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Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage
ARTICLE IN PRESS
ZOOLOGY Zoology 112 (2009) 471–477 www.elsevier.de/zool
Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage Roger A. Byrnea,, Simon D. Rundleb, Jennifer J. Smirthwaiteb, John I. Spicerb a
Department of Biology, State University of New York at Fredonia, 122 Jewett Hall, Fredonia, NY 14063, USA Marine Biology and Ecology Research Centre, School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK
b
Received 23 June 2008; received in revised form 12 January 2009; accepted 10 March 2009
Abstract Responses of freshwater organisms to environmental oxygen tensions (PO2) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO2 must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO2s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO2s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (‘‘stir-bar’’ theory of embryonic rotational behaviour). r 2009 Elsevier GmbH. All rights reserved. Keywords: Gastropods; Hypoxia; Hyperoxia; Oxygen sensor; Veliger
Introduction Environmental oxygen tensions (PO2) can vary dramatically within freshwater systems. Kemp and Dodds (2001), for example, demonstrated both hypoxia Corresponding author.
E-mail address: [email protected] (R.A. Byrne). 0944-2006/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2009.03.001
(associated with detrital material) and hyperoxia (associated with diatom mats and epilithic diatoms) in a single stream reach and also showed that these differences varied seasonally. Such microhabitat variation has also been documented previously in standing waters (e.g., Carlton and Wetzel, 1988) and can occur over short time scales depending on photosynthetic activity and temperature. Miranda et al. (2000) for
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example showed that conditions were hypoxic in macrophyte stands at dawn, but hyperoxic at the surface of stands at dusk. The ability of freshwater organisms to adjust to such fluctuations in PO2 is a key trait and there is a substantial literature documenting how the mature stages of aquatic animals are able to alter their rates of oxygen uptake, via physiological and behavioural means (see Mangum and Van Winkle, 1973; Burnett, 1997 for reviews). Much less well understood is how earlier developmental stages respond to different PO2s or the ecological consequences of exposure of early stages to different PO2s. The eggs of most freshwater invertebrates are attached to the substratum, and, hence, unable to move away from extremes in PO2. Although adult choice of oviposition sites might reduce the environmental influences on egg development, it is almost certainly the case that embryos must look to other forms of ‘‘behavioural’’ or physiological mechanisms for survival. Freshwater gastropods have been reasonably well studied in terms of their responses to different PO2s and are known to show varied interspecific responses (Hanley and Ultsch, 1999). Developing gastropod embryos in freshwaters display a characteristic rotational behaviour within their egg cases, which is hypothesised to serve a function of mixing fluids within the egg capsule and, thus, to facilitate diffusion of oxygen (Burggren, 1985; Hunter and Vogel, 1986; Goldberg et al., 2008). In the freshwater pulmonate snail Helisoma trivolvis, this behaviour is mediated by serotonergic sensory motor circuits that operate ciliary bands resulting in a characteristic suite of rotational behaviours (Diefenbach et al., 1991). Kuang et al. (2002) have subsequently shown that rotational behaviour is affected by altering environmental PO2 with a dosedependent, behavioural response resulting in higher rotational activities at lower PO2s. Recently, Goldberg et al. (2008) reported hypoxia-induced rotational behaviours in three families of basommatophoran gastropods, the Planorbidae (H. trivolvis), the Lymnaeidae (Lymnaea stagnalis) and the Physidae (Physa gyrina). Hence, it appears that embryonic snails are able to respond to low PO2 by altering their spinning behaviour. However, questions remain as to how general this response is. For example, little is known of whether changes in embryo rotation rates under hypoxia are dependent on developmental stage. It might be predicted that qualitative or quantitative adjustments to behaviour would occur as the embryo matures and enlarges, as oxygen demand is likely to be greater in the larger and more complex later stages. Moreover, the effect of hyperoxia on rotational behaviour has not been fully examined, yet the way that embryos respond to high PO2 could also be of importance. If, for example, we
assume that spinning behaviour is energetically demanding, and if spinning in embryos serves to increase diffusion of oxygen, it might be predicted that, under hyperoxia, spinning behaviour would decrease to reduce costs. Clearly, information on both the ontogenetic variation in the sensitivity of embryos to PO2 and the influence of hyperoxia as well as hypoxia is necessary for a complete understanding of how environmental conditions may affect development and, ultimately, the ecological success of gastropod species. Consequently, we investigated how exposure to different environmental PO2s affected rotational behaviour in embryos of the pond snail L. stagnalis. We tested the predictions: (1) that, in addition to increasing their rotational behaviour during hypoxia, embryos would decrease their spinning under acute hyperoxia and (2) that later developmental stages would show a greater level of response to both hypoxia and hyperoxia. To test the latter prediction, we used two well-defined developmental stages separated by 2 days development time (at 20 1C): the ‘‘veliger’’ and the later ‘‘hippo’’ stage (Morrill, 1982).
Material and methods Experimental animals Adults of L. stagnalis for producing eggs were collected using hand-held nets from South Drain, Somerset Levels, south-west England (Smirthwaite et al., 2007). Upon return to the laboratory snails were maintained in aquaria (volume ¼ 10 l) each containing continually aerated, ASTM pond water at 20 1C under a 12:12 L:D schedule. Snails were fed lettuce leaves ad libitum and pond water was replaced weekly. Snails laid egg masses on the inside surface of the aquaria, and these masses were carefully removed using a scalpel and transferred to aerated smaller containers prior to experimentation.
