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The long-term effects of acute exposure to ionising radiation on survival and fertility in Daphnia magna
Environmental Research 150 (2016) 138–143
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The long-term effects of acute exposure to ionising radiation on survival and fertility in Daphnia magna Elena I. Sarapultseva a,b, Yuri E. Dubrova c,d,n a
Department of Biology, Institute of Nuclear Power Engineering NRNU MEPhI, Studgorodok,1, Obninsk, Kaluga Region 249040, Russian Federation National Research Nuclear University “MEPhI”, Kashirskoe Highway, 31, Moscow 115409, Russian Federation c Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom d Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina Str. 3, 11933 Moscow, Russian Federation b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 March 2016 Received in revised form 17 May 2016 Accepted 26 May 2016 Available online 8 June 2016
The results of recent studies have provided strong evidence for the transgenerational effects of parental exposure to ionising radiation and chemical mutagens. However, the transgenerational effects of parental exposure on survival and fertility remain poorly understood. To establish whether parental irradiation can affect the survival and fertility of directly exposed organisms and their offspring, crustacean Daphnia magna were given 10, 100, 1000 and 10,000 mGy of acute γ-rays. Exposure to 1000 and 10,000 mGy significantly compromised the viability of irradiated Daphnia and their first-generation progeny, but did not affect the second-generation progeny. The fertility of F0 and F1 Daphnia gradually declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses. The effects of parental irradiation on the number of broods were only observed among the F0 Daphnia exposed to 1000 and 10,000 mGy, whereas the brood size was equally affected in the two consecutive generations. In contrast, the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. We propose that the decreased fertility observed among the F2 progeny of parents exposed to 10,000 mGy is attributed to transgenerational effects of parental irradiation. Our results also indicate a substantial recovery of the F2 progeny of irradiated F0 Daphnia exposed to the lower doses of acute γ-rays. & 2016 Elsevier Inc. All rights reserved.
Keywords: Radiation Fertility Viability Transgenerational Daphnia
1. Introduction The results of recent studies have clearly demonstrated the existence of non-targeted effects of ionising radiation (Kadhim et al., 2013). This phenomenon describes a number of effects, including transgenerational genomic instability where parental exposure to ionising radiation destabilises the genome of their offspring (Barber and Dubrova, 2006; Dubrova, 2013). As destabilisation of the offspring's genomes results in accumulation of mutations at proteincoding genes and chromosome aberrations, these observations imply that a number of fitness-related traits in the offspring may be affected by this process. Given that the abovementioned effects can manifest over a number of generations, they may therefore be regarded as an additional component of the genetic risk of ionising radiation (Barber and Dubrova, 2006; Dubrova, 2013; Little et al., 2013). Being initially established in mammals, the long-term effects of parental exposure to a number of environmental factors have also n Corresponding author at: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. E-mail address: [email protected] (Y.E. Dubrova).
http://dx.doi.org/10.1016/j.envres.2016.05.046 0013-9351/& 2016 Elsevier Inc. All rights reserved.
been described in other species – Arabidopsis thaliana (Suter and Widmer, 2013; Groot et al., 2016), zebrafish (King Heiden et al., 2009; Baker et al., 2014), rainbow trout (Smith et al., 2016), as well as in some invertebrates, including Caenorhabditis elegans (BuissetGoussen et al., 2014), the earthworm Eisenia fetida (Hertel-Aas et al., 2007, 2011) and crustaceans (Alonzo et al., 2008; Plaire et al., 2013; Parisot et al., 2015). It should be noted that the abovementioned studies on transgenerational instability in mice have so far provided little experimental evidence regarding the multigenerational effects of parental exposure on viability and fertility. As such analysis in mammals is extremely laborious, the introduction of new model organisms characterised by short life span and high fertility is clearly warranted. We and others have previously shown that the parthenogenetic crustacean Daphnia magna represents a useful and very sensitive experimental model for the analysis of long-term effects of exposure to ionising radiation (Sarapultseva and Bychkovskaya, 2010; Sarapultseva and Gorski, 2013; Alonzo et al., 2008; Massarin et al., 2010). Daphnia is characterised by relatively short life span which seldom exceeds 10–11 weeks. For most of the growth season females produce a clutch of at least 10 eggs every 3–4 days (Ebert, 2005). Moreover, Daphnia is an ecologically important organism
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well-studied in the context of evolution, ecology, ecotoxicology (Stollewerk, 2010) and genomics (Colbourne et al., 2011). Given the abovementioned advantages, D. magna has been used in numerous studies aimed to analyse the effects of ionising radiation on mortality and fecundity (reviewed in Dallas et al. (2012) and Fuller et al. (2015)). In a number of studies the effects of chronic exposure have been established across several successive generations of irradiated D. magna (Alonzo et al., 2008; Zeman et al., 2008; Massarin et al., 2010; Plaire et al., 2013; Parisot et al., 2015). However in our opinion, their results do not provide a definitive evidence for the manifestation of transgenerational effects in D. magna, as daphnids were continuously irradiated across all generations. It should be stressed that the manifestation of transgenerational effects can only be established by analysing the non-exposed progeny of irradiated parents. For example, in our recent study we have shown that acute parental irradiation at doses of 100 and 1000 mGy significantly reduces the life-span of directly exposed Daphnia and their F1 offspring (Sarapultseva and Gorski, 2013). In other words, our data suggest that the phenomenon of radiation-induced transgenerational inheritance manifests in Daphnia, affecting the F1 survival. Here we report that parental irradiation can also compromise the fertility of directly exposed Daphnia and their progeny.
