DSpace at VNU: Chronic effects of cyanobacterial toxins on Daphnia magna and their offspring

DSpace at VNU: Chronic effects of cyanobacterial toxins on Daphnia magna and their offspring tài liệu, giáo án, bài giản...

Chronic effects of cyanobacterial toxins on Daphnia magna and

their offspring

Thanh Son Daoa,b, Lan-Chi Do-Hongc, Claudia Wiegandb,d,*

a Institute for Environment and Resources, 142 To Hien Thanh Street, District 10, Ho Chi Minh City, Vietnam

b Leibniz Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 301, 12587 Berlin, Germany

c Vietnam National University- HoChiMinhCity, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam

d University of Southern Denmark, Institute of Biology, Campusvej 55, 5230 Odense M, Denmark

a r t i c l e i n f o

Article history:

Received 10 July 2009

Received in revised form 14 December 2009

Accepted 26 January 2010

Available online 2 February 2010

Keywords:

Chronic effects

Daphnia magna

Microcystin

Cyanobacterial crude extract

Life traits

Malformation

a b s t r a c t

The zooplankton grazer Daphnia magna endures living in water bodies up to moderate densities of cyanobacteria, such as Microcystis spp., known for producing toxic secondary metabolites Although daphnids are affected via decreased food filtering, inhibition of digestive proteases and lethality, development of tolerance against cyanobacterial toxins has also been observed Aim of our study was to investigate in detail chronic effects of cyanobacterial toxins, with emphasis on microcystin, on D magna The animals were exposed chronically for two generations to either microcystin-LR in 5 or 50mg L1, or to cyanobacterial crude extract containing the same amount of total microcystin, starting at neonate stadium Survival, growth, maturation and fecundity were observed for the first generation during two months In the offspring survival, maturation, and growth were followed for the first week

Low concentration of microcystin-LR slightly affected the growth and reproduction of parent daphnids Survivorship decreased during chronic exposure with increasing microcystin concentration Age to maturity of the offspring increased and their survival decreased after parent generation was exposed to the toxin, even if the offspring were raised in control medium Besides, cessation of the eggs/embryos was observed and malformation of neonates caused by cyanobacterial toxins was firstly recorded

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1 Introduction

In nature, cyanobacteria are not only low nutrient food

for zooplankton (Lampert, 1977; Mu¨ller-Navarra et al.,

2000; Von Elert et al., 2003) but also difficult to consume

due to commonly big colonial or long filamentous

forma-tion and mucilage producforma-tion (Rohrlack et al., 1999; Ebert,

2005) Furthermore, cyanobacteria are capable of

producing toxic metabolites and bioactive compounds such

as microcystin (MC), anatoxin-a, cylindrospermopsin,

microviridin J or microcin SF608 (Sivonen and Jones, 1999;

Banker and Carmeli, 1999; Rohrlack et al., 2003) Most of those bioactive metabolites are contained inside the cells and released into the water during cell lysis Zooplankton directly suffer from toxic cyanobacteria as they are primary consumers feeding on suspended particles, and phyto-plankton including small cyanobacteria Zoophyto-plankton accumulated MC at concentrations ranging from 0.3 to 16.4mg g1dried weight (DW) which is much higher than present in natural water, from undetectable 5.8 ng g1

DW (Ferra˜o-Filho et al., 2002a) The negative correlation between the density of cladoceran and the biomass of cyanobacteria producing MC was recorded in the field reflecting the nutritional and toxin effects on the animals (Ferra˜o-Filho et al., 2002b) Similarly,Hansson et al (2007)

found a clear negative correlation between MC in water and

* Corresponding author at: University of Southern Denmark, Institute

of Biology, Campusvej 55, 5230 Odense M, Denmark Tel.: þ45 6550 2785.

E-mail address: wiegand@biology.sdu.dk (C Wiegand).

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Toxicon

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total zooplankton biomass In addition, big cladoceran had

an apparently negative response while smaller cladoceran

seemed to have a weakly positive response to MC This was

assumed as the effects of toxic cyanobacteria which

induced a shift in zooplankton size and community

composition in nature (Hansson et al., 2007)

