Parts of this chapter have previously been published in Ernst et al. (2001): Presence of Planktothrix sp. and cyanobacterial toxins in lake Ammersee, Germany and their impact on whitefish (Coregonus lavaretus L.). Environmental Toxicology 16, 483-488
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BSTRACTExperimental investigations into the ichthyotoxicity of microcystin-containing Planktothrix rubescens suggest that coregonid exposure to P. rubescens can cause physiological stress and pathological damage, substantially affecting coregonid fitness and elevating mortality rates.
Adverse effects have been shown for P. rubescens densities known to occur in pre-alpine lakes, thus providing a possible explanation for recurrent slumps in coregonid yields observable in several of those lakes, e.g. in Lake Ammersee. Hence, this study aimed to elucidate whether there is evidence for P. rubescens exposure of coregonids in Lake Ammersee. Feral coregonids were obtained between 2001 and 2004 from monthly catches carried out with gill nets of various mesh sizes. The sample cohort was completed by samples from catches of local fishermen, including samples taken during bloom episodes in 1998 and 1999. Fish were investigated microscopically for accumulation of ingested P. rubescens filaments within the intestine. Gut contents were analysed for cyanobacterial biliprotein and gut contents as well as liver homogenates were analysed for microcystin accumulation. P. rubescens filaments were observable in the coregonids gut contents giving evidence for P. rubescens exposure of coregonids in Lake Ammersee. The results demonstrate this exposure to cause an accumulation of P. rubescens components within the coregonids intestine, as 8% of the investigated coregonids showed prominent blue colouration of gut contents probably resulting from a significant accumulation of cyanobacterial biliproteins.
From the coregonids sampled during bloom episodes in August 1998 and April 1999, four out of ten contained significant microcystin accumulations, which unambiguously demonstrate microcystin exposure of feral coregonids in Lake Ammersee. The detection of covalently-bound microcystin in liver tissue furthermore demonstrates microcystins to traverse the ileal membrane and to accumulate in the liver. This makes substantial detrimental effects on the coregonids appear inevitable and thus substantiates the initially proposed suggestion of a causal relationship between P. rubescens mass occurrence and challenged coregonid populations in pre-alpine lakes such as Lake Ammersee.
KEYWORDS: Cyanobacteria; Planktothrix; Microcystin; Coregonids; Fish
I
NTRODUCTIONOccurrences of Planktothrix rubescens blooms in pre-alpine lakes have been observed to coincide with recurrent slumps in coregonid yields causally associated with reduced fish weight and fitness (Braun, 1953; Ernst et al., 2001). The current knowledge on the ichthyotoxicity of cyanobacteria in general (summarised in Malbrouck & Kestemont, 2006) and recently published information specifically on the toxicity of P. rubescens on coregonids (Ernst et al., 2007; Ernst et al., 2006a), suggest that coregonid exposure to microcystin-containing P. rubescens can cause enhanced physiological stress as well as continuous organ damage. Elevated susceptibility to ectoparasitic infestations and increased mortality in coregonids experimentally exposed to P. rubescens filaments additionally corroborate effects on the coregonids fitness (Ernst et al., 2007). Hence, a causal relationship between the occurrence of P. rubescens containing microcystin and changes in growth and population dynamics of coregonids appears likely and provides a possible explanation for recurrent slumps in coregonid yields in pre-alpine lakes, such as in Lake Ammersee.
Compared to other cyanobacteria, Planktothrix sp. have been shown to contain very high amounts of microcystin per gram dry weight, primarily consisting of various demethylated variants of MC-RR (Blom et al., 2001; Ernst et al., submitted; Fastner et al., 1999a; Kurmayer et al., 2005). This has been shown to apply also to Lake Ammersee (Ernst et al., submitted), where more than 50% of monthly surveyed seston samples contained microcystin, the concentration of which correlated with the abundance of P. rubescens, thus indicating that in Lake Ammersee the appearance of P. rubescens coincides with measurable microcystin concentrations. This was confirmed by the observation of a concurrence between microcystin and biliproteins specific to cyanobacteria as demonstrated by a correlation of microcystin amounts and the seston sample phycoerythrin content (Ernst et al., submitted).