139
with 50 mL of water in each glass vials (Apparaturschik Ltd., Moscow Region). Experimental vials were checked daily during 21 days for survival and neonatal removal. Each day, neonates were removed and counted as well as all dead daphnids. To analyse the effects of parental exposure on the successive generation (F1), one-day-old neonates from the third broods of generation F0 were randomly taken from at least three females of irradiated or control groups and transferred to glass vials with 50 mL of water (one Daphnia per vial). Using the same protocol, a group of second-generation offspring (F2) was also established. Generations F1 and F2 were maintained as the original samples but without exposure to γ-irradiation. The survival and fecundity of the parthenogenetic progeny from these generations were measured on a daily basis over 21 days. 2.4. Data analysis The probability of survival was estimated using an algorithm proposed by Breslow and Day (1987). The data were analysed using ANOVA and the Kruskal-Wallis test. All statistical analyses were conducted using SYSTAT 13 version (Systat Software Inc., San Jose, CA, USA).
3. Results 2. Materials and methods 3.1. The effects of parental irradiation on survival 2.1. Daphnia maintenance The strain of Daphnia magna Straus used in our experiments was originally collected in the pond of the Moscow Zoo and maintained for several years at the laboratory in continuous parthenogenetic reproduction following the OECD guideline 211 (Organisation for Economic Co-operation and Development, 2012) with modifications. D. magna were reared at density of one animal per 50 mL in aerated dechlorinated filtered tap-water (рН 7.5–8.2, О2 9.0 mg/l; total mineralisation 6.4 mg/l, Ca:Mg 4:1, Fe 0.3 mg/l, Mn 0.1 mg/l) renewed twice a week. Daphnia were fed with green algae suspension (Chlorella vulgaris) at daily ration of 90 μg C per daphnid (4 105 cells/mL). Algae were cultured in high salt Tamiya medium (Anderson et al., 2005). Algal cell concentration was determined using Goryaeva camera (PJSC “Steklopribor” Ukraine) on an optical density meter (IPS-03, Ltd. OMIKRON, Krasnoyarsk, Russia). D. magna were incubated at 20 °C (7 0.5 °C) on a 12 h/12 h light/dark cycle photoperiod at light intensity 700–1200 lx (Climate Control model R2, LLC Omicron, Krasnoyarsk, Russia). Neonates were removed every weekday. 2.2. Irradiation One-day-old Daphnia from the third broods of at least five females were acutely γ-irradiated at the Lutch Facility (60Co source, Lutch Irradiator, Latenegro, Latvia) at Medical Radiological Research Center, Obninsk, Russia at doses of 10 mGy (for 21 s at 28 mGy min–1), 100 mGy (for 35 s at 170 mGy min–1), 1000 and 10,000 mGy (for 1 and 10 min, respectively, at 1000 mGy min–1). The absorbed dose was assessed using 27,012 and DKS-101 dosimeters. During irradiation, D. magna were kept in the plastic tubes containing 15 mL of water with 10 individuals. All corresponding non-treated control groups were placed in the same conditions but without irradiation. 2.3. Survival and fertility Control and irradiated daphnids (referred to as the exposed generation F0) were maintained individually, one per test vessel,
Fig. 1 summarises the effects of parental irradiation on survival of directly exposed daphnids and their first- and second-generation progeny. As the survival of control F0, F1 and F2 daphnids did not significantly differ (P 40.74), we therefore combined the control data for all three generations. The survival of F0 Daphnia exposed to 1000 and 10,000 mGy of acute γ-rays was significantly compromised (Fig. 1A). Parental irradiation with the same doses also affected the survival of their F1 progeny (Fig. 1B). In the meantime, the survival of F2 progeny of irradiated parents did not significantly differ from that in controls (Fig. 1C). 3.2. The effects of parental irradiation on fertility In all irradiated and control groups, daphnids started producing eggs at the age of 9 days independent of F0 generation dose and released 3–4 successive broods over the 21-day period. As exposure to ionising radiation compromised the viability, here we analysed the fertility of daphnids which survived to the end of the experiment. Table 1 presents the results of ANOVA analysis estimating the effects of parental irradiation on the number of progeny per Daphnia. The two-way ANOVA analysis revealed that the magnitude of the effects of parental irradiation on fertility differed across generations. Acute γ-irradiation also differentially affected the number of broods and brood size. According to the one-way ANOVA, the significant effects of irradiation on the number of progeny per Daphnia were observed in all generations. The detailed analysis of the effects of parental irradiation on fertility of daphnids is given in Table 2. According to our data, acute parental exposure to γ-rays equally compromised the total fertility of irradiated F0 Daphnia and their F1 progeny (Fig. 2A). The fertility of F0 and F1 Daphnia declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses. In contrast, the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. We also analysed the effects of parental irradiation on the brood size and number of broods. The number of broods was only affected among the F0 Daphnia exposed to 1000 and 10,000 mGy, whereas in the F1 and F2 progeny of irradiated parents it did not
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Fig. 1. The effects of parental irradiation on viability of F0 (A), F1 (B) and F2 daphnids (C). Probabilities for significant difference compared to control (log-rank statistics) are shown. Table 1 ANOVA analysis for the effects of irradiation on fertility. Factors
No progeny per Daphnia F (df)
P*
All three generations, n ¼361 Irradiation 25.23 (4, 1.55 10 11 346) Generation 14.10 (2, 1.29 10 6 346) Interaction 2.33 (8, 0.0189 346) Generation F0, n ¼133 Irradiation 12.39 (4, 1.52 10 8 128) Generation F1, n ¼134 Irradiation 13.34 (4, 3.98 10 9 129) Generation F2, n ¼94 Irradiation 6.59 (4, 0.0001 89) *
No broods
F
4.54
P*
0.0014
Table 2 The effects of irradiation on fertility in three generations of Daphnia. Brood size