Experimental setup Developmental stages Developmental stages were assigned following the procedures in Smirthwaite et al. (2007). Briefly, the ‘‘veliger’’ stage occurs approximately 4.5 days after egg laying under our temperature conditions, and is characterised by embryos with two similar lobes of tissue at either end of the viscera and is represented approximately by stage E5 in Cumin’s Normentafel (Cumin, 1972). The ‘‘hippo’’ stage (sensu Raven, 1966), equivalent to Cumin’s stage E7 (Cumin, 1972) occurs approximately 2 days after the veliger stage, and is represented by further development of the lobes and the
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visceral mass being covered completely by the shell. Embryonic developmental stages can also be expressed as proportional developmental units from laying (0) to hatching (100) with intermediate stages represented as a percentage of developmental time elapsed (e.g., in Kuang et al., 2002; Goldberg et al., 2008). Using this approach, the veliger occurs at approximately stage 30 and the hippo around stage 40 (derived from Smirthwaite et al., 2007). Experimental procedure Egg masses containing embryos of the appropriate developmental stage (i.e. veliger or hippo) were selected and placed in a specially-constructed flow-through chamber (dimensions 2.5 cm 5.0 cm 1.0 cm). Pond water PO2 was adjusted either by bubbling with compressed gas (room air for normoxia; N2 for hypoxia; O2 for hyperoxia) or by equilibrating a reservoir of pond water (volume ¼ 4 l) with bubbled gas from either room air, O2 or N2 in combination to achieve a target PO2. Gas-equilibrated pond water passed through the chamber driven by a peristaltic pump (Gilson Minipuls 3, Gilson Inc., Middleton, WI, USA) (approx. 10 ml min1) in a flow-through-to-waste setup. Actual PO2s in the inflow and the outflow were monitored using in-line PO2 electrodes (Strathkelvin 1302, Strathkelvin Instruments, Glasgow, UK) connected to an oxygen interface (Model 928, Strathkelvin Instruments, Glasgow, UK) coupled to a PC operating SI 928 recording software. The time lag from initiation of gas treatment to equilibration in the chamber was approximately 5 min. Behavioural measurements Embryonic behaviour was recorded using video capture (1 frame per second), via a camera (Pixera Corp., San Jose´, CA, USA) connected to the viewport of a stereo microscope (WPI 500053, 20–40 , World Precision Instruments, Inc., Sarasota, FL, USA). Video images for analysis were captured digitally in AVI format. Once the experiment commenced, the field of view was maintained so as to allow examination of the same eggs throughout. Between five and seven embryos were clearly visible in each field of view and the behaviours of 3–4 of these individuals, selected haphazardly, were monitored for the duration of the experiment, so that we would have a continuous recording of the same individuals throughout. Embryos were used only once in trials. Although a number of specific classes of rotational behaviour have been described in other species of gastropod (see Diefenbach et al., 1991) we simply measured the time to complete 3–5 rotations and expressed the rate of spinning as mean revolutions per minute. We used video playback ( 16) to enable clear visualisation of rotational behaviours. Behaviours were
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measured 10 min after equilibration, except in cases noted in the results. Two series of experiments were performed. Series one examined the effects of hyperoxia and return to normoxia on rotational behaviours. Rotational behaviour was recorded throughout and quantified after a 10 min acclimatisation period for each change in chamber PO2. Egg masses containing embryos of the appropriate developmental stage were incubated consecutively in the following treatments: 30 min normoxia (normoxia 1), 30 min hyperoxia (4300% air saturation; hyper), and 30 min normoxia (100% air saturation; normoxia 2). Measurements were made of 12 individual veliger stage embryos over four trials, and 9 individual hippo embryos over three trials. In the second series of experiments we investigated the effects of increasing hypoxia and return to normoxia on rotational behaviour. Embryos were exposed to the following consecutive series of gas treatments: 30 min 100% air saturation (normoxia), 30 min 50% air saturation (50% hypoxia), 30 min 20% air saturation (80% hypoxia), 30 min 0% air saturation (100% hypoxia), and 60 min 100% air saturation (normoxia). In this series, we measured the responses of 6 individual veliger stage embryos over two trials, and 5 individual hippo stage embryos also over two trials.
Data analyses We tested for differences between treatments using repeated-measures ANOVA (SigmaStat, Systat Software Inc., San Jose´, CA, USA). Data were first log10 transformed if necessary (i.e. if non-normal or if variances were unequal between treatments). If the ANOVA yielded significant results then a Holm–Sidak pair-wise multiple comparison post-hoc test was used to determine differences between treatment means. In a separate analysis examining the combined effects of clutch and gas treatment for each developmental stage, we found no significant gas treatment–clutch interaction in any of the experiments indicating that the direction of response (i.e. whether embryos increased or decreased their rates of rotation) was not affected by clutch.
Results Effects of hyperoxia There were significant effects of developmental stage and hyperoxia on spinning behaviour. The later ‘‘hippo’’ stage displayed a significantly higher rotation rate compared with the veliger stage (Fig. 1, Table 1): hippo embryos incubated under normoxic conditions rotated at a rate of 1.8770.24 min1 (mean7standard error)
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Hippo Veliger
rotational rates declined to a rate that was not significantly different from pre-treatment values. Responses of the hippo stage to hypoxia were broadly similar to those for the veliger stage with a general increase in rotation rate with decreasing PO2 (Fig. 2). There were, however, some subtle differences between stages. Firstly, hippos appeared more sensitive to low PO2 and showed a significant increase in rotation rate at 50% saturation compared with normoxia, and a dose response reaching a maximum of 2.8970.18 min1 at 0% air saturation (Fig. 2). There was also a different response on return to normoxia compared with veligers, leading to a significant gas treatment stage interaction (Fig. 2, Table 2): on return to normoxia, hippos showed an immediate decline in rotational rate and, after 5 min, rotational rates had returned to pre-treatment rates (Fig. 1), declining even further over the following 10–20 min with a complete cessation of rotational behaviour in several cases. Although not shown, mean rotation rates in hippos reverted to earlier normoxic levels after 20–30 min post-reintroduction to normoxic water.
