et al. (1993) and modified by Lapenna et al. (2001). Frozen aliquots (500 μL) were mixed with 1 mL of a cold working solution (15% w/v trichloroacetic acid, 0.25 N HCl, 0.67% w/v thiobarbituric acid, 2.25 mmol/L butylated hydroxytoluene solution and 0,1 mL of 8.1% SDS) and kept on ice during the process. Subsequently, samples (pH ~ 1.5) were heated for 30 min at 95 °C and then 3 mL of butanol were added. Finally, tubes were stirred for 5 min and then centrifuged for 10 min at 1500 g at room temperature. The organic layers were collected and placed in glass cuvettes. The thiobarbituric acid solution was replaced by 0.25 mmol/L of HCl for the blanks. Results were calculated to minimize background interference (Hermes-Lima and Storey, 1995a) and expressed as nmol of compound per gram of dry soft tissue (nmol/g). The spectrophotometric quantification of TBARS actually overestimates malondialdehyde in tissues but is considered an effective tool for comparative studies of oxidative stress (Lapenna and Cuccurullo, 1993; Hermes-Lima and Storey, 1995a; Ramos-Vasconcelos and Hermes-Lima, 2003). 2.6. Determination of uric acid and allantoin

groups of animals (active controls, estivating and 20 min and 24 h after arousal). Tissue samples were homogenized in an Ultra-Turrax® homogenizer (1 min at 4 °C) in 4 mL of buffer (86 mmol/L NaCl, 1.8 mmol/L KCl, 2.1 mmol/L CaCl2, 10 mmol/L HEPES, and 1 mmol/L dithioerythritol, pH 7.6). Finally, they were centrifuged at 10,500 g for 5 min at 4 °C. Supernatants were kept frozen until determinations were made. Determination was based on the disappearance of uric acid, as indicated by the decrease of absorbance at 292 nm (Mahler et al., 1955). Aliquots of 900 μL of supernatants were added to 5.1 mL of a 29.4 mM uric acid aqueous solution to make 25-mM the final uric acid concentration. The mixture was measured every 30 min for its absorbance at 292 nm, while being stirred at 30 °C; measurements were stopped when no further decrease in absorbance was observed. As controls, a mixture containing the assay buffer instead of the supernatant was similarly processed. Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. Sensitivity of the method was 2.5 μg per tube. Sensitivity for urate oxidase detection was 3.6 nmol of uric acid consumed per min. Results were expressed as the amount of uric acid consumed per minute and per milligram of protein (nmol/min/mg). 2.8. Statistics Changes in mortality rate with time of estivation were evaluated with the chi square test. Paired observations (as for body mass changes of the same animal at different times) were evaluated by Student's t test. For multigroup comparisons, the distribution of variables was first evaluated by Kolmogorov–Smirnov's normality test, and equal variance Bartlett's test was used to evaluate homogeneity of variances for each set of experimental variables. The data were square roottransformed if variances differed significantly and differences between groups were evaluated by one-way ANOVA and the Tukey test as a post-hoc analysis. Comparisons of urate oxidase activity, however, were made by using Kruskal–Wallis one-way ANOVA and the Dunn's test, since several null values were observed after estivation and arousal. In all cases, significance level was fixed at P b 0.05.

The visceral and head-foot masses were separately obtained from each animal, desiccated at 60 °C until constant weight and homogenized in an Ultra-Turrax® homogenizer for 5 min at 4 °C in 6.5 mL of 0.5% lithium carbonate, centrifuged at 3000 g for 5 min at 4 °C, and aliquots of supernatants were kept frozen until assaying. The extraction efficiency was controlled as described in Vega et al. (2007). Standard curves were run with and without adding 0.5% lithium carbonate, to control for presumptive biases of determinations, but such biases were not found. Uric acid was measured in 100 μL aliquots, which were treated with urate oxidase, and the amount of hydrogen peroxide formed was quantified by a peroxidase catalyzed reaction with 4-aminophenazone and chlorophenol, which produces a colored quinoneimine product (Trinder, 1969). Sensitivity of the method was 0.25 μg (1.5 nmol) per tube. Also, 100 μL aliquots of the excreta were used for uric acid determinations. Allantoin was measured in 1 mL aliquots using a colorimetric method described by Young and Conway (1942). In this procedure allantoin is hydrolyzed in a weak alkaline solution at 100 °C to allantoic acid, and then degraded to urea and glyoxylic acid in a weak acid solution. Glyoxylic acid reacts with phenylhydrazine hydrochloride to produce a phenylhydrazone. This product forms a chromophore with potassium ferricyanide which is read at 522 nm. Sensitivity of the method was 1.25 mg (7.9 mmol) per tube. Also, 1 mL aliquots of the excreta were used for allantoin determinations. Uric acid and allantoin tissue concentrations were calculated on a per snail bases, and were expressed as mmol of compound per gram of dry soft tissue (mmol/g).