Considerable numbers of laboratorial studies

investi-gated acute effects of cyanobacterial toxins on daphnids

concerning survival, food filtering, molting or enzyme

activities of the animals Survival of Daphnia spp was

rapidly reduced by toxic Microcystis aeruginosa (Nizan et al.,

1986; Ferra˜o-Filho et al., 2000) The 48-h lethal

concen-tration (LC50) of purified microcystin-LR (MC-LR) for three

Daphnia species ranged from 9.6 to 21.4 mg MC-LR

mL1(DeMott et al., 1991) Similarly, cyanobacterial crude

extract containing MC varied in concentrations of 48-h

LD50or 48-h effective concentration (EC50) ranging from 36

to 162.45mg mL1or 34.2 to 1380mg MC g1DW in Daphnia

pulicaria and Daphnia similis respectively (Jungmann and

Benndorf, 1994; Sotero-Santos et al., 2006; Okumura

et al., 2007) Hietala et al (1997a)showed a discrepancy

among the 48-h EC50concentration of toxic M aeruginosa

to ten clones of Daphnia pulex, varying from 0.022 to

>2.61 mg C L1 Hence, susceptibility to cyanobacterial

toxins differs not only between zooplankton species but

also within clones of Daphnia

The food filtering rate of Daphnia spp was inhibited when

the organisms were exposed to toxic cyanobacteria and

purified MC-LR (Lampert, 1982; Nizan et al., 1986; DeMott,

1999; Ferra˜o-Filho et al., 2000; Ghadouani et al., 2004)

Toxic M aeruginosa caused sudden stops in swimming and

filtering activity in daphnids (Rohrlack et al., 2001)

Defor-mation of carapace and interruption of molting process of

daphnids were induced by the cyanobacterial metabolite

microviridin J (Kaebernick et al., 2001; Rohrlack et al., 2004)

Physiological responses included elevated activities of

the biotransformation enzyme glutathione S-transferase in

D magna after exposure to MC-LR or Cylindrospermopsis

raciborskii but inhibition after exposure to microcin SF608

or Aphanizomenon issatschenkoi (Wiegand et al., 2002;

Nogueira et al., 2004a,b) In addition, the digestive

enzyme trypsin of daphnids was inhibited by

cyanobacte-rial metabolites obtained from a widely distributed

cya-nobacterial genus Planktothrix (Rohrlack et al., 2005)

In chronic and semi-chronic exposures, toxin

accumu-lation and detrimental influence on the animals such as

survival, growth, maturation and fecundity were observed

Exposed to toxic M aeruginosa, daphnids accumulated MC

at very high concentrations, up to 0.0712 mg MC per

Daphnia and 24.5mg MC g1DW of daphnids respectively

(Thostrup and Christoffersen, 1999; Mohamed, 2001)

Similar to acute exposures, survival of Daphnia was

decreased by toxic M aeruginosa, both at species and clone

levels (DeMott et al., 1991; Hietala et al., 1995, 1997b;

Rohrlack et al., 2001)

Body length of daphnids was significantly reduced

when they were fed with M aeruginosa (Lu¨rling, 2003;

Lu¨rling and Van der Grinten, 2003; Trubetskova and

Haney, 2006) Consequently, maturation and time to first

reproduction of daphnids were delayed if they fed on toxic

M aeruginosa (Hietala et al., 1995; Laure´n-Ma¨a¨tta¨ et al.,

1997) Toxic M aeruginosa also caused aborted eggs (Gustafsson et al., 2005) and reduced the fecundity of

D magna (Thostrup and Christoffersen, 1999; Lu¨rling and Van der Grinten, 2003; Trubetskova and Haney, 2006) or even completely inhibited the reproduction of D pulex (Hietala et al., 1997b) In contrast, dissolved MC-LR at the concentration of 3.5 mg L1had no effect on growth and reproduction of D magna (Lu¨rling and Van der Grinten, 2003)

By continuous exposure to low densities of M aeruginosa over generations, D magna increased their tolerance to the toxic cyanobacterium, had higher fitness and earlier reached the maturity age and first clutch of reproduction These adapta-tions were assumed as the result of maternal effects (Gustafsson and Hansson, 2004; Gustafsson et al., 2005) Overall, toxic cyanobacteria and their toxins severely impacted daphnids in both acute and chronic exposures The organisms were not only suppressed in their survival but impaired in development as well As mentioned above, investigations of the chronically detrimental influence on daphnids have been conducted with cyanobacterial cultures or pure cyanobacterial toxin In most of the mentioned chronic studies, exposures were performed with one generation of daphnids and lasted for around three weeks Gustafsson et al (2005) were conducting transgenerational experiments in which the F1 daphnids produced up to seven clutches; hence for the F1 generation the exposure lasted for around one month

In this study, chronic effects of dissolved MC-LR and

a cell free cyanobacterial crude extract containing MC on life history of D magna were investigated over their entire life duration of two months Their offspring were raised in either toxic or control medium until maturation to follow consequences of maternal exposure The observed life traits

of the daphnids were survival, growth, maturation, time to first reproduction and fecundity The investigation on chronic effects was carried out with two concentrations of

MC (5 and 50mg L1) which would fall within the range of dissolved MC in natural waters in the world, from trace concentration to 200mg L1(Sivonen and Jones, 1999) and have already impacted enzyme activities in D magna (Wiegand et al., 2002) In all exposure, Scenedesmus spp were provided as food source to exclude effects due to malnutrition, hence to be able to attribute effects to cya-nobacterial toxin or toxic compounds