Hyperoxia
Rotation rate (min-1)
2.2 2.0 1.8 1.6 1.4 1.2 1.0
Normoxia 1
Hyperoxia
Normoxia 2
Gas Treatment
Fig. 1. Effects of hyperoxia (4300 mmHg PO2) and return to normoxia on embryonic rotation rate in L. stagnalis, at two developmental stages: the earlier ‘‘veliger’’ stage and the later ‘‘hippo’’ stage. Symbols represent mean rates for repeatedly measured individuals. Error bars are standard error estimates.
while veliger embryos rotated at 1.5070.11 min1. Contrary to the prediction that rotational rates would decrease under hyperoxia, hippo embryos increased their rate of rotation significantly to 2.1670.26 min1, whereas veliger embryos showed no significant response to greater PO2s. Upon return to normoxia the rotational rate in hippo embryos declined to pre-treatment values and there was a significant difference compared with the hyperoxia treatment; veliger embryos once again showed no significant response.
Discussion The main aim of our study was to investigate the ability of snail embryos to ‘fine tune’ their rotational behaviour in response to environmental PO2. The behaviour of embryos of the freshwater pulmonate L. stagnalis supported our predictions for hypoxic conditions in that both stages increased their rotation rates with decreasing PO2. Subtle differences were also observed between developmental stages when returned to normoxia from hyperoxia: veligers resumed a ‘‘normal’’ rotation rate, whereas hippo rotation rates showed an almost complete cessation. Our prediction that spinning would reduce under hyperoxic conditions was not supported, however, with hippos increasing their rotational rate only slightly and veligers showing no significant change under conditions of hyperoxia.
Effects of hypoxia Embryos at both developmental stages increased rates of rotation in response to step-wise decreases in ambient PO2 (Fig. 2, Table 2). Embryos at the veliger stage again had a lower rotation rate than hippo stage embryos: mean rotation rates for normoxic veliger embryos were 1.0670.08 min1, increased significantly at 20% air saturation, and reached a maximum of 1.9370.18 min1 when embryos were incubated at 0% air saturation. Within 12 min of return to normoxia, veliger embryo
Table 1. Results of a two-factor repeated measures ANOVA testing the effects of developmental stage (veliger vs. hippo) and gas treatment (normoxia, hyperoxia, return to normoxia) for hyperoxia trials on embryonic rotational behaviour of Lymnaea stagnalis embryos. Source of variation
DF
SS
MS
F-ratio
P-value
Development stage Subject (development stage) Gas treatment Development stage gas treatment Residual Total
1 19 2 2 38 62
1.067 4.345 0.237 0.111 1.247 6.977
1.067 0.229 0.119 0.0553 0.0328 0.113
4.665
0.044
3.611 1.685
0.037 0.199
DF ¼ degrees of freedom; SS ¼ sum of squares; MS ¼ means squared.
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Fig. 2. Effects of step-wise hypoxia treatment and return to normoxia on embryonic rotation rate in L. stagnalis, at two developmental stages: the earlier ‘‘veliger’’ stage and the later ‘‘hippo’’ stage. Symbols represent mean rates for repeatedly measured individuals. Error bars are standard error estimates.
There was no clear evidence that hyperoxia reduced rotational behaviour in either veliger stage or hippo stage embryos. As rotational behaviour has the functional role of mixing the contents of the egg capsule and maximising diffusional gradients from the external medium by homogenising capsular gas tensions (Burggren, 1985; Hunter and Vogel, 1986; Goldberg et al., 2008), it appears that neither embryonic stage is able to decrease its rotational behaviour. Reduction in ventilation in response to elevated environmental PO2 has been documented in a number of ‘water breathing’ groups of animals, e.g. fish (Wood and Jackson, 1980; Berschick et al., 1987) and crayfish (Wheatley, 1989). It might be that rotation activity is not an excessively costly behaviour to embryos. Alternatively, embryos may not have sufficient mechanisms for fine tuning their response under conditions of hyperoxia and, hence, may
incur significant costs during such activity. As such periods of hyperoxic conditions are likely to exist in some habitats, embryos in such conditions may suffer significant costs that could have consequences for their fitness. Our study confirmed earlier studies by Kuang et al. (2002) on H. trivolvis, another pulmonate snail, that rotational behaviour increases with decreasing PO2 in a dose-dependent manner. Goldberg et al. (2008) also demonstrated that this response is more generalised throughout representative families of basommatophoran gastropods, including L. stagnalis, the subject of our study. Although qualitatively similar, the responses by the two developmental stages of L. stagnalis to declining PO2 are quantitatively different in that rotation rates in the hippo stage are approximately 1.3–1.6 times those of the less developed veliger stage. Although such an
Table 2. Results of a two-factor repeated measures ANOVA testing the effects of developmental stage (veliger vs. hippo) and gas treatment (normoxia, hyperoxia, return to normoxia) for hypoxia trials on embryonic rotational behaviour of Lymnaea stagnalis embryos. Source of variation
DF
SS
MS
F-ratio
Development stage Subject (development stage) Gas treatment Development stage gas treatment Residual Total
1 9 5 5 45 65
4.539 3.656 25.980 3.995 3.249 40.146
4.539 0.406 5.196 0.799 0.0722 0.618
11.17265
DF ¼ degrees of freedom; SS ¼ sum of squares; MS ¼ means squared.