The mortality rate significantly rose from zero on day 2 to 54% on day 75 of estivation (chi square test, Fig. 1A). A significant loss in body mass during estivation was observed in all the studied days when compared with pre-estivation values (Student's paired t test, Fig. 1B). Mean body mass loss during estivation increased from 18% on day 2 to 51% on day 45 (Fig. 1B) and statistically significant differences occurred (day 2 vs. days 15, 30 and 45; day 15 vs. day 45; one-way ANOVA, Tukey test). A 45 days period of estivation was chosen for subsequent experiments. In these conditions, body mass recovery occurred rather rapidly after arousal (Fig. 1C), since significant differences between pre-estivation and post-arousal values still occurred only 20 min and 1 day after arousal (paired t test). Multigroup comparisons between postarousal values yielded some significant differences (20 min vs. all the other groups, and day 1 vs. day 4; one-way ANOVA, Tukey test). No deaths were recorded during the observations.

2.7. Urate oxidase activity (EC 1.7.3.3)

3.2. Infestation by fly larvae

Approximately 100 mg samples of the midgut gland, the coiled gut, the posterior kidney and the foot were obtained for the four

Several fly's puparia were found in the surroundings of four dead snails that had been induced to estivate. Both the puparia and the

3. Results 3.1. Changes in mortality rate and body mass during experimental estivation and after arousal

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512 Table 1 Frequency (%) of behavioral events at different times after induction of estivation in P. canaliculata (N = 18). Hours after induction

Crawling

Attached and immobile

Retracted into shell

Operculum tightly adhered

2 4 6 8 10 24 48

72.2 55.6 33.3 11.1 5.6 0 0

0 22.2 33.3 55.6 22.2 22.2 0

27.8 22.2 33.3 33.3 72.2 27.78 0

0 0 0 0 0 50.0 100

509

acid was significantly elevated in animals after 45 days of estivation and it decreased after arousal. Additionally, no significant differences were observed between control and arousal groups. Allantoin levels in dried soft tissues (Fig. 3C) significantly increased during estivation as compared with the control group (one-way ANOVA, Tukey test). No significant increase as compared with the estivating snails was observed at 20 min after arousal, but a significant increase was observed at 24 h after arousal as compared with all other groups (one-way ANOVA, Tukey test). Neither uric acid nor allantoin was detected in the 24-h soluble or particulate excreta of aroused animals. 3.6. Urate oxidase activity in several organs