F
13.89 1.61 10 10 0.0071
2.02
0.2393
1.31
5.57
0.0004
4.31
1.09
0.3636
10.43 2.37 10 7
0.20
0.9384
4.86
Generation F0
mGy
n
Mean 7 sem
Generation F1
Generation F2
n
Mean 7 sem
n
Mean 7sem
29 29 28 24 24
66.86 7 1.66 65.107 1.65 56.17 2.84** 51.58 7 1.55**** 47.54 7 3.33****
20 19 19 18 18
67.25 7 1.21 66.637 2.17 67.53 7 2.14 65.50 7 2.16 55.56 7 1.94***
20 19 19 18 18
3.65 7 0.11 3.58 7 0.12 3.58 7 0.12 3.56 7 0.12 3.50 7 0.15
20 19 19 18 18
18.69 7 0.55 18.88 7 0.72 18.95 7 0.36 18.56 7 0.52 16.077 0.50*
P*
11.30 1.76 10 5 5.02 0.0434
Dose,
0.0026
0.0014
No progeny per Daphnia 0 29 66.727 1.98 10 27 65.337 2.61 100 27 56.96 7 2.98* 1000 25 46.727 4.19*** 10,000 25 42.047 3.68**** No broods 0 29 3.487 0.15 10 27 3.56 7 0.17 100 27 3.157 0.17 1000 25 2.647 0.25* 10,000 25 2.56 7 0.24* Brood size 0 29 19.63 7 0.55 10 27 18.687 0.53 100 27 17.58 7 0.81 1000 25 16.69 7 1.16* 10,000 25 14.88 7 1.25**
29 29 28 24 24 29 29 28 24 24
3.55 7 0.12 3.52 7 0.11 3.36 7 0.16 3.50 7 0.10 3.177 0.24 19.357 0.82 18.69 7 0.35 16.167 0.72* 14.93 7 0.52**** 14.077 1.00****
Significant values are given in bold. Bonferroni corrected probability of difference from control (Kruskal-Wallis test). *
differ from that in controls (Fig. 2B). On the other hand, the effects of parental γ-irradiation on the brood size were very close to those for the integrated measure of fertility estimated as the number of progeny per Daphnia (Fig. 2C). 4. Discussion The analysis of the effects of acute F0 γ-irradiation on viability
Po 0.05. P o 0.01. Po 0.001. **** P o0.0001. **
***
and fertility has revealed that: (i) the survival of F0 daphnids exposed to 1000 and 10,000 mGy of acute γ-rays and their F1 progeny were equally compromised; (ii) the F0 and F1 total fertility significantly decreased at lower parental dose of 100 mGy; (iii) the
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Fig. 2. Fertility of irradiated Daphnia F0 and their progeny. (A) Mean number of progeny per Daphnia. (B) Mean number of broods. (C) Mean brood size. 95% confidence intervals (CI) are shown. Significant difference compared to control (the Kruskal-Wallis test) is indicated by asterisks.
F2 viability did not significantly differ from that in controls and their fertility was only compromised among progeny of parents that received the highest dose of 10,000 mGy. Taken together, our results indicate a substantial recovery of the non-exposed F2 progeny of irradiated Daphnia. The data presented here assessing the effects of parental irradiation on viability are in line with the results of previous studies showing that a number of fitness-related traits in the offspring of irradiated parents are affected by this process. As already mentioned, our previous study showed that acute parental exposure to 100 and 1000 mGy of γ-rays significantly shortened the life span of exposed Daphnia and their F1 progeny (Sarapultseva and Gorski, 2013). The F0 and F1 data on survival presented here are therefore in line with our previous results. The results of our study also show that F0 irradiation can significantly affect the fertility of F0 daphnids and their progeny. However, these effects substantially differ across generations. Thus, exposure to γ-rays significantly compromises the number of broods and brood size in the F0 generation, whereas in the F1 and F2 progeny of irradiated parents the number of broods is close to that in controls. The decreased number of broods in F0 daphnids may be attributed to the cytotoxic effects of irradiation on their germ cells. Although the irradiated one-day-old Daphnia have not yet deposited eggs (Ebert, 2005), their primordial germ cells are affected which can either compromise their survival or ability to form eggs. The effects of irradiation on fertility of female childhood cancer survivors, mostly attributed to depletion of the number of primordial oocytes are well documented (Wallace et al., 2005). According to the results of some studies, the same effects also exist in invertebrate species. Thus, high-dose acute γ-
irradiation significantly decreases the number of eggs produced by soil invertebrate Folsomia candida (Nakamori et al., 2008) as well as marine copepods Paracyclopina nana (Won and Lee, 2014) and Tigriopus japonicas (Han et al., 2014). The same mechanisms may therefore explain the F0 decrease in the number of broods following γ-irradiation. As we analysed parthenogenic strain of D. magna, even early F0 irradiation resulted in exposure of primordial diploid eggs. In this respect, the design of our study is close to those aimed to analyse the effects of F0 in utero irradiation on the development of F1 embryos in mammals (Streffer et al., 2003). According to their results, the developing mammalian embryos show extremely high sensitivity to irradiation, the lethal effects of which substantially exceed those following exposure during adulthood. Our data also show that the effects of irradiation on the F0 fertility, which are partially attributed to the early embryonic mortality, considerably surpass those on the life span of exposed adult Daphnia and their F1 offspring. As far as the transgenerational effects of parental irradiation are concerned, our data provide a sufficient evidence for the manifestation of this phenomenon in the F2 progeny of irradiated parents. First of all, in contrast to the F1 offspring, the F2 progeny indeed have not been exposed to ionising radiation. Another line of evidence supporting this notion comes from the comparison of the effects of parental irradiation on fertility across three generations of exposed daphnids. As already mentioned the fertility of F0 and F1 Daphnia declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses, whereas the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. In