71.974 11.066
P-value 0.009 o0.001 o0.001
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increase in rotation rates could be due to developmental changes in number and morphology of rotational cilia, hippo stage embryos also appeared to have a more sensitive response to low PO2 in that they showed a significant increase in rotation rate even at 50% saturation. As the hippo stage is both anatomically more complex and larger than the veliger stage, it is reasonable to assume that metabolic oxygen demand would also be greater, thus requiring a more robust mixing movement and that embryos have a more sophisticated response system at this stage of development. Return to normoxia after hypoxic exposure was accompanied by a rapid reduction in rotational rates in both embryonic stages: movement in veliger embryos returned to normoxic rates within 12 min; however, hippo embryos reduced rotational rates significantly below pre-treatment rates and in some cases actually stopped moving entirely for a period of 20 min or more, before slowly resuming rotational behaviour. A similar cessation of rotational behaviour was noted in Helisoma embryos (Kuang et al., 2002) and by Goldberg et al. (2008) in L. stagnalis and P. gyrina; although inhibition of rotational behaviour was of lower magnitude in the lymnaeid species. Overstimulation of oxygen receptors was cited as the proximate cause resulting in shutdown of serotonergic pathways leading to ciliary movement (Kuang et al., 2002). We assume that a similar cause is in effect in Lymnaea embryos, although Goldberg et al. (2008) postulate that the controlling ciliary movement pathways in Lymnaea are more likely to be dopaminergic. It is also evident that the response is developmentally determined as veliger stage embryos do not respond by cessation of rotational behaviour on return to normoxia. The ontogeny of neural systems of control in later hippo stage embryos may permit a more systemic response to hypoxia and mediate a higher level of response upon return to normoxia. Incubation in a virtually anoxic environment resulted in an approximate doubling of rotational rates both for veliger and hippo stage embryos. Although L. stagnalis is a pulmonate and thus breathes air, it has long been known that adults of this species and other pulmonates can exchange gasses across the integument as well as by way of the lung (Jones, 1972; Graham, 1990) as a means of supplementing pulmonary gas exchange. The embryonic response to severe hypoxia appears to be a classic compensatory response in that the assumed ‘‘purpose’’ is to maintain tissue PO2. Goldberg et al. (2008) have coined the term ‘‘embryo stir-bar’’ to describe how rotational behaviour in pulmonate embryos in response to hypoxia functions to effectively mix the egg capsular contents, thereby achieving a uniform capsular PO2 and maintaining a diffusive gradient for oxygen to enter into the egg capsule from the external environment.
In adult L. stagnalis the response to hypoxia is to increase ventilation, achieved by increasing the opening time of the pneumostome at the air–water surface (Jones, 1961; Hermann and Bulloch, 1998; Taylor et al., 2003). Interestingly, L. stagnalis reared without access to an air–water interface can maintain metabolism by means of integumentary gas exchange, and also will not display air-breathing behaviours even under hypoxic conditions (Hermann and Bulloch, 1998). The stimulus for eliciting ventilation behaviour seems to arise from external oxygen receptors, rather than from any central source (Inoue et al., 1996). Thus it seems that the response to hypoxia in embryonic L. stagnalis matches the compensatory response seen in hatched individuals even though the behaviour is fundamentally different in form.
Acknowledgements We thank Julie Soane for her technical expertise and advice in setting up our experimental system. Support was provided by NERC Grant # NER/B/S/2001/00843 (to SDR and JIS), and a SUNY travel award (to RAB).
References Berschick, P., Bridges, C.R., Grieshaber, M.K., 1987. The influence of hyperoxia, hypoxia and temperature on the respiratory physiology of the intertidal rockpool fish Gobius cobitis Pallas. J. Exp. Biol. 130, 368–387. Burggren, W.W., 1985. Gas exchange, metabolism and ventilation in gelatinous frog egg masses. Physiol. Zool. 58, 503–514. Burnett, L.E., 1997. The challenges of living in hypoxic and hypercapnic aquatic environments. Am. Zool. 37, 633–640. Carlton, R.G., Wetzel, R.G., 1988. Phosphorus flux from lake sediments: effects of epipelic algal oxygen production. Limnol. Oceanogr. 33, 562–570. Cumin, R., 1972. Normentafel zur Organogenese von Lymnaea stagnalis (Gastropoda, Pulmonata) mit besonderer Beru¨cksichtigung der Mitteldarmdru¨se. Rev. Suisse Zool. 79, 709–774. Diefenbach, T.J., Koehncke, N.K., Goldberg, J.I., 1991. Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin. J. Neurobiol. 22, 922–934. Goldberg, J.I., Doran, S.A., Shartau, R.B., Pon, J.R., Ali, D.W., Tam, R., Kuang, S., 2008. Integrative biology of an embryonic respiratory behaviour in pond snails: the ‘embryo stir-bar hypothesis’. J. Exp. Biol. 211, 1729–1736. Graham, J.E., 1990. Ecological, evolutionary, and physical factors influencing aquatic animal respiration. Am. Zool. 30, 137–146. Hanley, R.W., Ultsch, G.R., 1999. Ambient oxygen tension, metabolic rate and habitat selection in freshwater snails. Arch. Hydrobiol. 114, 195–214.