2 Materials and methods 2.1 Microcystin-LR and cyanobacterial crude extract Microcystin-LR and a cyanobacterial crude extract con-taining MC were used for the exposures The MC-LR was purchased from Axxora (Germany), and the cyanobacterial crude extract was produced from our batch cultures of toxic

M aeruginosa The cyanobacterium, strain AB 2005/47, was isolated from Chaohu Lake in China in 2005 and has been cultivated as single strain in the laboratory of the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany since then The crude extract was prepared according toFastner et al (1998)with modification Briefly, the biomass of cultures on GF/C filters (Whatman) were homogenized and firstly extracted over night in 70% MeOH

(Carl Roth, Germany) containing 5% acetic acid (Merck,

Germany) and 0.1% triflouracetic acid (TFA; Merck,

Germany) followed by 2  60 min in MeOH 90% containing

5% acetic acid and 0.1% TFA with 30 s sonication during the

last extraction Each extraction step was followed by

centrifugation (4500 rpm, 10 min, 4C) The supernatants of

all extractions were pooled, dried at 35C, re-dissolved in

MeOH (100%) and centrifuged at 14 000 rpm, 1 C for

10 min The supernatant was kept at 20C prior to

high-performance liquid chromatography (HPLC) analysis

Pflugmacher et al (2001)by high-performance liquid

chro-matography (HPLC, Waters Alliance, Eschborn, Germany)

system equipped with a reverse phase column (RP 18; 5mM

LIChroSpher 100) and photodiode array detection Injection

volume of the samples was 80mL Separation was achieved at

40C by a gradient of Milli-Q water and acetonitrile (ACN,

Rathburn, Walkerburn, UK), both enriched with 0.1% (v/v)

TFA The flow rate was 1 mL min1, starting at 35% ACN,

linearly increasing to 55% ACN within 15 min, followed by

a cleaning step of 100% ACN and 10 min equilibration to start

conditions MC-LR was used as the reference toxin The HPLC

analysis shown that there were several different variants of

MC and their total concentration in the crude extract was

6.92mg mL1MC-LR equivalent

Mass spectrometer measurements for the

cyanobacte-rial crude extract were performed using an API 165 mass

spectrometer (Applied Biosystems, Foster City, CA, USA)

with an atmospheric pressure ionization source operating

in turbo ion-spray mode Quantitative determination of the

toxins was carried out by comparing peak areas in sample

obtained from the pure toxins Proportion of the main

cyanobacterial toxins in the crude extract was as follows,

MC-RR, 71.09%; MC-LR, 24%; MC-LA, 3.09%; MC-YR, 1.79%

and MC-LF, 0.03%Kru¨ger et al., (submitted for publication)

2.2 Experimental organisms

The test organism was D magna Straus, obtained from

MicroBioTests Inc Belgium and has been maintained in the

laboratory of the Leibniz Institute of Freshwater Ecology

and Inland Fisheries, Berlin, for several generations The

Daphnia medium provided by MicroBioTests Inc Belgium

consisted of CaCl2, KCl, NaHCO3 and MgSO2 The living

green algae Scenedesmus spp were used as the sole food for

D magna The algae were cultivated in Z8 medium (Kotai,

1972) with continuous aeration Both culturing of

Scene-desmus and Daphnia as well as the exposures were

con-ducted at a temperature of 20  1C and a photoperiod of

14 h light and 10 h dark

Prior start of the experiments, fifteen adolescent female

D magna were incubated in a 500 mL beaker and fed with

Scenedesmus spp for 2–3 weeks Offspring from the second

to fifth clutch of these D magna were used for experiments

and hereafter referred to as mother daphnids Thirty

neonates less than 24 h old were randomly selected for

each chronic exposure (Adema, 1978) and individually

incubated in 50 mL beakers containing 20 mL of medium

The animals were fed 4  106Scenedesmus spp cells per

daphnid per day equaling a cell density of 2  105cells

mL1(approximately 3 mg C L1) Density of Scenedesmus cells was adjusted daily The food density and availability were chosen according toLampert (1977)andGustafsson

et al (2005)respectively, in which individual animal was fed with 6  104Scenedesmus cells mL1in 60 mL beakers, equaling 3.6  106cells per daphnid per day

2.3 Experimental setup For exposure, MC-LR or cell free cyanobacterial crude extract containing MC was added into Daphnia medium In total, there were four different exposures: 5 and 50mg L1of purified MC-LR, and 5 and 50mg L1of total MC in crude extract, hereafter referred as M5, M50, E5 and E50 respec-tively The control was implemented twice (C1 and C2) because the food was changed between exposure M5 and the others Daphnids of C1 and M5 treatments were fed with green alga Scenedesmus vacuolatus Due to unavailability of S vacuolatus, daphnids were fed with another Scenedesmus species, Scenedesmus armatus for the other exposures (M50, E5 and E50 respectively) Consequently a new control (C2) was conducted in which daphnids were fed with S armatus

in order to get the same supply of nutrient quality Results of daphnids incubated in the same food regime were compared only (C1–M5; C2–M50, E5 and E50) Twice a week, half of toxic and non-toxic Daphnia medium was renewed whereas food density was kept constant on a daily basis Each expo-sure and control experiment lasted for two months 2.4 Life history traits