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other words, our data imply that, in sharp contrast to the direct effects of irradiation on the F0 and F1 daphnids where their fertility and the decline of fertility is proportional to the dose of exposure, the F2 effects are only triggered by a very high dose of parental irradiation. These results are in line with our previous mouse data showing that relatively high-dose paternal exposure to acute γrays can only destabilise the F1 genome (Mughal et al., 2012). The effects of parental exposure to ionising radiation across several successive generations have also been analysed in invertebrates, including D. magna. As already mentioned, the design of multi-generational studies on D. magna differed from that used in here, as in these studies daphnids were continuously chronically irradiated across all generations (Alonzo et al., 2008; Zeman et al., 2008; Massarin et al., 2010; Plaire et al., 2013; Parisot et al., 2015), whereas we did not expose the F1 and F2 progeny of irradiated parents. That is why their results cannot be compared with our data. In contrast, in a number of recent multi-generational studies on other invertebrate species the transgenerational effects were investigated among non-exposed progeny of irradiated parents. Thus Hertel-Aas et al. (2011) investigated the effects of high-dose chronic γ-irradiation on reproduction in the earthworm Eisenia fetida. According to the results of this study, the fertility of nonexposed F1 progeny of irradiated worms did not significantly differ from that in controls. It should be noted that in mice relatively high-dose chronic paternal γ-irradiation does not destabilise the F1 genomes (Mughal et al., 2012). It therefore remains to establish whether high-dose acute irradiation can affect the F1 fertility in earthworms. The results of recent study on nematode Caenorhabditis elegans show that parental acute X-irradiation can destabilise the genomes of their non-exposed progeny (Huumonen et al., 2012). On the other hand, the results of another multigenerational study on the same species cannot be compared with our data, as, similar to the abovementioned studies on D. magna, nematodes were continuously irradiated across all generations (Buisset-Goussen et al., 2014). The manifestation of transgenerational instability was also observed in the first- and second-generation progeny of irradiated pea plants (Zaka et al., 2004). Taken together, our current data and the results of abovementioned multigenerational studies on invertebrates show that the effects of acute parental exposure can manifest over a number of generations. As far as the results of our study on the transgenerational effects of parental irradiation on viability are concerned, it should be noted that we did not observe a significant shortening of the life span among the F2 progeny. In this respect, it is worth mentioning the results of transgenerational study by Luning et al. (1976), showing that paternal irradiation can significantly compromise the viability of F2 embryos. According to our results, the decreased fertility observed among the F2 offspring of parents exposed to 10,000 mGy of acute γ-rays is almost exclusively attributed to the reduction of their brood size (Fig. 2C). As the decline in brood size is directly related to early embryonic mortality, the results of our study are therefore in line with the data reported by Luning et al. (1976). It would appear that the transgenerational effects of parental irradiation in daphnids may predominantly manifest during early development, compromising the survival of embryos. According to the results of mouse studies, the transgenerational effects of radiation observed in the offspring of exposed males are an epigenetic phenomenon (Barber and Dubrova, 2006; Dubrova, 2013). It has been suggested that paternal irradiation may alter the epigenetic landscape of male germ cells and that such epigenetic marks, following the transmission to subsequent generations, can affect the offspring genomes (Barber and Dubrova, 2006; Dubrova, 2013). The results of our recent study show that paternal irradiation can dramatically alter the pattern of gene expression in their F1 offspring, thus implying that epigenetic deregulation of a
number of signalling pathways can be regarded as one of the mechanisms underlying transgenerational effects (Gomes et al., 2015). The results of number of recent studies on invertebrates, including D. magna, also provide a strong evidence for the effects of environmental factors, such as exposure to chemicals and metals, on transgenerational epigenetic alterations in the progeny (Vandegehuchte et al., 2010; Norouzitallab et al., 2014). Although to date little is known about the epigenetic mechanisms operating in the Daphnia genome (Harris et al., 2012), the results of a recent in-depth analysis of global methylation (Asselman et al., 2015) and transcriptome (Orsini et al., 2016) in this species provide a solid foundation for future studies in this area. In conclusion, our data show that parental acute γ-irradiation can substantially shorten the life span and compromise fertility of exposed daphnids and their F1 offspring. We have also shown that in the fertility of non-exposed second-generation progeny of irradiated Daphnia is affected, thus implying the manifestation of transgenerational effects of parental irradiation in this species. Our data raise a number of important questions regarding the mechanisms underlying the direct (observed in generations F0 and F1) and non-targeted (transgenerational) long-term effects of irradiation on viability and fertility. The results of our study also show that crustacean D. magna represent a useful and very sensitive system for monitoring the effects of environmental pollution, including radioactive contamination. It should be noted that our study covers environmentally relevant doses (10 and 100 mGy). As according to our results, exposure to 100 mGy marginally reduces the fertility of F0 and F1 daphnids and does not affect their F2 progeny, it would appear that the environmental impact of acute γ-irradiation for this species is likely to be negligent.
Funding source This work was partially supported by the Russian Foundation for Basic Research (14-48-03002) to E.I.S.
Acknowledgments We thank M. Busygin and D. Ouskalova for expert care of Daphnia, A. Brovin and T. Kolesnikova for help with irradiation and dosimetry. We also thank anonymous reviewers for helpful comments and suggestions.