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Hermann, P.M., Bulloch, A.G.M., 1998. Developmental plasticity of respiratory behavior in Lymnaea. Behav. Neurosc. 112, 656–667. Hunter, T., Vogel, S., 1986. Spinning embryos enhance diffusion through gelatinous egg masses. J. Exp. Mar. Biol. Ecol. 96, 303–308. Inoue, T., Haque, T.Z., Takasaki, M., Lukowiak, K., Syed, N.I., 1996. Hypoxia induced respiratory patterned activity in Lymnaea originates at the periphery: role of the dopamine cell. Soc. Neurosci. Abstr. 22, 1142. Jones, J., 1961. Aspects of respiration in Planorbis corneus and Lymnaea stagnalis (Gastropoda: Pulmonata). Comp. Biochem. Physiol. 12, 283–295. Jones, J.D., 1972. Comparative Physiology of Respiration. Special Topics in Biology Series. Edward Arnold Publishers, London. Kemp, M.J., Dodds, W.K., 2001. Centimeter-scale patterns in dissolved oxygen and nitrification rates in a prairie stream. J. North Am. Benthol. Soc. 20, 347–357. Kuang, S., Doran, S.A., Wilson, R.J.A., Goss, G.G., Goldberg, J.I., 2002. Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond snail embryos. J. Neurobiol. 52, 73–83. Mangum, C., van Winkle, W., 1973. Responses of aquatic invertebrates to declining oxygen conditions. Am. Zool. 13, 529–541.
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Miranda, L.E., Driscoll, M.P., Allen, M.S., 2000. Transient physicochemical microhabitats facilitate fish survival in inhospitable aquatic plant stands. Freshwater Biol. 44, 617–628. Morrill, J.B., 1982. Development of the pulmonate gastropod, Lymnaea. In: Harrison, F.W., Cowden, R.R. (Eds.), Developmental Biology of Freshwater Invertebrates. Liss Publishers, New York, pp. 399–483. Raven, C.P., 1966. Morphogenesis: The Analysis of Molluscan Development, 2nd Ed. Biling and Sons Ltd., London. Smirthwaite, J.J., Rundle, S.D., Bininda-Emonds, O.R.P., Spicer, J.I., 2007. An integrative approach identifies developmental sequence heterochronies in freshwater basommatophoran snails. Evol. Dev. 9, 122–130. Taylor, B.E., Harris, M.B., Burk, M., Smyth, K., Lukowiak, K., Remmers, J.E., 2003. Nitric oxide mediates metabolism as well as respiratory and cardiac responses to hypoxia in the snail Lymnaea stagnalis. J. Exp. Zool. 295A, 37–46. Wheatley, M.G., 1989. Physiological responses of the crayfish Pacifastacus leniusculus to environmental hyperoxia. I. Extracellular acid-base and electrolyte status and transbranchial exchange. J. Exp. Biol. 143, 33–51. Wood, C.M., Jackson, E.B., 1980. Blood acid-base regulation during environmental hyperoxia in the rainbow trout (Salmo gairdneri). Respir. Physiol. 42, 351–372.
ZOOLOGY Zoology 112 (2009) 471–477 www.elsevier.de/zool
Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage Roger A. Byrnea,, Simon D. Rundleb, Jennifer J. Smirthwaiteb, John I. Spicerb a
Department of Biology, State University of New York at Fredonia, 122 Jewett Hall, Fredonia, NY 14063, USA Marine Biology and Ecology Research Centre, School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK
b
Received 23 June 2008; received in revised form 12 January 2009; accepted 10 March 2009
Abstract Responses of freshwater organisms to environmental oxygen tensions (PO2) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO2 must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO2s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO2s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (‘‘stir-bar’’ theory of embryonic rotational behaviour). r 2009 Elsevier GmbH. All rights reserved. Keywords: Gastropods; Hypoxia; Hyperoxia; Oxygen sensor; Veliger
Introduction Environmental oxygen tensions (PO2) can vary dramatically within freshwater systems. Kemp and Dodds (2001), for example, demonstrated both hypoxia Corresponding author.
E-mail address: [email protected] (R.A. Byrne). 0944-2006/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2009.03.001
(associated with detrital material) and hyperoxia (associated with diatom mats and epilithic diatoms) in a single stream reach and also showed that these differences varied seasonally. Such microhabitat variation has also been documented previously in standing waters (e.g., Carlton and Wetzel, 1988) and can occur over short time scales depending on photosynthetic activity and temperature. Miranda et al. (2000) for
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example showed that conditions were hypoxic in macrophyte stands at dawn, but hyperoxic at the surface of stands at dusk. The ability of freshwater organisms to adjust to such fluctuations in PO2 is a key trait and there is a substantial literature documenting how the mature stages of aquatic animals are able to alter their rates of oxygen uptake, via physiological and behavioural means (see Mangum and Van Winkle, 1973; Burnett, 1997 for reviews). Much less well understood is how earlier developmental stages respond to different PO2s or the ecological consequences of exposure of early stages to different PO2s. The eggs of most freshwater invertebrates are attached to the substratum, and, hence, unable to move away from extremes in PO2. Although adult choice of oviposition sites might reduce the environmental influences on egg development, it is almost certainly the case that embryos must look to other forms of ‘‘behavioural’’ or physiological mechanisms for survival. Freshwater gastropods have been reasonably well studied in terms of their responses to different PO2s and are known to show varied interspecific responses (Hanley and Ultsch, 1999). Developing gastropod embryos in freshwaters display a characteristic rotational behaviour within their egg cases, which is hypothesised to serve a function of mixing fluids within the egg capsule and, thus, to facilitate diffusion of oxygen (Burggren, 1985; Hunter and Vogel, 1986; Goldberg et al., 2008). In the freshwater pulmonate snail Helisoma trivolvis, this behaviour is mediated by serotonergic sensory motor circuits that operate ciliary bands resulting in a characteristic suite of rotational behaviours (Diefenbach et al., 1991). Kuang et al. (2002) have subsequently shown that rotational behaviour is affected by altering environmental PO2 with a dosedependent, behavioural response resulting in higher rotational activities at lower PO2s. Recently, Goldberg et al. (2008) reported hypoxia-induced rotational behaviours in three families of basommatophoran gastropods, the Planorbidae (H. trivolvis), the Lymnaeidae (Lymnaea stagnalis) and the Physidae (Physa gyrina). Hence, it appears that embryonic snails are able to respond to low PO2 by altering their spinning behaviour. However, questions remain as to how general this response is. For example, little is known of whether changes in embryo rotation rates under hypoxia are dependent on developmental stage. It might be predicted that qualitative or quantitative adjustments to behaviour would occur as the embryo matures and enlarges, as oxygen demand is likely to be greater in the larger and more complex later stages. Moreover, the effect of hyperoxia on rotational behaviour has not been fully examined, yet the way that embryos respond to high PO2 could also be of importance. If, for example, we
assume that spinning behaviour is energetically demanding, and if spinning in embryos serves to increase diffusion of oxygen, it might be predicted that, under hyperoxia, spinning behaviour would decrease to reduce costs. Clearly, information on both the ontogenetic variation in the sensitivity of embryos to PO2 and the influence of hyperoxia as well as hypoxia is necessary for a complete understanding of how environmental conditions may affect development and, ultimately, the ecological success of gastropod species. Consequently, we investigated how exposure to different environmental PO2s affected rotational behaviour in embryos of the pond snail L. stagnalis. We tested the predictions: (1) that, in addition to increasing their rotational behaviour during hypoxia, embryos would decrease their spinning under acute hyperoxia and (2) that later developmental stages would show a greater level of response to both hypoxia and hyperoxia. To test the latter prediction, we used two well-defined developmental stages separated by 2 days development time (at 20 1C): the ‘‘veliger’’ and the later ‘‘hippo’’ stage (Morrill, 1982).