For life history study, the survival, growth, maturation, time to first reproduction and fecundity of mother Daphnia were observed daily for two months Simultaneously, the survival, growth and maturation of their offspring were also recorded for one week Death of the animal was defined as the stop of heartbeat confirmed by microscopic observation (Olympus TH3) Maturation of Daphnia was defined as time point of first egg occurrence in the brood chamber The time to first reproduction was at first offspring release from brood chamber during molting Fecundity of animals was recorded as the number of clutches and number of offspring per clutch produced by every mother Daphnia during exposure time In case of release of decomposed eggs, embryos or neonates, the offspring number of that clutch was assumed as zero Twice a week, the growth of test organisms was moni-tored by measuring the distance from top of the head to the base of tail spine (body length) by photographing the animals under a microscope (Olympus SZX7) equipped with a digital camera (Olympus XC50) The body length of Daphnia was measured exactly to 1mm based on the soft-ware DT5 analysis (Soft Imaging System – Olympus) Neonates (<24 h old, called F1 Daphnia) were randomly selected for survival, growth and maturation study of the offspring F1 Daphnia from control experiment were raised

in either non-toxic or in the four different kinds of toxic media Similarly, F1 daphnids from toxin exposed mother

D magna, were split in two groups: a) raised under continuous exposure in the same toxic medium and b) raised in control medium (Fig 1) One hundred F1

daphnids were selected for each treatment in which every

ten neonates were transferred into a beaker containing

100 mL of exposure or control medium Half of the medium

was renewed twice a week The animals were fed with

Scenedesmus ad libitum The survival and body length of

the offspring were monitored until maturation The

experiment terminated when the last offspring matured

2.5 Statistical analysis

Sigmastat, version 2.0, SPSS Inc was used for data

treatment One-way Analysis of Variance (ANOVA) and

Tukey test Post Hoc were applied to calculate statistically

significant difference of the body length of adult D magna

Kruskal–Wallis was applied for calculation of statistically

significant difference of the maturation, time to first

reproduction and body length of offspring D magna due to

lacking normality or distributed homogeneity of the

samples

3 Results

3.1 Mother D magna life traits

3.1.1 Survival

After two months of exposure, the concentration of

5 mg L1 MC-LR or total MC in crude extract slightly

decreased the survival of adult D magna, at about 10% of

total animals (Fig 2) However, those of 50mg L1caused

the reduction of 60 and 55% of total daphnids in M50 and

E50 treatments respectively The number of daphnids in

M50 and E50 were quite stable within the first three weeks

of incubation but quickly descended afterwards The

number of daphnids in C1 decreased 13% whereas that in

C2 did 37% during two months There was little difference

between the survival of C1 and M5 treatments

Unexpect-edly, the number of daphnids in C2 reduced more than

those in E5 by the end of exposure However, survival

proportion of M50 and E50 incubations was 23 and 17%

respectively lower than that of C2 incubation (Fig 2)

3.1.2 Growth performance

Animals in C1 and C2 were fed with different

Scene-desmus and those in C2 grew somewhat larger (Fig 3)

Daphnids of the treatments M5, M50 and E50 grew faster at

first but then they developed slower than the control animals, C1 and C2 It resulted in significantly longer bodies

of the M5, M50 and E50 treated daphnids compared to their controls during the first 4, 15 and 32 days respectively (ANOVA followed by Tukey’s test, p < 0.05;Fig 3A,C,D) During the age from 15 to 57 days daphnids in M5 were smaller with some fluctuation compared to those in control (Fig 3A) The animals in M50 and E50 treatments stayed significantly smaller than the control from the age of 39 and 43 days to the end of incubation (ANOVA followed by Tukey’s test, p < 0.05;Fig 3C,D) Contrastingly, the body length of one day old animals in E5 was smaller than that in C2 However, three days later to the end of exposure the daphnids in E5 grew faster and were significantly bigger than the C2 (ANOVA followed by Tukey’s test, p < 0.001;

Fig 3B) The data of E5, 32 days old and M50, 11 days old were missed due to camera failure

Fig 1 Experimental setup for the Daphnia magna from mothers (n ¼ 30) to offspring (n ¼ 100) incubated in non-toxic and toxic medium Mother Daphnia exposure to: C1 and C2, controls; M5 and M50, to 5 or 50mg L 1 of MC-LR; E5 and E50, to 5 or 50mg L 1 of total MC from cyanobacterial crude extract respectively Offspring (F1) exposure: neonates from each mother Daphnia were split into groups and either continuously exposed as their mothers or raised in control medium (C1 or C2) Offspring from control mother Daphnia were split and those from C1 raised in either C1 or M5, whereas from C2 raised in C2 or M50, E5 or E50.