In Lake Ammersee, P. rubescens has been shown to persist over several years (Ernst et al., submitted), whereby P. rubescens filaments were evenly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer. Adverse effects on coregonids have been shown experimentally for P. rubescens cell densities greater than 1500 cells/ml (Ernst et al., 2007). Such P. rubescens cell densities were observed in Lake Ammersee during 47% of the total observation period of 261 weeks (Ernst et al., submitted), including periods with filaments distributed over the entire water column. This consequently implies that exposure levels employed in the laboratory study occur naturally in Lake Ammersee.
The aim of this study thus was to determine whether there is evidence for P. rubescens exposure of feral coregonids in Lake Ammersee, via examination of the uptake of toxic P. rubescens and microcystin accumulations in wild catch coregonids.
M
ATERIAL&
METHODSInvestigation of the Gut Content of Lake Ammersee Coregonids
Gut contents of Lake Ammersee coregonids obtained from random samples from catches of the local fishery cooperative were examined microscopically for P. rubescens filament accumulation.
From June 2001 to December 2004, Lake Ammersee coregonids were further regularly examined for elevated biliprotein (i.e. phycocyanin and allophycocyanin) concentrations in gut content.
Required coregonids were obtained from monthly catches carried out (in cooperation with the fisheries advisory board of Upper-Bavaria, Germany) with gill nets in the pelagic zone of the lake.
Fish were caught in nets with mesh sizes of 20, 25, 30, 35, 40 and 45 mm (length: 100 m per mesh size) and thus representative of the coregonid population structure in the lake (with the exception of yearlings which are normally caught in mesh sizes <20 mm). Previous coregonid gut content samples, taken during bloom episode in August 1998 and April 1999, were included in the sample cohort.
At least six individuals per month (maximum 14 fish) were assessed, except for December 2002 (4 fish), April 1999 (2 fish), March 2003 (3 fish), March (5 fish) and October 2004 (4 fish), as at these time points the gut contents of the majority of coregonids were insufficient to allow accurate assessment. In December 2001, January 2003, February 2003, January 2004 and February 2004 guts of the coregonids were totally empty and thus no assessment could be carried out.
Coregonids were dissected and gut contents were removed from the intestine, dried via speed vac evaporation and stored at –20 °C and darkness until extraction and biliprotein analysis.
Biliprotein concentrations in the lyophilised gut content samples were determined via extraction of defined sample quantities (≤250 mg dw) in phosphate buffered saline (≤30 ml/mg dw) by two freeze-thaw cycles using liquid nitrogen. Each extract was centrifuged (15 min, 16,000 x g) and the absorption (A) of the resulting supernatants was determined at wavelengths of 615 nm and 652 nm. Absorption was additionally determined at 750 nm for nullification (N). The optical density for the respective wavelength (ODxxx) was calculated as ODxxx = A – N. Phycocyanin (PC) and allophycocyanin (APC) concentrations were calculated according to the description of Tandeau de Marsac (1977) using the following equations:
PC [mg/l] = (OD615 – 0.747 x OD652) / 5.34 APC [mg/l] = (OD652 – 0.208 x OD615) / 5.09
The analytical protocol provided for a quantification limit of ≥0.9 µg/mg dw and ≥0.75 µg/mg dw for phycocyanin and allophycocyanin, respectively. The procedure was carried out once for each gut content sample.