emergent adults were identified as of the scuttle fly Megaselia scalaris (Loew, 1866) (Diptera, Phoridae) (see Acknowledgements). 3.3. Behavioral changes after induction of estivation and arousal When the animals were induced to estivate (Table 1), they first crawled on the dry surface, and later became immobile. Seventy two percent of them had already retracted into the shell by 10 h after being left on the dry surface; however, the latter were all keeping a slit between the shell aperture and the operculum. 24 h after induction, however, half of the animals had the elastic operculum tightly adhered to the shell, while the others were either still attached and immobile (22%) or already retracted into the shell (28%). All animals had the operculum tightly closed at 48 h after the beginning of observations. The arousal response was induced by water exposure (Table 2). Detachment of the operculum was first seen in 28% of animals 5 min after induction (Fig. 2B). Extrusion of an exploring tentacle was occurring in 72% of animals 20 min after induction (Fig. 2C), and the head and foot was starting to deploy and both tentacles were being turned outward in 56% of animals after 30 min (Fig. 2D), while 11% of animals were either still attached to the vessel wall or were already crawling (Fig. 2E). At 5 h, 17% of animals had already eaten lettuce, and 83% of them at 10 h (Fig. 2F). 3.4. Lipid peroxidation Changes in TBARS concentration in dried soft tissues of P. canaliculata (Fig. 3A), as indicative of lipid peroxidation, were evaluated by one-way ANOVA followed by the Tukey test. A twofold and statistically significant increase in animals after 45 days of estivation was observed as compared with the control group of active animals. Also, TBARS significantly dropped to values slightly above the control levels at 20 min and 24 h after operculum detachment. The latter low values, however, were still significantly higher than the active control group. 3.5. Uric acid and allantoin concentrations Uric acid concentration in dried soft tissues showed parallel changes to those of TBARS (one-way ANOVA, Tukey test; Fig. 3B): uric

Enzyme activity was found in active animals (Table 3) both in organs containing urate tissues (midgut gland and coiled gut) and in some which do not (posterior kidney and foot) (Giraud-Billoud et al., 2008). No significant differences in enzyme activity between tissues were found (one-way ANOVA, Tukey test). After 45 days of estivation, urate oxidase activity dropped dramatically in all the studied tissues (statistically significant differences; one-way ANOVA, Tukey test). Actually all these cases yielded undetectable values except for a single kidney sample. Enzyme activity showed a tendency to recover after arousal, but a sustained recovery (both at 20 min and 24 h after arousal) reached statistical significance for the midgut gland only (one-way ANOVA, Tukey test). 4. Discussion A central objective of the current paper was to shed light on the possible role/s of uric acid and of urate storing tissues during and after estivation in P. canaliculata (Vega et al., 2007; Giraud-Billoud et al., 2008). Uric acid in ampullariid snails was first discovered in the genus Pila (Lal and Saxena, 1952; Saxena, 1955) and was found to increase in tissues during estivation (Meenakshi, 1964; Reddy et al., 1974; Chaturvedi and Agarwal, 1981). We have suggested some possible physiological roles for this purine (Vega et al., 2007; Giraud-Billoud et al., 2008) and the results of the current paper may be relevant for two of them: (1) the action of uric acid as an antioxidant and oxyradical scavenger, and (2) the role of urate tissues as a store of combined nitrogen, to be recycled into proteins and nucleic acids. 4.1. Uric acid as an antioxidant and oxyradical scavenger Adaptive strategies involving antioxidant enzymes and glutathione have evolved to minimize the effects of oxyradical production during the resumption of normal metabolic rates (Hermes-Lima et al., 1998; Hermes-Lima and Zenteno-Savin, 2002) in both land-living (Otala lactea, Helix aspersa) and freshwater (Biomphalaria tenagophila) pulmonate snails. With the exception of Helix pomatia (Nowakowska et al., 2009) pulmonate gastropods increase their levels of these endogenous antioxidants during estivation (Hermes-Lima et al., 1998; Ferreira et al., 2003; Ramos-Vasconcelos and Hermes-Lima,

Table 2 Frequency (%) of behavioral events at different times after induction of arousal in P. canaliculata (N = 18). Time after induction

Attached operculum

Operculum detachment

First tentacle extrusion

Foot starts to deployment

Attaching/crawling

Feeding

5 min 10 min 20 min 30 min 50 min 5h 10 h

77.8 27.8 11.1 0 0 0 0

27.8 77.8 16.7 5.6 0 0 0

0 0 72.2 38.9 0 0 0

0 0 0 44.4 44.4 0 0

0 0 0 11.1 55.6 83.3 16.7

– – – – – 16.7a 83.3

a

Lettuce was available for feeding since 90 min.