Fig 2 Survival of mother Daphnia magna during exposure time (n ¼ 30 at the start) Abbreviation as in Fig 1

Fig 3 Body length of mother Daphnia magna (mean value  SD of n ¼ 30 at the start) during the incubation Asterisks indicate significant differences by ANOVA

3.1.3 Maturation

D magna exposed to pure toxin or crude extract at both

5 and 50mg MC L1reached their maturity around the age

of 6 days, significantly earlier than the control, which took

around 7 days (Kruskal–Wallis test, p < 0.01; Fig 4A)

Subsequently, the toxin exposed animals were significantly

younger at first reproduction (Kruskal–Wallis test,

p < 0.001) except that those of M5 and C1 had a similar age

at first reproduction (Fig 4B)

3.1.4 Fecundity

During two months of incubation, the mother D magna

produced 16–17 clutches of offspring The accumulative

number of neonates from the control daphnids was highest

in the end (Fig 5) Compared to the control accumulative

offspring, those from the M5, E5 and E50 raised higher

within the first 8, 4 and 12 clutches respectively followed

by a slower development In the E50 treatment,

reproduc-tion only marginally proceeded after the 10th clutch

(Fig 5) In total, the concentration of 5mg L1MC-LR or both

concentrations of MC in crude extract resulted in a

reduc-tion of around 10% neonates compared to the control The

concentration of 50mg L1MC-LR caused a decline of more

than 60% of offspring (Table 1)

3.1.5 Malformation

Exposures to the pure toxin or crude extract induced the

abortion of eggs/embryos via decomposition and death

already in the brood chamber of D magna (Fig 6A,B)

Furthermore, it caused the malformation of neonates

including the incomplete development of the antennae and

un-rejected tail spine (Fig 6C) The proportion of

mal-formed and dead neonates was higher in the M50 (7.1% and

0.7% respectively) and E5 (6.6% and 0.5%) incubations

compared to the M5 (0.3% and 0.09%) and E50 (0.05% and 0%) exposures (Table 1) The abortion proportion of exposed D magna was highest in M50 (21.9%), followed by E5 (15.1%), E50 (9.8%) and lowest in M5 (0.7%) Similarly, the percentage of aborting females was highest in M50 (83.3%), E5 (80%), E50 (66.7%) and lowest in M5 (10%) 3.2 Offspring Daphnia magna life traits

3.2.1 Survival The survival of offspring from control mother D magna ranged from 97 to 100% in both control and toxin expo-sures However, those of exposed mother daphnids suffered from mortalities within their first week post natal (death proportion of 6–47%), regardless, if raised in control medium or under continuing exposure (Fig 7) Survival proportion was lower if the offspring of pure toxin expe-rienced mothers were continuously incubated in toxin medium compared to raised in control medium In detail, the survival proportion of M5M5 (86%) was 8% lower than that of M5C1 (94%) Similarly, the survival proportion of M50M50 (53%) was 7% lower than that of M50C2 (60%) (Fig 7A) However, the E50E50 offspring survived slightly better than the E50C2 ones, 94% compared to 88% (Fig 7B) Only offspring of E5 mothers did not significantly decrease their survival after eight days of exposure (Fig 7B) Microcystin-LR had stronger negative effects on the survi-vorship of the offspring than the crude extract did Mortality rate of daphnids correlated directly to toxin concentration (Fig 7)

3.2.2 Growth performance Offspring of D magna previously exposed to pure toxin

or crude extract containing MC dealt with a significant decrease of their body length during the first week of incubation in non-toxic or toxic medium (Kruskal–Wallis test, p < 0.001;Fig 8) In addition, the growth of offspring

of mother daphnids previously exposed to 50mg MC L1

was more inhibited than that of mother exposed to

5 MCmg L1(Kruskal–Wallis test, p < 0.05;Fig 8) 3.2.3 Maturation

Offspring from D magna exposed to 5 and 50mg L1of MC-LR or total MC from crude extract significantly delayed

Fig 4 A, Maturation and B, Time to first reproduction of mother Daphnia

magna (mean value  SD of n as indicated in the columns), asterisks indicate

significant difference by Kruskal–Wallis test (**, P < 0.01; ***, P < 0.001).