The gut content samples, taken in August 1998 and April 1999, were additionally analysed for microcystin contamination. Gut content extracts were obtained by sonication in 100% methanol (60 min) and subsequent centrifugation (68,000 x g, 60 min). Methanol was removed via
speed-vac evaporation and the resulting extract re-dissolved in water for further purification by solid phase extraction (see Ernst et al. 2005). The resulting eluents were finally dissolved in a defined volume of water. Microcystins were quantified via anti-Adda ELISA in comparison with internal MC-LR standards (Alexis, Switzerland) and given as the microcystin-LR equivalent (MC-LRequiv.) concentration. The MC-LRequiv. concentration in Lake Ammersee coregonid gut contents were compared using a one-way ANOVA and Tukey’s multiple comparison test. Gut content samples were classified to be microcystin-positive when significant elevations were determined at the p <0.05 level.
Determination of Microcystin-Adducts in Liver Homogenates
Lake Ammersee coregonids were additionally investigated for microcystin accumulation in liver via random examination of fish liver homogenates. Fish, caught in August and November 1998, as well as April and August 1999, were dissected and liver tissue was homogenised in extraction buffer containing 1 mM PMSF, 5 mM EDTA, 1 mM DTT, 140 mM NaCl, 1% Triton X-100 and 10 mM Tris (pH 7.5). For qualitative detection of covalently-bound microcystin adducts, liver homogenates were separated via 10% SDS PAGE in accordance with Laemmli (1970). Separated proteins were transferred onto a nitrocellulose membrane via Western blot technique. The membranes were blocked using TTBS + 1% BSA for 30 min and MC-LR adducts were detected via incubation with polyclonal sheep anti-Adda serum (diluted 1:1000 in blocking buffer; see also Fischer et al., 2001) at room temperature for one hour according to Fischer & Dietrich (2000).
Membranes were washed using TTBS (3x5 min) and incubated with secondary antibody (anti sheep IgG-AP, Sigma-Aldrich, Germany, diluted 1:5000 in TTBS) at room temperature for one hour. After washing with TTBS (3x5 min) and TBS (1x15 min), specific bands were visualised using AEC chromogen (BioGenex, USA) according to the manufacturer’s instructions. The molecular weights of detected adducts were estimated by comparison with full range rainbow marker proteins RPN 800 (Amersham, UK).
R
ESULTSP. rubescens filaments were observable in blue coloured gut content of Lake Ammersee coregonids (Fig 4.10). Coregonids showing prominent blue colouration of gut contents contained biliprotein (i.e. phycocyanin and allophycocyanin) concentrations above the respective limits of quantification. Altogether, 8% of the analysed gut content samples (n=289) contained significant amounts of biliproteins. The 95% confidence interval of the mean phycocyanin (PC) and allophycocyanin (APC) concentrations determined in biliprotein-positive gut contents were 1.5-2.6 µg/mg and 1.2-2.2 µg/mg for PC and APC, respectively. The highest biliprotein concentrations detected were 5.4 µg PC/mg and 5.7 µg APC/mg in a single coregonid caught in May 2002. Biliproteins were observed in gut content samples in all years investigated, whereby gut content samples never contained biliproteins during November, January and February, but
Fig. 4.10: Lake Ammersee coregonid (a) and copepod from a Lake Ammersee seston sample (b) containing conspicuous blue coloured gut content (inserts). Microscopical examination of gut contents (c & d) further demonstrates ingestion of P. rubescens filaments (P) by Lake Ammersee coregonids and indicates biliprotein release during filament decomposition within the coregonids intestine (d).
regularly and with highest frequency in April and May (Tab. 4.4). A sporadic microscopical investigation of seston samples demonstrated that in Lake Ammersee also copepods may exhibit conspicuous blue gut colouration (Fig. 4.10).
From the coregonids sampled during the bloom episodes in August 1998 and April 1999, four out of ten samples contained significantly elevated microcystin levels corresponding to a mean concentration of 30 ±3 µg MC-LRequiv./g dw (Fig. 4.11).