510

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

Fig. 2. Behavioral events after induction of arousal in P. canaliculata (N = 18). A. Estivating snail which had just been exposed to water and stills shows the operculum adhered to the shell. B. Operculum detachment, first sign of arousal after water contact. C. A tentacle is being extruded. D. Deployment of head and foot. E. The snail is already attached to the vessel's wall and it may crawl. F. A snail with fully deployed head and foot while eating lettuce (see Table 2 for the timing of these events).

2003) and Hermes-Lima et al. (1998) have proposed that this may be a crucial adaptation to minimize the potential injury due to oxidative stress during arousal, since the end of estivation by water exposure is unpredictable in the field, and the animal has to rapidly cope with the effects of reoxygenation. If we extend the hypothesis of Hermes-Lima et al. (1998) to include uric acid as an antioxidant system, one may expect (1) an accumulation of uric acid during estivation, and (2) a consumption of uric acid stores (and an increase in allantoin) if uric acid serves as an antioxidant defense during the arousal re-oxygenation. In agreement with that, (1) uric acid concentration showed a more than two-fold increase in dry soft tissues during the 45-days estivation period, which was followed by a remarkably rapid return to pre-estivation values within 20 min of arousal induction, and which were maintained at 24 h, and (2) the concentration of allantoin produced by uric acid oxidation showed a steady rise during the whole period of observation, reaching a four-fold increase respective to pre-estivation levels by 24 h after induction of arousal. Meanwhile, neither uric acid nor allantoin was detected in the excreta, strongly suggesting that depletion of uric acid deposits participates in the response to the oxidative stress of arousal, and that the allantoin formed is retained (i.e., is not excreted). The simultaneous rise in both uric acid and TBARS which occurred in soft tissues of P. canaliculata during the 45 days of estivation should be related to the synthesis of this purine from hypoxanthine and xanthine, since the involved reactions result in the generation of superoxide and hydroxyl radicals. Accumulation of uric acid during estivation has also been shown in the pulmonate gastropod O. lactea at a rate of 55-90 nmol/day (Speeg and Campbell, 1968). A similar mean figure (82 nmol/day) can be calculated from our data on Fig. 3B. The oxidative damage associated to uric acid synthesis has not been estimated in O. lactea but TBARS concentrations in P. canaliculata indicate that it was maximal by the end of estivation (Fig. 3A). Remarkably, TBARS suddenly decreased within 20 min after arousal induction, and they remained so 24 h after induction (Fig. 3A). TBARS in arousing snails were slightly but significantly above those in the

active control snails. This drastic decrease in peroxidation damage during arousal is indicative of the powerful antioxidant mechanisms (including uric acid) which should be put to work at the time. One may wonder why accumulated uric acid does not protect the estivating snail from oxidative damage (as the elevated TBARS indicate). We hypothesize that the newly formed uric acid is being removed from the circulation (where it could only act as an antioxidant) and stored in crystalloids in the urocytes (Vega et al., 2007; Giraud-Billoud et al., 2008). However, as indicated above, the accumulated purine may be rapidly used later as an antioxidant during post-estivation arousal. Statistically significant differences were found between control and estivating animals (*), estivating and aroused animals (**) and control and aroused animals (▲) (ANOVA I, Kruskal-Wallis test; Dunn's test was used for post hoc analysis; P b 0.05). ND = not detected. N was 8 for the active control group and 6 for the other groups. 4.2. Uric acid as an available store of combined nitrogen Uric acid nitrogen recycling into proteins and nucleic acids has been shown in insects and crustaceans (Potrikus & Breznak, 1981; Cochran, 1985; Buckner et al., 1985; Linton & Greenaway, 1997; Dillaman et al., 1999; López-Sánchez et al., 2009). The first step in the recycling chain of events requires the activity of urate oxidase, which was here shown in different organs of active snails (Table 3). After 45 days of estivation, however, urate oxidase activity dropped dramatically in all studied tissues, and only a partial and sustained recovery was observed after arousal in the midgut gland. The results support the possibility of uric acid nitrogen reutilization, but this would have to be determined directly. 4.3. Estivation in nature and in laboratory models P. canaliculata shows remarkable behavioral and ecological plasticity which facilitates its rapid invasion of new areas (Lach