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The long-term effects of acute exposure to ionising radiation on survival and fertility in Daphnia magna Elena I. Sarapultseva a,b, Yuri E. Dubrova c,d,n a
Department of Biology, Institute of Nuclear Power Engineering NRNU MEPhI, Studgorodok,1, Obninsk, Kaluga Region 249040, Russian Federation National Research Nuclear University “MEPhI”, Kashirskoe Highway, 31, Moscow 115409, Russian Federation c Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom d Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina Str. 3, 11933 Moscow, Russian Federation b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 March 2016 Received in revised form 17 May 2016 Accepted 26 May 2016 Available online 8 June 2016
The results of recent studies have provided strong evidence for the transgenerational effects of parental exposure to ionising radiation and chemical mutagens. However, the transgenerational effects of parental exposure on survival and fertility remain poorly understood. To establish whether parental irradiation can affect the survival and fertility of directly exposed organisms and their offspring, crustacean Daphnia magna were given 10, 100, 1000 and 10,000 mGy of acute γ-rays. Exposure to 1000 and 10,000 mGy significantly compromised the viability of irradiated Daphnia and their first-generation progeny, but did not affect the second-generation progeny. The fertility of F0 and F1 Daphnia gradually declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses. The effects of parental irradiation on the number of broods were only observed among the F0 Daphnia exposed to 1000 and 10,000 mGy, whereas the brood size was equally affected in the two consecutive generations. In contrast, the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. We propose that the decreased fertility observed among the F2 progeny of parents exposed to 10,000 mGy is attributed to transgenerational effects of parental irradiation. Our results also indicate a substantial recovery of the F2 progeny of irradiated F0 Daphnia exposed to the lower doses of acute γ-rays. & 2016 Elsevier Inc. All rights reserved.
Keywords: Radiation Fertility Viability Transgenerational Daphnia
1. Introduction The results of recent studies have clearly demonstrated the existence of non-targeted effects of ionising radiation (Kadhim et al., 2013). This phenomenon describes a number of effects, including transgenerational genomic instability where parental exposure to ionising radiation destabilises the genome of their offspring (Barber and Dubrova, 2006; Dubrova, 2013). As destabilisation of the offspring's genomes results in accumulation of mutations at proteincoding genes and chromosome aberrations, these observations imply that a number of fitness-related traits in the offspring may be affected by this process. Given that the abovementioned effects can manifest over a number of generations, they may therefore be regarded as an additional component of the genetic risk of ionising radiation (Barber and Dubrova, 2006; Dubrova, 2013; Little et al., 2013). Being initially established in mammals, the long-term effects of parental exposure to a number of environmental factors have also n Corresponding author at: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. E-mail address: [email protected] (Y.E. Dubrova).
http://dx.doi.org/10.1016/j.envres.2016.05.046 0013-9351/& 2016 Elsevier Inc. All rights reserved.
been described in other species – Arabidopsis thaliana (Suter and Widmer, 2013; Groot et al., 2016), zebrafish (King Heiden et al., 2009; Baker et al., 2014), rainbow trout (Smith et al., 2016), as well as in some invertebrates, including Caenorhabditis elegans (BuissetGoussen et al., 2014), the earthworm Eisenia fetida (Hertel-Aas et al., 2007, 2011) and crustaceans (Alonzo et al., 2008; Plaire et al., 2013; Parisot et al., 2015). It should be noted that the abovementioned studies on transgenerational instability in mice have so far provided little experimental evidence regarding the multigenerational effects of parental exposure on viability and fertility. As such analysis in mammals is extremely laborious, the introduction of new model organisms characterised by short life span and high fertility is clearly warranted. We and others have previously shown that the parthenogenetic crustacean Daphnia magna represents a useful and very sensitive experimental model for the analysis of long-term effects of exposure to ionising radiation (Sarapultseva and Bychkovskaya, 2010; Sarapultseva and Gorski, 2013; Alonzo et al., 2008; Massarin et al., 2010). Daphnia is characterised by relatively short life span which seldom exceeds 10–11 weeks. For most of the growth season females produce a clutch of at least 10 eggs every 3–4 days (Ebert, 2005). Moreover, Daphnia is an ecologically important organism
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well-studied in the context of evolution, ecology, ecotoxicology (Stollewerk, 2010) and genomics (Colbourne et al., 2011). Given the abovementioned advantages, D. magna has been used in numerous studies aimed to analyse the effects of ionising radiation on mortality and fecundity (reviewed in Dallas et al. (2012) and Fuller et al. (2015)). In a number of studies the effects of chronic exposure have been established across several successive generations of irradiated D. magna (Alonzo et al., 2008; Zeman et al., 2008; Massarin et al., 2010; Plaire et al., 2013; Parisot et al., 2015). However in our opinion, their results do not provide a definitive evidence for the manifestation of transgenerational effects in D. magna, as daphnids were continuously irradiated across all generations. It should be stressed that the manifestation of transgenerational effects can only be established by analysing the non-exposed progeny of irradiated parents. For example, in our recent study we have shown that acute parental irradiation at doses of 100 and 1000 mGy significantly reduces the life-span of directly exposed Daphnia and their F1 offspring (Sarapultseva and Gorski, 2013). In other words, our data suggest that the phenomenon of radiation-induced transgenerational inheritance manifests in Daphnia, affecting the F1 survival. Here we report that parental irradiation can also compromise the fertility of directly exposed Daphnia and their progeny.