Tab. 4.4: Monthly catches showing coregonids in Lake Ammersee with (+) and without (–) biliprotein-positive gut contents. Sampling was carried out from June 2001 to December 2004. Previous coregonid gut content samples, taken during bloom episodes in August 1998 and April 1999, were included in the sample cohort
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
-1999 +1998
2001 + - + + - - -
2002 - - + + + - - - +
2003 - - + + + - - - - + - -
a b
d c
P P 200 µm
50 µm 25 µm
2 cm
Fig. 4.11: Determination of microcystin in blue coloured gut contents of Lake Ammersee coregonids caught during Planktothrix bloom episodes in 1998 and 1999 (error bars = 95% CI, n = 10). Gut content samples were classified as microcystin-positive when significant elevations were determined at the *p <0.05 significance level using ANOVA and Tukey’s Multiple Comparison test.
Previously published inEnvironmental Toxicology 16: 483-488, 2001
Qualitative detection of covalently bound microcystin in liver tissue of Lake Ammersee coregonids sampled during bloom episodes in August 1998 and 1999 furthermore revealed microcystin adducts in liver homogenates. Those adducts had molecular weights between 28 and 39 kD, which is characteristically for microcystin covalently bound to protein phosphatases (Fig. 4.12).
D
ISCUSSIONThe documentation of P. rubescens filaments in gut content of Lake Ammersee coregonids demonstrates that feral coregonids indeed ingest P. rubescens from seston, thus giving evidence for natural exposure of coregonids to P. rubescens in Lake Ammersee.
Once ingested it appears likely that P. rubescens cells were ruptured by the coregonid digestive process. However, rupture of cyanobacterial cells in the intestine of fish has been demonstrated to vary between different cyanobacteria species with cells of various Aphanizomenon sp. being almost totally broken while others, predominantly cyanobacteria species with cell walls including
Fig. 4.12: Detection of microcystin-binding-protein adducts in liver homogenates of Lake Ammersee coregonids by immunoblotting. Samples of August 1998 and 1999 show bands with a molecular weight of 38 kD which is characteristically for microcystin covalently bound to protein phosphatases.
Aug 98 Marker protein Nov 99 Apr 99 Aug 99
38 kD 33 kD 29 kD 0,00
0,01 0,02 0,03 0,04 0,05
Aug 98 - 1 Aug 98 - 2 Aug 98 - 3 Aug 98 - 4 Aug 98 - 5 Aug 98 - 6 Aug 98 - 7 Aug 98 - mix Apr 99 - 1 Apr 99 - 2
µg MC / mg
* * * *
//
50 40 30 20 10 0 µg MC-LRequiv. /g dw
an exopolysaccharide sheath (e.g. Microcystis sp.), remaining largely intact (Carbis et al., 1997;
Cazenave et al., 2005; Gavel et al., 2004; Kamjunke et al., 2002a; Kamjunke et al., 2002b; Lewin et al., 2003). P. rubescens lacks a mucilaginous sheath (Anagnostides & Komárek, 1988;
Feuillade, 1994) and should thus in principal be susceptible to rupture in the fish intestine.
The results of this study confirm the breakdown of P. rubescens filaments within the gastrointestinal tract of feral coregonids, as demonstrated by the regular detection of intracellular biliproteins specific for cyanobacteria (i.e. phycocyanin and allophycocyanin) in the gut content.
This indicates that P. rubescens exposure of Lake Ammersee coregonids indeed results in a release of cyanobacterial components within the coregonid intestine thus allowing exposure of coregonids to toxic P. rubescens components.
Analyses of coregonid gut contents further demonstrated that gut contents showing prominent blue colouration contained biliprotein concentrations above the respective limits of quantification.
Corroborated by microscopical examinations, thus, the blue colouration of gut content appears to be predominantly caused by the presence of cyanobacterial biliproteins, released following the rupture of filaments within the gastrointestinal tract of coregonids. As such, this may represent a marker for the exposure of feral coregonids to P. rubescens.