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

511

water levels and extreme temperatures (above 35 °C and below 10 °C) (Wada and Yoshida, 2000). Snail behaviors both during the experimental induction of estivation and after arousal from this state were studied by Little (1968) in Pomacea lineata and in the current paper for P. canaliculata (Tables 1 and 2, and Fig. 2). The behavioral patterns observed were similar for both species, but the timing was different, for instance, P. lineata (Little, 1968) lasted 4 to 8 days to tightly close the operculum, while this occurs within 2 days in P. canaliculata. We are not aware of any study of the mechanisms of estivation and arousal in the invasive P. canaliculata, but some studies in noninvasive ampullariid species have been published (Lal and Saxena, 1952; Saxena, 1955; Coles, 1968; Little, 1968; Burky et al., 1972; Horne, 1979; Chaturvedi and Agarwal, 1979, 1981; Athawale and Reddy, 2002). However, they have not received an attention comparable to that of estivation in pulmonate gastropods (HermesLima and Storey, 1995a,b; Hermes-Lima et al., 1998; Guppy et al., 2000; Hermes-Lima and Zenteno-Savin, 2002; Ferreira et al., 2003; Ramos-Vasconcelos and Hermes-Lima, 2003; Ramos-Vasconcelos et al., 2005; Nowakowska et al., 2009; Ferreira-Cravo et al., 2010). The mortality rate observed in the current study was similar to that observed by Yusa et al. (2006) in animals kept in dry conditions and approximately of the same size as those of the current study. Pomacea canaliculata seems to withstand desiccation much longer than P. paludosa (=P. depressa; Little, 1968) but not more than P. lineata (same paper). Comparison with other non-invasive congeneric species is difficult, since most authors preferred to report the extreme survival times observed rather than the changes in the mortality rate with time. About a 50% of body mass loss was observed after 45 days of estivation in P. canaliculata, which was accompanied by a relatively low mortality rate (17%). This loss should be mostly water loss, since about 73% of body mass was recovered within 20 min of water exposure (with no food). Similar figures of body mass loss were reported by Little (1968) in P. lineata. Fig. 3. Changes in the concentration of thiobarbituric acid reactive substances (TBARS) (panel A), uric acid (panel B) and allantoin (panel C) in dried soft tissues of control active, estivating and aroused P. canaliculata. Values are means ± SE (N = 6). Statistically significant differences were found between control and estivating groups (*), estivating and aroused groups (**) and control and aroused groups (▲) (ANOVA I, Tukey test).

et al., 2000; Estebenet and Martín, 2002; Cazzaniga, 2006). Particularly, its ability to estivate after self burial during the dry season is a critical aspect for the maintenance of population size, particularly in crops which alternate periods of flooding and dryness, as it occurs in Japan paddy fields (Wada and Yoshida, 2000; Watanabe et al., 2000; Syobu et al., 2001; Yusa et al., 2006). The factors inducing burrowing behavior preceding both estivation and hibernation were studied in P. canaliculata in semi-field conditions and it was found that burrowing was induced by both low

Table 3 Urate oxidase activity in organs of active, estivating and aroused P. canaliculata.

Midgut gland Posterior kidney Coiled gut Foot muscle

Active Control

Estivating

Aroused, 20 minutes

Aroused, 24 hours

59.3 ± 10.5 59.6 ± 12.7 101.0 ± 32.9 96.8 ± 23.4

ND * 20.5 ± 20.5 * ND * ND *

46.8 ± 17.5 ND ▲ 2.1 ± 2.1 ▲ 62.4 ± 30.4 **

25.9 ± 15.9 ND ▲ 14.7 ± 9.0 8.2 ± 5.2 ▲

Rates are given in nmol/min/mg (mean ± SE). Statistically significant differences were found between control and estivating animals (*), estivating and aroused animals (**) and control and aroused animals (▲) (ANOVA I, Kruskal–Wallis test; Dunn's test was used for post hoc analysis; P b 0.05). ND = not detected. N was 8 for the active control group and 6 for the other groups.