Abbreviation as in Fig 1

Fig 5 Accumulative fecundity of mother Daphnia magna during exposure time as proportion of neonates in relation to the total neonates of control Abbreviation as in Fig 1

maturity age compared to their mothers, by 1–4 days

(Kruskal–Wallis test, p < 0.001;Fig 9)

4 Discussion

In this study, daphnids in each treatment were fed with

either S vacuolatus or S armatus at 4  106Scenedesmus

spp cells per daphnid per day (around 3 mg C L1), that is

a density providing surplus food, as cells were not

completely consumed every day Unexpectedly, differences

between the control groups occurred, as C2 grew bigger but

suffered lower survival rates for unknown reasons Other

differences were not significant Therefore, life trait

differ-ences between control and toxin exposed D magna were

compared within the groups fed with the same species of

Scenedesmus only and could then be attributed to the

cya-nobacterial toxins applied

The molecular mechanism of MC-LR toxicity is mediated

(MacKintosh et al., 1990) thereby leading to deregulation in

cells Furthermore, MC-LR has been found binding to ATP

synthetase beta-subunit affecting the energy sustaining

ability of the cells (Mikhailov et al., 2003) Another more

general effect of cyanobacterial compounds is via

provoking of oxidative stress (Wiegand and Pflugmacher,

2005) Cyanobacterial bioactive metabolites caused the

suppression of trypsin-like activities, which together with

chymotrypsin enzymes account for 75–83% of proteolytic

activities in Daphnia sp gut contents (Von Elert et al., 2004; Czarnecki et al., 2006) Furthermore, MC-LR inhibited D pulicaria filtering activity, hence impaired food uptake (Ghadouani et al., 2004) Summing up, cyanobacteria and their compounds decrease the energy re-establishing process in daphnids at various points: by malnutrition, by impairment of feeding and digestion, by cellular processes; all in turn will sum up to lower energy availability for maintenance, growth and reproduction (scope for growth) Under exposure to toxin, additional energy is spent for physiological reactions, such as biotransformation and antioxidant enzyme activities, toxin excretion, or mecha-nisms of repairing damages In our study a lower scope of growth was evidenced by growth inhibition, fecundity failure and mortality increase

Survival of daphnids in treatments with low toxin concentration was not much affected in our study This is in agreement with the investigation ofLu¨rling and Van der Grinten (2003)in which D magna exposed to 3.5mg L1

dissolved MC-LR showed no adverse effect However, at

a ten times higher concentration (50mg MC L1) survival of mother daphnids was strongly decreased by either pure MC-LR or cyanobacterial crude extract (Fig 2B) These records were in line with previous studies (Hietala et al.,

1995, 1997b; Lu¨rling, 2003; Semyalo et al., 2009) in which daphnids were treated with toxic cyanobacterial cultures Hence, chronic exposure to a concentration of 50mg MC L1,

a concentration that is about 400 times lower than the 48-h

Table 1

Fecundity of mother Daphnia magna and malformation and abortion of their offspring.

Exposure Total neonates

relative to control

Malformed neonates

Dead neonates Number of decomposed clutches Aborting females

M, empty carapace; Me, removed carapace with decomposed eggs/embryos inside; Mn, removed carapace with dead neonates.

Fig 6 Malformation of eggs/embryos and offspring Daphnia magna (arrows) A, decomposed eggs/embryos; B, dead neonates; C, normal neonate (left) and

LC50values (9.6–21.4mg MC-LR mL1DeMott et al., 1991), is

sufficiently potent to cause a declined survival over the life

span of D magna

In cyanobacterial crude extracts not only variants of MC

but also other cyanobacterial bioactive compounds

adversely affected daphnids (Reinikainen et al., 2001;

Wiegand et al., 2002; Rohrlack et al., 2004) In our study,

cyanobacterial crude extract contained five different MC

congeners of which MC-RR and MC-LR were mainly

dominant, gaining 71.09% and 24% of total MC respectively The daphnid survival was concentration dependently decreased by the cyanobacterial crude extract used, but not

as much as by the MC-LR treatment due to the lower toxicity and higher hydrophilicity of MC-RR compared to MC-LR (Atencio et al., 2008) Despite the concentrations of

50mg L1dissolved MC used in our study adversely affected daphnids’ biotransformation and antioxidant enzymes (Wiegand et al., 2002) the detrimental impact on their life history was only revealed after three weeks of exposure (M50 and E50 treatments) In contrast, during toxic Microcystis cell treatments Daphnia pulex, Daphnia long-ispina, D pulicaria, D magna and Daphnia lumholtzi started

to significantly decrease their survival already after the first week (Lampert, 1981; Hietala et al., 1995, 1997b; Trubetskova and Haney, 2006; Semyalo et al., 2009) However, if it could be considered that the average toxin content per cell of Microcystis is 0.2 pg (Falconer et al., 1999) and 1 mg C L1 of this species corresponds to around 1.4  105cells mL1(Gustafsson and Hansson, 2004) the concentration of MC in the cyanobacterial cultures used for exposures of previous investigations ranged from 0.24 to

28mg L1, which is within a similar range of MC concen-tration used in our study, 5 and 50 mg L1 Besides the specific toxic effects of cyanobacterial compounds, cyano-bacterial cells affect daphnids also by malnutrition and impairment of feeding The availability of green algae as alternative food source always increased survival in those studies