139
with 50 mL of water in each glass vials (Apparaturschik Ltd., Moscow Region). Experimental vials were checked daily during 21 days for survival and neonatal removal. Each day, neonates were removed and counted as well as all dead daphnids. To analyse the effects of parental exposure on the successive generation (F1), one-day-old neonates from the third broods of generation F0 were randomly taken from at least three females of irradiated or control groups and transferred to glass vials with 50 mL of water (one Daphnia per vial). Using the same protocol, a group of second-generation offspring (F2) was also established. Generations F1 and F2 were maintained as the original samples but without exposure to γ-irradiation. The survival and fecundity of the parthenogenetic progeny from these generations were measured on a daily basis over 21 days. 2.4. Data analysis The probability of survival was estimated using an algorithm proposed by Breslow and Day (1987). The data were analysed using ANOVA and the Kruskal-Wallis test. All statistical analyses were conducted using SYSTAT 13 version (Systat Software Inc., San Jose, CA, USA).
3. Results 2. Materials and methods 3.1. The effects of parental irradiation on survival 2.1. Daphnia maintenance The strain of Daphnia magna Straus used in our experiments was originally collected in the pond of the Moscow Zoo and maintained for several years at the laboratory in continuous parthenogenetic reproduction following the OECD guideline 211 (Organisation for Economic Co-operation and Development, 2012) with modifications. D. magna were reared at density of one animal per 50 mL in aerated dechlorinated filtered tap-water (рН 7.5–8.2, О2 9.0 mg/l; total mineralisation 6.4 mg/l, Ca:Mg 4:1, Fe 0.3 mg/l, Mn 0.1 mg/l) renewed twice a week. Daphnia were fed with green algae suspension (Chlorella vulgaris) at daily ration of 90 μg C per daphnid (4 105 cells/mL). Algae were cultured in high salt Tamiya medium (Anderson et al., 2005). Algal cell concentration was determined using Goryaeva camera (PJSC “Steklopribor” Ukraine) on an optical density meter (IPS-03, Ltd. OMIKRON, Krasnoyarsk, Russia). D. magna were incubated at 20 °C (7 0.5 °C) on a 12 h/12 h light/dark cycle photoperiod at light intensity 700–1200 lx (Climate Control model R2, LLC Omicron, Krasnoyarsk, Russia). Neonates were removed every weekday. 2.2. Irradiation One-day-old Daphnia from the third broods of at least five females were acutely γ-irradiated at the Lutch Facility (60Co source, Lutch Irradiator, Latenegro, Latvia) at Medical Radiological Research Center, Obninsk, Russia at doses of 10 mGy (for 21 s at 28 mGy min–1), 100 mGy (for 35 s at 170 mGy min–1), 1000 and 10,000 mGy (for 1 and 10 min, respectively, at 1000 mGy min–1). The absorbed dose was assessed using 27,012 and DKS-101 dosimeters. During irradiation, D. magna were kept in the plastic tubes containing 15 mL of water with 10 individuals. All corresponding non-treated control groups were placed in the same conditions but without irradiation. 2.3. Survival and fertility Control and irradiated daphnids (referred to as the exposed generation F0) were maintained individually, one per test vessel,
Fig. 1 summarises the effects of parental irradiation on survival of directly exposed daphnids and their first- and second-generation progeny. As the survival of control F0, F1 and F2 daphnids did not significantly differ (P 40.74), we therefore combined the control data for all three generations. The survival of F0 Daphnia exposed to 1000 and 10,000 mGy of acute γ-rays was significantly compromised (Fig. 1A). Parental irradiation with the same doses also affected the survival of their F1 progeny (Fig. 1B). In the meantime, the survival of F2 progeny of irradiated parents did not significantly differ from that in controls (Fig. 1C). 3.2. The effects of parental irradiation on fertility In all irradiated and control groups, daphnids started producing eggs at the age of 9 days independent of F0 generation dose and released 3–4 successive broods over the 21-day period. As exposure to ionising radiation compromised the viability, here we analysed the fertility of daphnids which survived to the end of the experiment. Table 1 presents the results of ANOVA analysis estimating the effects of parental irradiation on the number of progeny per Daphnia. The two-way ANOVA analysis revealed that the magnitude of the effects of parental irradiation on fertility differed across generations. Acute γ-irradiation also differentially affected the number of broods and brood size. According to the one-way ANOVA, the significant effects of irradiation on the number of progeny per Daphnia were observed in all generations. The detailed analysis of the effects of parental irradiation on fertility of daphnids is given in Table 2. According to our data, acute parental exposure to γ-rays equally compromised the total fertility of irradiated F0 Daphnia and their F1 progeny (Fig. 2A). The fertility of F0 and F1 Daphnia declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses. In contrast, the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. We also analysed the effects of parental irradiation on the brood size and number of broods. The number of broods was only affected among the F0 Daphnia exposed to 1000 and 10,000 mGy, whereas in the F1 and F2 progeny of irradiated parents it did not
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Fig. 1. The effects of parental irradiation on viability of F0 (A), F1 (B) and F2 daphnids (C). Probabilities for significant difference compared to control (log-rank statistics) are shown. Table 1 ANOVA analysis for the effects of irradiation on fertility. Factors
No progeny per Daphnia F (df)
P*
All three generations, n ¼361 Irradiation 25.23 (4, 1.55 10 11 346) Generation 14.10 (2, 1.29 10 6 346) Interaction 2.33 (8, 0.0189 346) Generation F0, n ¼133 Irradiation 12.39 (4, 1.52 10 8 128) Generation F1, n ¼134 Irradiation 13.34 (4, 3.98 10 9 129) Generation F2, n ¼94 Irradiation 6.59 (4, 0.0001 89) *
No broods
F
4.54
P*
0.0014
Table 2 The effects of irradiation on fertility in three generations of Daphnia. Brood size
F
13.89 1.61 10 10 0.0071
2.02
0.2393