The occurrence of significant biliprotein concentrations in Lake Ammersee coregonid gut contents and thus exposure of coregonids to P. rubescens components varied with season. Indeed, no biliprotein-positive gut content samples were observed in samples taken in November, January and February. This appears to be due to the naturally reduced activity and lower food intake due to spawning behaviour and slowed winter metabolism. This is in agreement with previous observations (Enz et al., 2001; Mookerji et al., 1998; Skurdal et al., 1985) and is additionally corroborated by the predominantly empty coregonid guts during the winter months. In contrast to this, biliproteins were observable in the coregonids gut contents with highest frequency and concentration during April and May. This applied to all years investigated. Interestingly, this was also true for 2002 and 2003, when P. rubescens cell densities in Lake Ammersee were very low and never exceed 3000 cells/ml (Ernst et al., submitted). The field observations carried out in this study thus demonstrate that biliprotein accumulations in coregonids gut content occurred independently from annual variations in P. rubescens abundances and thus to a certain extent also independent of prevailing P. rubescens cell densities. This indicates that P. rubescens exposure of Lake Ammersee coregonids is not solely dependent on P. rubescens abundance and that biliprotein accumulation in gut contents and accordingly P. rubescens exposure of Lake Ammersee coregonids might also originate from sources other than direct ingestion of P. rubescens.
An additional source for the incorporation of P. rubescens components could be copepods that may temporarily play an important role in the coregonid diet (Enz et al., 2001; Mayr, 1998). As observations of this study demonstrate that not only Lake Ammersee coregonids, but also copepods in Lake Ammersee seston samples exhibit conspicuous blue gut colouration, it is likely
that copepods also accumulate P. rubescens components within their digestive tract. It thus appears possible, that Lake Ammersee coregonids may be also exposed to P. rubescens components via feeding on copepods.
The determination of biliproteins in gut content of Lake Ammersee coregonids suggests that other cyanobacterial components, including toxic metabolites (e.g. microcystins), may also accumulate in the digestive tract of exposed coregonids. This was confirmed by the detection of significantly elevated microcystin levels in coregonid gut contents sampled during P. rubescens bloom episodes in 1998 and 1999 which are in agreement with previous investigations demonstrating microcystin contaminations in faeces of cyanobacterial exposed fish (Chen et al., 2007; Xie et al., 2004). These results thus prove that feral coregonids in Lake Ammersee are exposed to P. rubescens and in consequence also to microcystins.
Microcystin exposure of Lake Ammersee coregonids was additionally confirmed by the presence of Adda-positive bands in liver homogenates indicating the presence of covalently-bound microcystin adducts in livers (Fischer & Dietrich, 2000; Hitzfeld et al., 1999; Mikhailov et al., 2003). These hepatic microcystin adducts furthermore revealed that microcystins are not only released within the coregonid intestine, but also traverse the ileal membrane and enter the coregonids metabolism. The exposure of feral Lake Ammersee coregonids to microcystin containing P. rubescens can thus account for the experimentally observed toxicological effects (i.e.
physiological stress and organ pathology, effects fish growth and fitness and enhanced fish mortality) previously reported (Ernst et al., 2007; Ernst et al., 2006a).
In summary, this study gives evidence for naturally occurring exposure of coregonids in Lake Ammersee to P. rubescens. The results demonstrate this exposure to cause an accumulation of P. rubescens components within the coregonid gastrointestinal tract, by direct ingestion and subsequent rupture of P. rubescens filaments and/or feeding of coregonids on copepods which have accumulated P. rubescens components. The P. rubescens components accumulating within the coregonids intestine have been shown not only to include biliproteins, which apparently cause a prominent blue colouration of coregonid faeces, but also microcystins. This unambiguously demonstrates the exposure of feral coregonids in Lake Ammersee to ichthyotoxic microcystins.
Since microcystins have also been demonstrated to cross the ileal membrane and to accumulate in the liver of Lake Ammersee coregonids, substantial detrimental effects on coregonids seem inevitable.
The results presented here thus substantiate the initially-proposed suggestion of a causal relationship between P. rubescens mass occurrence and challenged coregonid populations in pre-alpine lakes such as Lake Ammersee.