4.4. Infestation by fly larvae An interesting observation in the current study was the occasional infestation of snails by the scuttle fly Megaselia scalaris, whose larvae are saprophages but are also known to exert facultative parasitoidism (on spiders and insects) and parasitism (myiasis in amphibians, reptiles and humans). Also, the extraordinary capacity of these flies to penetrate into or escape from seemingly closed containers (Disney, 2008) would make them able to find any buried estivating snails. As reviewed by the latter author, these flies frequently choose defenseless animals to lay their eggs, which suggest the possibility that female flies may have laid their eggs on living estivating snails. 5. Conclusions It is concluded that uric acid stores increase during estivation and that they may be consumed during arousal as a significant antioxidant defense against the damaging effects of reoxygenation. The sustained increase in the uric acid oxidative product (allantoin) which occurs during arousal, appears more easily related to non-enzymatic oxidation than to the action of urate oxidase. In fact, the activity of this enzyme shut down during estivation in all the studied tissues while a partial though sustained recovery was only observed in the midgut gland. The hypothesis of a physiological role of uric acid as a non enzymatic antioxidant should be complemented with studies of other enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (glutathione) antioxidant defences in P. canaliculata. Also, the possible role of urate oxidase in nitrogen recycling in active snails should be further studied in this species.

512

M. Giraud-Billoud et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 506–512

Acknowledgements The authors wish to thank three anonymous reviewers for valuable observations. The identification of scuttle flies was kindly made for us by R. Henry L. Disney, Ph.D., D.Sc. (University of Cambridge, UK). This work was supported by grants from Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Fondo Nacional de Ciencia y Técnica (FONCYT) of Argentina. References Ames, B.N., Cathcart, R., Schwiers, E., Hochstein, P., 1981. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc. Natl Acad. Sci. USA 78, 6858–6862. Athawale, M., Reddy, S., 2002. Storage excretion in the Indian apple snail, Pila globosa (Swainson), during aestivation. Indian J. Exp. Biol. 40, 1304–1306. Becker, B.F., 1993. Towards the physiological function of uric acid. Free Radic. Biol. Med. 14, 615–631. Buckner, J.S., Caldwell, J.M., Knoper, J.A., 1985. Subcellular localization of uric acid storage in the fat body of Manduca sexta during the larval-pupal transformation. J. Insect Physiol. 