Material and methods Experimental animals Adults of L. stagnalis for producing eggs were collected using hand-held nets from South Drain, Somerset Levels, south-west England (Smirthwaite et al., 2007). Upon return to the laboratory snails were maintained in aquaria (volume ¼ 10 l) each containing continually aerated, ASTM pond water at 20 1C under a 12:12 L:D schedule. Snails were fed lettuce leaves ad libitum and pond water was replaced weekly. Snails laid egg masses on the inside surface of the aquaria, and these masses were carefully removed using a scalpel and transferred to aerated smaller containers prior to experimentation.
Experimental setup Developmental stages Developmental stages were assigned following the procedures in Smirthwaite et al. (2007). Briefly, the ‘‘veliger’’ stage occurs approximately 4.5 days after egg laying under our temperature conditions, and is characterised by embryos with two similar lobes of tissue at either end of the viscera and is represented approximately by stage E5 in Cumin’s Normentafel (Cumin, 1972). The ‘‘hippo’’ stage (sensu Raven, 1966), equivalent to Cumin’s stage E7 (Cumin, 1972) occurs approximately 2 days after the veliger stage, and is represented by further development of the lobes and the
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visceral mass being covered completely by the shell. Embryonic developmental stages can also be expressed as proportional developmental units from laying (0) to hatching (100) with intermediate stages represented as a percentage of developmental time elapsed (e.g., in Kuang et al., 2002; Goldberg et al., 2008). Using this approach, the veliger occurs at approximately stage 30 and the hippo around stage 40 (derived from Smirthwaite et al., 2007). Experimental procedure Egg masses containing embryos of the appropriate developmental stage (i.e. veliger or hippo) were selected and placed in a specially-constructed flow-through chamber (dimensions 2.5 cm 5.0 cm 1.0 cm). Pond water PO2 was adjusted either by bubbling with compressed gas (room air for normoxia; N2 for hypoxia; O2 for hyperoxia) or by equilibrating a reservoir of pond water (volume ¼ 4 l) with bubbled gas from either room air, O2 or N2 in combination to achieve a target PO2. Gas-equilibrated pond water passed through the chamber driven by a peristaltic pump (Gilson Minipuls 3, Gilson Inc., Middleton, WI, USA) (approx. 10 ml min1) in a flow-through-to-waste setup. Actual PO2s in the inflow and the outflow were monitored using in-line PO2 electrodes (Strathkelvin 1302, Strathkelvin Instruments, Glasgow, UK) connected to an oxygen interface (Model 928, Strathkelvin Instruments, Glasgow, UK) coupled to a PC operating SI 928 recording software. The time lag from initiation of gas treatment to equilibration in the chamber was approximately 5 min. Behavioural measurements Embryonic behaviour was recorded using video capture (1 frame per second), via a camera (Pixera Corp., San Jose´, CA, USA) connected to the viewport of a stereo microscope (WPI 500053, 20–40 , World Precision Instruments, Inc., Sarasota, FL, USA). Video images for analysis were captured digitally in AVI format. Once the experiment commenced, the field of view was maintained so as to allow examination of the same eggs throughout. Between five and seven embryos were clearly visible in each field of view and the behaviours of 3–4 of these individuals, selected haphazardly, were monitored for the duration of the experiment, so that we would have a continuous recording of the same individuals throughout. Embryos were used only once in trials. Although a number of specific classes of rotational behaviour have been described in other species of gastropod (see Diefenbach et al., 1991) we simply measured the time to complete 3–5 rotations and expressed the rate of spinning as mean revolutions per minute. We used video playback ( 16) to enable clear visualisation of rotational behaviours. Behaviours were
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measured 10 min after equilibration, except in cases noted in the results. Two series of experiments were performed. Series one examined the effects of hyperoxia and return to normoxia on rotational behaviours. Rotational behaviour was recorded throughout and quantified after a 10 min acclimatisation period for each change in chamber PO2. Egg masses containing embryos of the appropriate developmental stage were incubated consecutively in the following treatments: 30 min normoxia (normoxia 1), 30 min hyperoxia (4300% air saturation; hyper), and 30 min normoxia (100% air saturation; normoxia 2). Measurements were made of 12 individual veliger stage embryos over four trials, and 9 individual hippo embryos over three trials. In the second series of experiments we investigated the effects of increasing hypoxia and return to normoxia on rotational behaviour. Embryos were exposed to the following consecutive series of gas treatments: 30 min 100% air saturation (normoxia), 30 min 50% air saturation (50% hypoxia), 30 min 20% air saturation (80% hypoxia), 30 min 0% air saturation (100% hypoxia), and 60 min 100% air saturation (normoxia). In this series, we measured the responses of 6 individual veliger stage embryos over two trials, and 5 individual hippo stage embryos also over two trials.