1.31
5.57
0.0004
4.31
1.09
0.3636
10.43 2.37 10 7
0.20
0.9384
4.86
Generation F0
mGy
n
Mean 7 sem
Generation F1
Generation F2
n
Mean 7 sem
n
Mean 7sem
29 29 28 24 24
66.86 7 1.66 65.107 1.65 56.17 2.84** 51.58 7 1.55**** 47.54 7 3.33****
20 19 19 18 18
67.25 7 1.21 66.637 2.17 67.53 7 2.14 65.50 7 2.16 55.56 7 1.94***
20 19 19 18 18
3.65 7 0.11 3.58 7 0.12 3.58 7 0.12 3.56 7 0.12 3.50 7 0.15
20 19 19 18 18
18.69 7 0.55 18.88 7 0.72 18.95 7 0.36 18.56 7 0.52 16.077 0.50*
P*
11.30 1.76 10 5 5.02 0.0434
Dose,
0.0026
0.0014
No progeny per Daphnia 0 29 66.727 1.98 10 27 65.337 2.61 100 27 56.96 7 2.98* 1000 25 46.727 4.19*** 10,000 25 42.047 3.68**** No broods 0 29 3.487 0.15 10 27 3.56 7 0.17 100 27 3.157 0.17 1000 25 2.647 0.25* 10,000 25 2.56 7 0.24* Brood size 0 29 19.63 7 0.55 10 27 18.687 0.53 100 27 17.58 7 0.81 1000 25 16.69 7 1.16* 10,000 25 14.88 7 1.25**
29 29 28 24 24 29 29 28 24 24
3.55 7 0.12 3.52 7 0.11 3.36 7 0.16 3.50 7 0.10 3.177 0.24 19.357 0.82 18.69 7 0.35 16.167 0.72* 14.93 7 0.52**** 14.077 1.00****
Significant values are given in bold. Bonferroni corrected probability of difference from control (Kruskal-Wallis test). *
differ from that in controls (Fig. 2B). On the other hand, the effects of parental γ-irradiation on the brood size were very close to those for the integrated measure of fertility estimated as the number of progeny per Daphnia (Fig. 2C). 4. Discussion The analysis of the effects of acute F0 γ-irradiation on viability
Po 0.05. P o 0.01. Po 0.001. **** P o0.0001. **
***
and fertility has revealed that: (i) the survival of F0 daphnids exposed to 1000 and 10,000 mGy of acute γ-rays and their F1 progeny were equally compromised; (ii) the F0 and F1 total fertility significantly decreased at lower parental dose of 100 mGy; (iii) the
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Fig. 2. Fertility of irradiated Daphnia F0 and their progeny. (A) Mean number of progeny per Daphnia. (B) Mean number of broods. (C) Mean brood size. 95% confidence intervals (CI) are shown. Significant difference compared to control (the Kruskal-Wallis test) is indicated by asterisks.
F2 viability did not significantly differ from that in controls and their fertility was only compromised among progeny of parents that received the highest dose of 10,000 mGy. Taken together, our results indicate a substantial recovery of the non-exposed F2 progeny of irradiated Daphnia. The data presented here assessing the effects of parental irradiation on viability are in line with the results of previous studies showing that a number of fitness-related traits in the offspring of irradiated parents are affected by this process. As already mentioned, our previous study showed that acute parental exposure to 100 and 1000 mGy of γ-rays significantly shortened the life span of exposed Daphnia and their F1 progeny (Sarapultseva and Gorski, 2013). The F0 and F1 data on survival presented here are therefore in line with our previous results. The results of our study also show that F0 irradiation can significantly affect the fertility of F0 daphnids and their progeny. However, these effects substantially differ across generations. Thus, exposure to γ-rays significantly compromises the number of broods and brood size in the F0 generation, whereas in the F1 and F2 progeny of irradiated parents the number of broods is close to that in controls. The decreased number of broods in F0 daphnids may be attributed to the cytotoxic effects of irradiation on their germ cells. Although the irradiated one-day-old Daphnia have not yet deposited eggs (Ebert, 2005), their primordial germ cells are affected which can either compromise their survival or ability to form eggs. The effects of irradiation on fertility of female childhood cancer survivors, mostly attributed to depletion of the number of primordial oocytes are well documented (Wallace et al., 2005). According to the results of some studies, the same effects also exist in invertebrate species. Thus, high-dose acute γ-
irradiation significantly decreases the number of eggs produced by soil invertebrate Folsomia candida (Nakamori et al., 2008) as well as marine copepods Paracyclopina nana (Won and Lee, 2014) and Tigriopus japonicas (Han et al., 2014). The same mechanisms may therefore explain the F0 decrease in the number of broods following γ-irradiation. As we analysed parthenogenic strain of D. magna, even early F0 irradiation resulted in exposure of primordial diploid eggs. In this respect, the design of our study is close to those aimed to analyse the effects of F0 in utero irradiation on the development of F1 embryos in mammals (Streffer et al., 2003). According to their results, the developing mammalian embryos show extremely high sensitivity to irradiation, the lethal effects of which substantially exceed those following exposure during adulthood. Our data also show that the effects of irradiation on the F0 fertility, which are partially attributed to the early embryonic mortality, considerably surpass those on the life span of exposed adult Daphnia and their F1 offspring. As far as the transgenerational effects of parental irradiation are concerned, our data provide a sufficient evidence for the manifestation of this phenomenon in the F2 progeny of irradiated parents. First of all, in contrast to the F1 offspring, the F2 progeny indeed have not been exposed to ionising radiation. Another line of evidence supporting this notion comes from the comparison of the effects of parental irradiation on fertility across three generations of exposed daphnids. As already mentioned the fertility of F0 and F1 Daphnia declined with the dose of parental exposure and significantly decreased at dose of 100 mGy and at higher doses, whereas the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000 mGy. In