31, 741–753. Burky, A.J., Pacheco, J., Pereyra, E., 1972. Temperature, water, and respiratory regimes of an amphibious snail, Pomacea urceus (Müller), from the Venezuelan savannah. Biol. Bull. 143, 304–316. Cazzaniga, N., 2006. Pomacea canaliculata: harmless and useless in its natural realm (Argentina). In: Joshi, R., Sebastian, L. (Eds.), Global advances in the ecology and management of golden apple snails. Philippine Rice Research Institute, Nueva Ecija, Philippines, pp. 37–60. Chaturvedi, M., Agarwal, R., 1979. Excretion, accumulation and site of synthesis of urea in the snail Pila globosa during active and dormant periods. J. Physiol. Paris 75, 233–237. Chaturvedi, M., Agarwal, R., 1981. Comparative study of storage pattern and site of synthesis of uric acid in the snails Viviparus bengalensis (Lamarck) and Pila globosa (Swainson), during active and dormant periods. Indian J. Exp. Biol. 19, 130–134. Cochran, D.G., 1985. Nitrogen excretion. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Regulation: Digestion, Excretion, Nutrition. : Comprehensive Insect Physiology Biochemistry and Pharmacology, 4. Pergamon Press, New York, pp. 467–506. Coles, G., 1968. The termination of aestivation in the large fresh-water snail Pila ovata (Ampullariidae) — I: Changes in oxygen uptake. Comp. Biochem. Physiol. 25, 517–522. Cowie, R., 2002. In: Baker, G.M. (Ed.), Apple snails (Ampullariidae) as agricultural pests: their biology, impacts and management. CABI, Wallingford, pp. 145–192. d'Orbigny, A., 1847. Voyage dans l'Amérique Méridionale. Tome 5. Mollusques. C.P. Bertrand, Paris, 711. Dillaman, R.M., Greenaway, P., Linton, S.M., 1999. Role of the midgut gland in purine excretion in the robber crab, Birgus latro (Anomura:Coenobitidae). J. Morphol. 241, 227–235. Disney, R., 2008. Natural history of the scuttle fly, Megaselia scalaris. Annu. Rev. Entomol. 53, 39–60. Estebenet, A., Martín, P., 2002. Pomacea canaliculata (Gastropoda: Ampullariidae): lifehistory traits and their plasticity. Biocell 26, 83–89. Ferreira, M., Alencastro, A., Hermes-Lima, M., 2003. Role of antioxidant defenses during estivation and anoxia exposure in freshwater snails Biomphalaria tenagophila (Orbigny, 1835). Can. J. Zool. 81, 1239–1248. Ferreira-Cravo, M., Welker, A., Hermes-Lima, M., 2010. The Connection between Oxidative Stress and Estivation in Gastropods and Anurans. In: Navas, C.A., Carvalho, J.E. (Eds.), Progress in Molecular and Subcellular Biology, pp. 47–61. Giraud-Billoud, M., Koch, E., Vega, I.A., Gamarra-Luques, C., Castro-Vazquez, A., 2008. Urate cells and tissues in the South American apple-snail Pomacea canaliculata. J. Molluscan Stud. 74, 259–266. Guppy, M., Withers, P., 1999. Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol. Rev. Camb. Philos. Soc. 74, 1–40. Guppy, M., Reeves, D.C., Bishop, T., Withers, P., Buckingham, J.A., Brand, M.D., 2000. Intrinsic metabolic depression in cells isolated from the hepatopancreas of estivating snails. FASEB J. 14, 999–1004. Halliwell, B., 2009. The wanderings of a free radical. Free Radic. Biol. Med. 46, 531–542. Hermes-Lima, M., Storey, K.B., 1995a. Antioxidant defenses and metabolic depression in a pulmonate land snail. Am. J. Physiol. Regul. Integr. Comp. Physiol. 268, 1386–1393. Hermes-Lima, M., Storey, K.B., 1995b. Xanthine oxidase and xanthine dehydrogenase from an estivating land snail. Z Naturforsch. C. J. Biosci. 50, 685–694. Hermes-Lima, M., Zenteno-Savin, T., 2002. Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp. Biochem. Physiol., C 133, 537–556.