Data analyses We tested for differences between treatments using repeated-measures ANOVA (SigmaStat, Systat Software Inc., San Jose´, CA, USA). Data were first log10 transformed if necessary (i.e. if non-normal or if variances were unequal between treatments). If the ANOVA yielded significant results then a Holm–Sidak pair-wise multiple comparison post-hoc test was used to determine differences between treatment means. In a separate analysis examining the combined effects of clutch and gas treatment for each developmental stage, we found no significant gas treatment–clutch interaction in any of the experiments indicating that the direction of response (i.e. whether embryos increased or decreased their rates of rotation) was not affected by clutch.
Results Effects of hyperoxia There were significant effects of developmental stage and hyperoxia on spinning behaviour. The later ‘‘hippo’’ stage displayed a significantly higher rotation rate compared with the veliger stage (Fig. 1, Table 1): hippo embryos incubated under normoxic conditions rotated at a rate of 1.8770.24 min1 (mean7standard error)
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Hippo Veliger
rotational rates declined to a rate that was not significantly different from pre-treatment values. Responses of the hippo stage to hypoxia were broadly similar to those for the veliger stage with a general increase in rotation rate with decreasing PO2 (Fig. 2). There were, however, some subtle differences between stages. Firstly, hippos appeared more sensitive to low PO2 and showed a significant increase in rotation rate at 50% saturation compared with normoxia, and a dose response reaching a maximum of 2.8970.18 min1 at 0% air saturation (Fig. 2). There was also a different response on return to normoxia compared with veligers, leading to a significant gas treatment stage interaction (Fig. 2, Table 2): on return to normoxia, hippos showed an immediate decline in rotational rate and, after 5 min, rotational rates had returned to pre-treatment rates (Fig. 1), declining even further over the following 10–20 min with a complete cessation of rotational behaviour in several cases. Although not shown, mean rotation rates in hippos reverted to earlier normoxic levels after 20–30 min post-reintroduction to normoxic water.
Hyperoxia
Rotation rate (min-1)
2.2 2.0 1.8 1.6 1.4 1.2 1.0
Normoxia 1
Hyperoxia
Normoxia 2
Gas Treatment
Fig. 1. Effects of hyperoxia (4300 mmHg PO2) and return to normoxia on embryonic rotation rate in L. stagnalis, at two developmental stages: the earlier ‘‘veliger’’ stage and the later ‘‘hippo’’ stage. Symbols represent mean rates for repeatedly measured individuals. Error bars are standard error estimates.
while veliger embryos rotated at 1.5070.11 min1. Contrary to the prediction that rotational rates would decrease under hyperoxia, hippo embryos increased their rate of rotation significantly to 2.1670.26 min1, whereas veliger embryos showed no significant response to greater PO2s. Upon return to normoxia the rotational rate in hippo embryos declined to pre-treatment values and there was a significant difference compared with the hyperoxia treatment; veliger embryos once again showed no significant response.
Discussion The main aim of our study was to investigate the ability of snail embryos to ‘fine tune’ their rotational behaviour in response to environmental PO2. The behaviour of embryos of the freshwater pulmonate L. stagnalis supported our predictions for hypoxic conditions in that both stages increased their rotation rates with decreasing PO2. Subtle differences were also observed between developmental stages when returned to normoxia from hyperoxia: veligers resumed a ‘‘normal’’ rotation rate, whereas hippo rotation rates showed an almost complete cessation. Our prediction that spinning would reduce under hyperoxic conditions was not supported, however, with hippos increasing their rotational rate only slightly and veligers showing no significant change under conditions of hyperoxia.
Effects of hypoxia Embryos at both developmental stages increased rates of rotation in response to step-wise decreases in ambient PO2 (Fig. 2, Table 2). Embryos at the veliger stage again had a lower rotation rate than hippo stage embryos: mean rotation rates for normoxic veliger embryos were 1.0670.08 min1, increased significantly at 20% air saturation, and reached a maximum of 1.9370.18 min1 when embryos were incubated at 0% air saturation. Within 12 min of return to normoxia, veliger embryo
Table 1. Results of a two-factor repeated measures ANOVA testing the effects of developmental stage (veliger vs. hippo) and gas treatment (normoxia, hyperoxia, return to normoxia) for hyperoxia trials on embryonic rotational behaviour of Lymnaea stagnalis embryos. Source of variation
DF
SS
MS
F-ratio
P-value
Development stage Subject (development stage) Gas treatment Development stage gas treatment Residual Total
1 19 2 2 38 62
1.067 4.345 0.237 0.111 1.247 6.977
1.067 0.229 0.119 0.0553 0.0328 0.113
4.665
0.044
3.611 1.685
0.037 0.199
DF ¼ degrees of freedom; SS ¼ sum of squares; MS ¼ means squared.
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Fig. 2. Effects of step-wise hypoxia treatment and return to normoxia on embryonic rotation rate in L. stagnalis, at two developmental stages: the earlier ‘‘veliger’’ stage and the later ‘‘hippo’’ stage. Symbols represent mean rates for repeatedly measured individuals. Error bars are standard error estimates.