Survival of daphnids in E50 treatment strongly decreased whereas that in E5 only slightly declined from the third week to the end of exposure (Fig 2B) In F1 daphnids, cyanobacterial crude extract caused both death

Fig 7 Survival of offspring Daphnia magna during the first week of

incu-bation, starting with 100 neonates for each exposure Abbreviation as in

Fig 1

Fig 8 Body length of exposed mother Daphnia magna and their offspring (mean value  SD of n ¼ 30 for mother and 100 for offspring at the start) during the first week of incubation Asterisks indicate significant differences by Kruskal–Wallis test (***, P < 0.001) The symbol # indicates significant difference by Kruskal–

and malformation occurring from the third clutch of

mother daphnids In total, E50 treatment induced higher

mortality for mother daphnids but lower malformed

neonates and decomposed clutches than E5 Apparently,

the lower crude extract concentration weakened the

mother daphnids to the expense of their offspring, whereas

the higher directly affected the mother daphids survival

The M50 treatment not only affected survival of the mother

daphnids, but in addition the offspring suffered

malfor-mation and abortion

Although dissolved toxins were less harmful than

cell-bound toxins to daphnids, exposure to dissolved toxins as

in our study provides a more evenly contact to the animals

On the other hand, our results may extrapolate the toxicity

of dissolved cyanobacterial toxins to the survival of

daph-nids in nature because the animals are almost unable to

filter big colonies or long filaments of cyanobacteria in

blooms

Unexpectedly, our study revealed that daphnids in E5

incubation grew faster and significantly bigger than the

control animals during the exposure Possibly, the toxin

concentration of 5 mg L1 did not inhibit the animals’

growth, as also evidenced byLu¨rling and Van der Grinten

(2003), but beside toxic metabolites there might be

nutri-tional compounds in the crude extract contributing to

growth enhancement of the daphnids However, MC

concentration of 50 mg L1 was sufficiently potent to

suppress the growth of daphnids by the last weeks of

exposure Hence, despite growth stimulation at the

beginning of the treatment by cyanobacterial toxins it was

limited afterwards The inhibition of ingestion process and

activity of digestive enzyme trypsin in daphnids (Rohrlack

et al., 2003; Czarnecki et al., 2006) resulted in starvation

which induced adverse effects on the animals’ life traits

including the decrease of growth It could be inferred that

in our study the D magna might withstand the toxin at first

due to the constant Scenedesmus availability as food source

However, the animals were impaired by the toxin

after-wards Still the mechanisms by which daphnids in M50

grew faster than those in control within the first two weeks

of experiment remained unknown

Contrasting to other studies, where treatment with

toxic Microcystis delayed the age to first reproduction in D

pulex (Hietala et al., 1995), we found earlier maturation and start of reproduction in treated D magna Ebert (1992)

found that maturity of D magna is positively related to their body length and maturation is initiated above

a threshold size In our study exposed daphnids grew faster during the first weeks of incubation (Fig 3) Therefore, the exposed animals matured and reproduced sooner than the control according to the hypothesis of threshold size of

D magna

Our results are in agreement with previous investiga-tions evidencing a reduction or completely inhibition of daphnid reproduction due to cyanobacterial compounds (Lu¨rling, 2003; Lu¨rling and Van der Grinten, 2003; Gustafsson et al., 2005; Trubetskova and Haney, 2006) The M5, E5 and E50 adults had a reproducing reduction of around 10% of total neonates whereas the fecundity of M50 had a severe decrease of more than 60% of total offspring by the end of incubation.Gustafsson et al (2005)showed that offspring number of daphnids in a mixed food treatment (Microcystis and Scenedesmus, ratio of 5/95 biomass) were lower than those of control in every clutch In our study MC-LR at 50 mg L1depressed the reproduction during lifetime of the animals, whereas the other treatments had some fluctuation in the beginning, before fecundity decreased However, the total numbers of neonates of all exposures were lower than that of their controls

The decrease of offspring happened via two possibili-ties: (1) 10–83.3% of exposed mother aborted their eggs/ embryos (Table 1) and the abortion happened repeatedly in several females; (2) in addition to dead neonates in brood chamber or malformed neonates, incomplete development

of the antennae and un-rejected tail spine were observed in many offspring, which usually died some hours after birth Abortion of D magna were observed when they were treated with toxic M aeruginosa (Gustafsson et al., 2005) or fungicides such as prochloraz, fenarimol and pyrifenox (Hassold and Backhaus, 2009) In addition, malformations

of neonates were described after fungicide application (Hassold and Backhaus, 2009) Our study traced back malformations of D magna offspring to be caused by cya-nobacterial toxins

