Microb Ecol DOI 10.1007/s00248-014-0541-z
SOIL MICROBIOLOGY
The Effects of Temperature Variation on the Sensitivity to Pesticides: a Study on the Slime Mould Dictyostelium discoideum (Protozoa) Andrea Amaroli
Received: 16 April 2014 / Accepted: 25 November 2014 # Springer Science+Business Media New York 2014
Abstract Slime moulds live in agricultural ecosystems, where they play an important role in the soil fertilization and in the battle against crop pathogens. In an agricultural soil, the amoebae are exposed to different stress factors such as pesticides and weather conditions. The use of pesticides increased up from 0.49 kg per hectare in 1961 to 2 kg in 2004, and the global greenhouse gas emission has grown 70 % between 1970 and 2004 leading to a global fluctuation of average surface temperature. Therefore, the European Directive 2009/128/EC has led to a new approach to agriculture, with the transition from an old concept based on high use of pesticides and fossil fuels to an agriculture aware of biodiversity and health issues. We studied the effects of temperature variations and pesticides on Dictyostelium discoideum. We measured the fission rate, the ability to differentiate and the markers of stress such as the activity and presence of pseudocholinesterase and the presence of heat shock protein 70. Our results highlight how the sensitivity to zinc, aluminium, silver, copper, cadmium, mercury, diazinon and dicofol changes for a 2 °C variation from nothing/low to critical. Our work suggests considering, in future regulations, about the use of pesticides as their toxic effect on non-target organisms is strongly influenced by climate temperatures. In addition, there is a need for a new consideration of the protozoa, which takes into account recent researches about the presence in this microorganism of classical neurotransmitters that, similar to those in animals, make protozoa an innocent target of neurotoxic pesticides in the battle against the pest crops.
A. Amaroli (*) Dipartimento di Scienze della Terra dell’Ambiente e della Vita (DISTAV), University of Genoa, Corso Europa 26, 16132 Genoa, Italy e-mail:
[email protected]
Keywords Soil protozoa . Integrated pest management . Heavy metals . Cholinesterase activity . Heat shock protein . Organophosphate pesticides
Introduction In the last years, the government regulation of agriculture paid special attention to the transition from an old concept of agriculture based on high use of both pesticides and fossil fuels to an agriculture more and more aware and careful to biodiversity and health issues. The use of pesticides in the agricultural field increased precipitously from 0.49 kg per hectare in 1961 to 2 kg in 2004 [1], and the global greenhouse gas emission has grown 70 % between 1970 and 2004, leading to a global change of average surface temperature [2]. In Europe, the summer average temperature has increased as well as the number and duration of heatwaves [2]. Supporting the future decisions of administrations about the integrated pest management following the directive 2009/128/ EC [3] and understanding the effects of the new environmental scenarios, generated by anthropogenic activities, on organisms and ecological communities are the great challenges of the current biological and ecological researches. Though during the past decades the focus on these scenarios has increased, there is still scanty information about the effects of co-exposures to these environmental perturbations. In this context, knowledge of the possible effects of both climate change and the use of pesticides on agricultural soil organisms is particularly relevant because of agriculture impact on both economy and public health issues. The soil is a very dynamic system where biological activities and nutrient cycles are carried out by soil organisms, sustaining terrestrial ecosystems. In fact, the soil organisms play a major role in both soil formation and soil structural development forming a biotic pore and a basic role in the nutrient cycling as primary
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producers of organic materials (e.g. phototrophic algae and bacteria) or, more commonly, as heterotrophic consumers of pre-formed organic materials [4]. The soil organisms can generally be subdivided into three groups, micro-, meso-, and macro-fauna, but the very small soil organisms could be defined “the nano-fauna”, analogous to nanoplankton [5]. Soil organic composition is mainly the product of micro- and nano-fauna (bacteria and protozoa) activity [4] because, if compared to the meso- and macro-fauna, the micro- and nanofauna consume more food per mass unit, have a shorter generation and lifetime and reproduce faster [6]. Traditionally, soil protozoa are classified as flagellates, ciliates, testate amoebae and naked amoebae. In the agricultural soil, ciliates and testate amoebae have gained more attention, probably because they are larger so easier to handle and examine [6]. Nevertheless, heterotrophic flagellates and naked amoebae, including the sporulating groups of slime moulds, constitute the most important part of the soil food system because they are able to enter into pore necks as small as 3 μm, consuming bacteria and fungi in narrow spaces where larger predators are unable to enter [5]. One of the most studied sporulating naked amoebae is the slime mould Dictyostelium discoideum. Despite that D. discoideum is not a specific soil agricultural organism, other dictyostelid genera live in this ecosystem [7] and the single-cell amoebae stage in the life cycle of slime moulds resembles other naked amoebae both in morphology and ecology in general [5]. D. discoideum combines a very simple organization with a complex developmental cycle. The life cycle of D. discoideum starts from the reproduction phase by binary fission of single-cell amoebae feeding on bacteria. Depletion of the food source triggers the developmental phase that culminates with the formation of a multicellular fruiting body in which the apex is the spores [8, 9]. Up to now, the laboratory culture procedures are more time standardized [10, 11], making D. discoideum a reliable model for our investigation. In previous works about this organism, we showed the presence of pseudocholinesterase activity, named propionylcholinesterase (PrChE) [12, 13]. We assumed it was involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation of the developmental phase of life cycle [12, 14, 15]. A remarkable finding of our investigations was that like the cholinesterase activities of the higher organisms, the variation of PrChE activity was found to be a valuable stress index that could represent a general biomarker of stress induced by environmental perturbations [16–19]. For this reason, in this work, we chose D. discoideum to study the effects of temperature variations and pesticides used worldwide for agriculture pest control and potentially harmful for non-target wildlife organisms. We measured the fission rate, the ability to eat bacteria and to culminate in a fruiting body formation as well as the marker of stress such as the PrChE activity and the presence
of a molecule immunologically recognized by anti-BChE and anti-HSP70 antibodies.