There was no clear evidence that hyperoxia reduced rotational behaviour in either veliger stage or hippo stage embryos. As rotational behaviour has the functional role of mixing the contents of the egg capsule and maximising diffusional gradients from the external medium by homogenising capsular gas tensions (Burggren, 1985; Hunter and Vogel, 1986; Goldberg et al., 2008), it appears that neither embryonic stage is able to decrease its rotational behaviour. Reduction in ventilation in response to elevated environmental PO2 has been documented in a number of ‘water breathing’ groups of animals, e.g. fish (Wood and Jackson, 1980; Berschick et al., 1987) and crayfish (Wheatley, 1989). It might be that rotation activity is not an excessively costly behaviour to embryos. Alternatively, embryos may not have sufficient mechanisms for fine tuning their response under conditions of hyperoxia and, hence, may
incur significant costs during such activity. As such periods of hyperoxic conditions are likely to exist in some habitats, embryos in such conditions may suffer significant costs that could have consequences for their fitness. Our study confirmed earlier studies by Kuang et al. (2002) on H. trivolvis, another pulmonate snail, that rotational behaviour increases with decreasing PO2 in a dose-dependent manner. Goldberg et al. (2008) also demonstrated that this response is more generalised throughout representative families of basommatophoran gastropods, including L. stagnalis, the subject of our study. Although qualitatively similar, the responses by the two developmental stages of L. stagnalis to declining PO2 are quantitatively different in that rotation rates in the hippo stage are approximately 1.3–1.6 times those of the less developed veliger stage. Although such an
Table 2. Results of a two-factor repeated measures ANOVA testing the effects of developmental stage (veliger vs. hippo) and gas treatment (normoxia, hyperoxia, return to normoxia) for hypoxia trials on embryonic rotational behaviour of Lymnaea stagnalis embryos. Source of variation
DF
SS
MS
F-ratio
Development stage Subject (development stage) Gas treatment Development stage gas treatment Residual Total
1 9 5 5 45 65
4.539 3.656 25.980 3.995 3.249 40.146
4.539 0.406 5.196 0.799 0.0722 0.618
11.17265
DF ¼ degrees of freedom; SS ¼ sum of squares; MS ¼ means squared.
71.974 11.066
P-value 0.009 o0.001 o0.001
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increase in rotation rates could be due to developmental changes in number and morphology of rotational cilia, hippo stage embryos also appeared to have a more sensitive response to low PO2 in that they showed a significant increase in rotation rate even at 50% saturation. As the hippo stage is both anatomically more complex and larger than the veliger stage, it is reasonable to assume that metabolic oxygen demand would also be greater, thus requiring a more robust mixing movement and that embryos have a more sophisticated response system at this stage of development. Return to normoxia after hypoxic exposure was accompanied by a rapid reduction in rotational rates in both embryonic stages: movement in veliger embryos returned to normoxic rates within 12 min; however, hippo embryos reduced rotational rates significantly below pre-treatment rates and in some cases actually stopped moving entirely for a period of 20 min or more, before slowly resuming rotational behaviour. A similar cessation of rotational behaviour was noted in Helisoma embryos (Kuang et al., 2002) and by Goldberg et al. (2008) in L. stagnalis and P. gyrina; although inhibition of rotational behaviour was of lower magnitude in the lymnaeid species. Overstimulation of oxygen receptors was cited as the proximate cause resulting in shutdown of serotonergic pathways leading to ciliary movement (Kuang et al., 2002). We assume that a similar cause is in effect in Lymnaea embryos, although Goldberg et al. (2008) postulate that the controlling ciliary movement pathways in Lymnaea are more likely to be dopaminergic. It is also evident that the response is developmentally determined as veliger stage embryos do not respond by cessation of rotational behaviour on return to normoxia. The ontogeny of neural systems of control in later hippo stage embryos may permit a more systemic response to hypoxia and mediate a higher level of response upon return to normoxia. Incubation in a virtually anoxic environment resulted in an approximate doubling of rotational rates both for veliger and hippo stage embryos. Although L. stagnalis is a pulmonate and thus breathes air, it has long been known that adults of this species and other pulmonates can exchange gasses across the integument as well as by way of the lung (Jones, 1972; Graham, 1990) as a means of supplementing pulmonary gas exchange. The embryonic response to severe hypoxia appears to be a classic compensatory response in that the assumed ‘‘purpose’’ is to maintain tissue PO2. Goldberg et al. (2008) have coined the term ‘‘embryo stir-bar’’ to describe how rotational behaviour in pulmonate embryos in response to hypoxia functions to effectively mix the egg capsular contents, thereby achieving a uniform capsular PO2 and maintaining a diffusive gradient for oxygen to enter into the egg capsule from the external environment.
In adult L. stagnalis the response to hypoxia is to increase ventilation, achieved by increasing the opening time of the pneumostome at the air–water surface (Jones, 1961; Hermann and Bulloch, 1998; Taylor et al., 2003). Interestingly, L. stagnalis reared without access to an air–water interface can maintain metabolism by means of integumentary gas exchange, and also will not display air-breathing behaviours even under hypoxic conditions (Hermann and Bulloch, 1998). The stimulus for eliciting ventilation behaviour seems to arise from external oxygen receptors, rather than from any central source (Inoue et al., 1996). Thus it seems that the response to hypoxia in embryonic L. stagnalis matches the compensatory response seen in hatched individuals even though the behaviour is fundamentally different in form.
Acknowledgements We thank Julie Soane for her technical expertise and advice in setting up our experimental system. Support was provided by NERC Grant # NER/B/S/2001/00843 (to SDR and JIS), and a SUNY travel award (to RAB).
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