Hermes-Lima, M., Storey, J.M., Storey, K.B., 1998. Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails. Comp. Biochem. Physiol., B 120, 437–448. Horne, F., 1979. Comparative aspects of estivating metabolism in the gastropod, Marisa. Comp. Biochem. Physiol., A 64, 309–311. Lach, L., Britton, D., Rundell, R., Cowie, R., 2000. Food preference and reproductive plasticity in an invasive freshwater snail. Biol. Invasions 2, 279–288. Lal, M.B., Saxena, B.B., 1952. Uricotelism in the common Indian apple-snail, Pila globosa (Swainson). Nature 170, 1024. Lapenna, D., Cuccurullo, F., 1993. TBA test and ‘free’ MDA assay in evaluation of lipid peroxidation and oxidative stress in tissue systems. Am. J. Physiol. 265, H1030–H1032. Lapenna, D., Ciofani, G., Pierdomenico, S.D., Giamberardino, M.A., Cuccurullo, F., 2001. Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma. J. Free Radic. Biol. Med. 31, 331–335. Linton, S.M., Greenaway, P., 1997. Intracellular purine deposits in the gecarcinid land crab Gecarcoidea natalis. J. Morphol. 231, 101–110. Little, C., 1968. Aestivation and ionic regulation in two species of Pomacea (Gastropoda, Prosobranchia). J. Exp. Biol. 48, 569–585. López-Sánchez, M., Neef, A., Peretó, J., Patiño-Navarrete, R., Pignatelli, M., Latorre, A., Moya, A., 2009. Evolutionary convergence and nitrogen metabolism in Blattabacterium strain Bge, primary endosymbiont of the cockroach Blattella germanica. PLoS Genet. 5, 1–11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mahler, H.R., Hübscher, G., Baum, H., 1955. Studies on uricase — I. Preparation, purification and properties of a cuproprotein. J. Biol. Chem. 216, 625–641. McCord, J.M., 2000. The evolution of free radicals and oxidative stress. Am. J. Med. 108, 652–659. Meenakshi, V., 1964. Aestivation in the Indian apple snail Pila. I. Adaptation in natural and experimental conditions. Comp. Biochem. Physiol. 11, 379–386. Nowakowska, A., Swiderska-Kolacz, G., Rogalska, J., Caputa, M., 2009. Antioxidants and oxidative stress in Helix pomatia snails during estivation. Comp. Biochem. Physiol., C 150, 481–486. Potrikus, C., Breznak, J., 1981. Gut bacteria recycle uric acid nitrogen in termites: a strategy for nutrient conservation. Proc. Natl Acad. Sci. USA 78, 4601–4605. Rahman, K., 2007. Studies on free radicals, antioxidants, and co-factors. Clin. Interv. Aging 2, 219. Ramos-Vasconcelos, G., Hermes-Lima, M., 2003. Hypometabolism, antioxidant defenses and free radical metabolism in the pulmonate land snail Helix aspersa. J. Exp. Biol. 206, 675–685. Ramos-Vasconcelos, G., Cardoso, L., Hermes-Lima, M., 2005. Seasonal modulation of free radical metabolism in estivating land snails Helix aspersa. Comp. Biochem. Physiol., C 140, 165–174. Reddy, Y.S., Rao, P.V., Swami, K.S., 1974. Probable significance of urea and uric acid accumulation during aestivation in the gastropod, Pila globosa (Swainson). Indian J. Exp. Biol. 12, 454–456. Saxena, B., 1955. Physiology of excretion in the common Indian apple-snail, Pila globosa (Swainson). Part I. J. Anim. Morphol. Physiol. (Bombay) 3, 87–95. Speeg, K.V., Campbell, J.W., 1968. Purine biosynthesis and excretion in Otala (= Helix) lactea: An evaluation of the nitrogen excretory potential. Comp. Biochem. Physiol. 26, 579–595. Storey, K.B., 2002. Life in the slow lane: molecular mechanisms of estivation. Comp. Biochem. Physiol., A 133, 733–754. Syobu, S., Mikuriya, H., Yamaguchi, J., Matsuzaki, M., Zen, S., Wada, T., 2001. Estimating the overwintering mortality of the apple snail, Pomacea canaliculata (Lamarck) (Gastropoda: Ampullariidae) in a paddy field of southern Japan using temperature data. Appl. Entomol. Zool. (Japan) 45, 203–208. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Ann. Clin. Biochem. 6, 24–27. Vega, I., Giraud-Billoud, M., Koch, E., Gamarra-Luques, C., Castro-Vazquez, A., 2007. Uric acid accumulation within intracellular crystalloid corpuscles of the midgut gland in Pomacea canaliculata (Caenogastropoda, Ampullariidae). Veliger 48, 276–283. Wada, T., Yoshida, K., 2000. Burrowing by the apple snail, Pomacea canaliculata (Lamarck); daily periodicity and factors affecting burrowing. Kyushu Pl. Prot. Res. 46, 88–93. Wasowicz, W., Neve, J., Peretz, A., 1993. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clin. Chem. 39, 2522–2526. Watanabe, T., Tanaka, K., Higuchi, H., Miyamoto, K., Kiyonaga, T., Kiyota, H., Suzuki, Y., Wada, T., 2000. Emergence of the apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae), after irrigation in a paddy. Appl. Entomol. Zool. (Japan) 35, 75–79. Young, E., Conway, C., 1942. On the estimation of allantoin by the Rimini–Schryver reaction. J. Biol. Chem. 142, 839. Young, I., Woodside, J., 2001. Antioxidants in health and disease. J. Clin. Pathol. 54, 176. Yusa, Y., Wada, T., Takahashi, S., 2006. Effects of dormant duration, body size, self-burial and water condition on the long-term survival of the apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae). Appl. Entomol. Zool. (Jpn.) 41, 627–632.