Gustafsson and Hansson (2004)found that offspring of toxic Microcystis exposed mother D magna survived better

if fed with Microcystis than offspring of mothers fed with Scenedesmus However, our study displayed a controversial result that more than 97% of offspring of control mothers survived the first week in toxic or non-toxic medium In contrast, offspring of toxin treated mothers suffered from mortalities even if reared in non-toxic medium Addition-ally, abortion of daphnids, decomposed eggs and embryos, and dead neonates were observed after toxin treatment It was commonly observed that egg proteins of exposed mother daphnids coagulated and eggs broke or decom-posed inside the brood chamber Hence, there must be adverse effects of MC already on the egg/embryos devel-opment in brood chamber.Wiegand et al (2009)proved that MC was not only absorbed by the gut cells but also occurred in developing eggs in the brood chamber of adult

D magna even within 48 h exposure to the toxin Possibly, the neonates of MC exposed mother daphnids suffered from toxic stress and energy cost for detoxification before

Fig 9 Maturation of exposed mother Daphnia magna and their offspring

(mean value  SD of n ranging between 28 and 97), asterisks indicate

significant difference by Kruskal–Wallis test (***, P < 0.001) n as indicated in

the columns Abbreviation as in Fig 1

they were released As a consequence they became more

vulnerable and their survival was decreased even if they

were raised in non-toxic medium

In exposure scenarios with cyanobacterial cells, the

amount of toxin that reaches physiological targets in

daphnids may be lower than the total available cell-bound

toxin of the cyanobacteria because of decreased food

filtering and impaired digestion (Rohrlack et al., 2001;

Czarnecki et al., 2006) Concentrations of cell-bound MC

used for pre-trial exposure to daphnids in the study of

Gustafsson and Hansson (2004) were increased from

1.2mg L1at the first week to 4.8mg L1at the fourth week

of incubation Differently, daphnids in our experiments

were constantly exposed to 5 and 50 mg L1 starting at

neonate stadium Possibly, slowly increasing toxin

concentration from 1mg L1to 4.8mg L1induced maternal

effects (Gustafsson and Hansson, 2004; Gustafsson et al.,

50mg L1MC from new born age might be already too high

In addition, out of the three clones of daphnids tested by

Gustafsson and Hansson (2004), only two developed

tolerance against toxic Microcystis Therefore, different

toxin concentrations at start of exposure and clone

susceptibilities to toxin could be the root of the different

results

Contrasting toGustafsson et al (2005), where offspring

of previously exposed D magna to toxic Microcystis

increased their fitness, attributed to genetic inheritance,

our study revealed significant and concentration

depen-dent decrease of body length (Fig 8) and increased

dura-tion to maturity (Fig 9) of F1 of exposed mother daphnids

The reduction of fitness in our case might be caused by

damage of offspring before hatching as mentioned above

A good evidence for that is that the offspring developed

slower and suffered from mortalities even if raised in

non-toxic medium

Despite exposed mother D magna reached maturity

earlier than the control, the offspring of exposed animals

significantly delayed the maturation compared to their

mother, due to slower growth so that they later reached the

threshold size of maturation according to the hypothesis of

Ebert (1992) The postponement of maturation raises an

additional vulnerability of sustainable population in the

field, e.g by predation

In summary, chronically exposed to MC-LR or

cyano-bacterial crude extract the D magna clone used in our study

strongly decreased survivorship from generation to

generation without inheritance of toxin tolerance In

addition, development of the exposed animals was

stimu-lated at first but inhibited afterwards However, the

explanation for the growth stimulation remains unknown

Besides, maturation and time to first reproduction was

reached sooner in exposed mother daphnids, but total

fecundity was decreased Egg/embryo and neonate

devel-opment in brood chamber of D magna was strongly

influ-enced by cyanobacterial toxin inducing abortion or

malformation of neonates The offspring of previously

exposed D magna were not only repressed in development

and delayed in maturation compared to their mothers; they

also suffered mortalities, even if raised in non-toxic

medium

In consequence, population and community of this investigated D magna would decrease or become rare if continuously affected by cyanobacterial toxins in nature Maternal effects of daphnids could be induced with cya-nobacterial toxin concentration of around 1 mg L1 (Gustafsson et al., 2005) but might not be at 5–50 times higher concentration at the neonate stadium exposure Study on chronic effects of dissolved cyanobacterial toxins

on different clones of D magna should be implemented as daphnid clones have different susceptibilities to the toxins Further investigations are in need for understanding the mechanisms of abortion and malformation in daphnids caused by cyanobacterial toxins

Acknowledgement The authors would like to thank Dr Jorge Nimptsch, Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany, for providing biomass of toxic Microcystis aeruginosa culture and preparing cyanobacterial crude extract for this study Rafael Ortiz, IGB, is acknowledged for discussion on data treatment Thanh Son Dao’s financial support by The Swiss Agency for Development and Coop-eration Project is gratefully acknowledged

Conflict of interest None

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