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other words, our data imply that, in sharp contrast to the direct effects of irradiation on the F0 and F1 daphnids where their fertility and the decline of fertility is proportional to the dose of exposure, the F2 effects are only triggered by a very high dose of parental irradiation. These results are in line with our previous mouse data showing that relatively high-dose paternal exposure to acute γrays can only destabilise the F1 genome (Mughal et al., 2012). The effects of parental exposure to ionising radiation across several successive generations have also been analysed in invertebrates, including D. magna. As already mentioned, the design of multi-generational studies on D. magna differed from that used in here, as in these studies daphnids were continuously chronically irradiated across all generations (Alonzo et al., 2008; Zeman et al., 2008; Massarin et al., 2010; Plaire et al., 2013; Parisot et al., 2015), whereas we did not expose the F1 and F2 progeny of irradiated parents. That is why their results cannot be compared with our data. In contrast, in a number of recent multi-generational studies on other invertebrate species the transgenerational effects were investigated among non-exposed progeny of irradiated parents. Thus Hertel-Aas et al. (2011) investigated the effects of high-dose chronic γ-irradiation on reproduction in the earthworm Eisenia fetida. According to the results of this study, the fertility of nonexposed F1 progeny of irradiated worms did not significantly differ from that in controls. It should be noted that in mice relatively high-dose chronic paternal γ-irradiation does not destabilise the F1 genomes (Mughal et al., 2012). It therefore remains to establish whether high-dose acute irradiation can affect the F1 fertility in earthworms. The results of recent study on nematode Caenorhabditis elegans show that parental acute X-irradiation can destabilise the genomes of their non-exposed progeny (Huumonen et al., 2012). On the other hand, the results of another multigenerational study on the same species cannot be compared with our data, as, similar to the abovementioned studies on D. magna, nematodes were continuously irradiated across all generations (Buisset-Goussen et al., 2014). The manifestation of transgenerational instability was also observed in the first- and second-generation progeny of irradiated pea plants (Zaka et al., 2004). Taken together, our current data and the results of abovementioned multigenerational studies on invertebrates show that the effects of acute parental exposure can manifest over a number of generations. As far as the results of our study on the transgenerational effects of parental irradiation on viability are concerned, it should be noted that we did not observe a significant shortening of the life span among the F2 progeny. In this respect, it is worth mentioning the results of transgenerational study by Luning et al. (1976), showing that paternal irradiation can significantly compromise the viability of F2 embryos. According to our results, the decreased fertility observed among the F2 offspring of parents exposed to 10,000 mGy of acute γ-rays is almost exclusively attributed to the reduction of their brood size (Fig. 2C). As the decline in brood size is directly related to early embryonic mortality, the results of our study are therefore in line with the data reported by Luning et al. (1976). It would appear that the transgenerational effects of parental irradiation in daphnids may predominantly manifest during early development, compromising the survival of embryos. According to the results of mouse studies, the transgenerational effects of radiation observed in the offspring of exposed males are an epigenetic phenomenon (Barber and Dubrova, 2006; Dubrova, 2013). It has been suggested that paternal irradiation may alter the epigenetic landscape of male germ cells and that such epigenetic marks, following the transmission to subsequent generations, can affect the offspring genomes (Barber and Dubrova, 2006; Dubrova, 2013). The results of our recent study show that paternal irradiation can dramatically alter the pattern of gene expression in their F1 offspring, thus implying that epigenetic deregulation of a
number of signalling pathways can be regarded as one of the mechanisms underlying transgenerational effects (Gomes et al., 2015). The results of number of recent studies on invertebrates, including D. magna, also provide a strong evidence for the effects of environmental factors, such as exposure to chemicals and metals, on transgenerational epigenetic alterations in the progeny (Vandegehuchte et al., 2010; Norouzitallab et al., 2014). Although to date little is known about the epigenetic mechanisms operating in the Daphnia genome (Harris et al., 2012), the results of a recent in-depth analysis of global methylation (Asselman et al., 2015) and transcriptome (Orsini et al., 2016) in this species provide a solid foundation for future studies in this area. In conclusion, our data show that parental acute γ-irradiation can substantially shorten the life span and compromise fertility of exposed daphnids and their F1 offspring. We have also shown that in the fertility of non-exposed second-generation progeny of irradiated Daphnia is affected, thus implying the manifestation of transgenerational effects of parental irradiation in this species. Our data raise a number of important questions regarding the mechanisms underlying the direct (observed in generations F0 and F1) and non-targeted (transgenerational) long-term effects of irradiation on viability and fertility. The results of our study also show that crustacean D. magna represent a useful and very sensitive system for monitoring the effects of environmental pollution, including radioactive contamination. It should be noted that our study covers environmentally relevant doses (10 and 100 mGy). As according to our results, exposure to 100 mGy marginally reduces the fertility of F0 and F1 daphnids and does not affect their F2 progeny, it would appear that the environmental impact of acute γ-irradiation for this species is likely to be negligent.
Funding source This work was partially supported by the Russian Foundation for Basic Research (14-48-03002) to E.I.S.
Acknowledgments We thank M. Busygin and D. Ouskalova for expert care of Daphnia, A. Brovin and T. Kolesnikova for help with irradiation and dosimetry. We also thank anonymous reviewers for helpful comments and suggestions.
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