Methods D. discoideum Reproduction and Development The developmental cycle of D. discoideum includes two phases. The reproduction phase consists of growth and multiplication by binary fission of single-cell amoebae feeding on bacteria. Nutrient starvation triggers the developmental phase. The amoebae aggregate in streams and migrate, creating the fruiting body, anchored to the substratum, where two different cell populations are differentiated, the somatic-like stalk cells and the generative spore cells. The phase of reproduction was obtained in a laboratory, according to the following procedure. The growth and multiplication were induced by inoculating one eighth of the fruiting bodies, grown on a Petri dish, 9-cm diameter, into a 50-ml sterile Falcon flask containing 15 ml of AX-2 axenic medium. The culture was later transferred into an Excella E24 incubator shaker at 20 °C and kept shaken at 150 rmp. The developmental phase was induced by transferring eight drops, each containing 10 μl of the aforementioned AX-2 culture, onto an Escherichia coli B2 monolayer growing on nutrient agar-N plates. The plates were incubated in a moist chamber for 2 days at 20 °C. When the fruiting bodies had developed, the plates were kept at 6 °C [10]. Detection of Temperature Variation Effect on Fission Rate The 15 ml of cells in the reproduction phase was diluted into a 500-ml sterile flask containing 200 ml of AX-2 axenic medium. At a concentration of 5×104 cells/ml, the culture was subdivided into smaller volumes of 10 ml, experimental samples. The experimental samples were exposed for 72 h to the following temperatures: 4, 6, 8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 32 or 34 °C. An experimental sample was exposed at the temperature of 20 °C for 72 h and utilized as control. All the experiments were conducted for 72 h, and the cell density was determined every 24 h by counting up the number of cells using a Neubauer chamber. The binary fission rate was computed as log2 nx/n0, where nx and n0 were the cell densities checked, respectively, at time 24 h (t24h) and t0h, t48h and t24h, and t72h and t48h. Detection of Pesticide Effect on Fission Rate The 15 ml of cells in the reproduction phase was diluted into a 500-ml sterile flask containing 200 ml of AX-2 axenic medium. At a concentration of 5×104 cells/ml, the culture was subdivided into smaller volumes of 10 ml, experimental samples. The experimental samples were exposed for 72 h at the
Temperature Variation and Slime Mould Sensitivity to Pesticides
temperature of 20 °C to the following pesticides: cadmium chloride (10 μM), mercury(II) chloride (1 μM), silver nitrate (5 μM), zinc sulphate (10 μM), aluminium potassium sulphate (5 μM), copper sulphate (10 μM), dicofol (0.1 μM) and diazinon (0.1 μM). An experimental sample was exposed at the temperature of 20 °C, without pesticides, for 72 h and utilized as control. All the experiments were conducted for 72 h, and the cell density was determined every 24 h by counting up the number of cells using a Neubauer chamber. The binary fission rate was computed as log2 nx/n0, where nx and n0 were the cell densities checked, respectively, at time t24h and t0h, t48h and t24h, and t72h and t48h. The pesticides were diluted in AX-2 medium or DMSO according to their water solubility. DMSO at the concentrations used in our experiment ( M
20°C
32°C
34°C
Hg
8°C+Cd 30°C+Cd 30°C+Cu
Fig. 6 BChE-related molecules evidenced by immunoblot analysis. M marker, 20°C control
120 100 80 60 40 20 0
b
O.D. HSP70
O.D. BChE
Fig. 5 Image analysis on BChErelated molecules (a) and HSP70related molecules (b) evidenced by immunoblot analysis. OD optical density
32 and 34 °C, to form fruiting bodies during the 360 h of observation. The formation of the fruiting body is a crucial step of the Dictyostelium developmental cycle that consists in the differentiation of its resistance form, the spore. Therefore, a delay or an impediment in the spore formation exposes amoebae to greater stressful conditions or death. The amoebae appear particularly sensitive to high temperatures, in agreement with the results on the markers of stress such as activity and presence of pseudocholinesterase and presence of HSP70. In fact, low temperatures do not affect the activity and the presence of pseudocholinesterase and reduce the presence of HSP70 while high temperatures lead to an increase in the activity of pseudocholinesterase linked to the formation of heavy forms of the enzyme and to the increase in the presence of HSP70. It is known that the increase and/or the formation of heavy forms of cholinesterase is correlated to alterations of the cell growth and induction of cellular death [15, 24, 25]. The HSP70 is known to be a stress factor related to warm temperature. Dictyostelium shows an apparent resistance to Zn, Ag, Al and Cu at the concentration tested. This ability of Dictyostelium can be related to its various detoxification systems, such as catalase, glutathione, superoxide dismutase [26], Cu-ATPase [27] and HSP [28]. However, Cd, Hg, diazinon and dicofol affect the fission rate and the cell ability to eat and differentiate. The pesticides have different ways of action, affecting target proteins and ion channels or inducing general cytotoxic effects. It is known that Cd and Hg have a toxic effect via alteration of calcium flow [29, 30] as well as the ability to bind the sulphhydryl groups of proteins [31], altering them, and in the case of cholinesterase, to form heavy chains of the enzyme [32]. So, the Cd and Hg effect could be related to the important role of both calcium [33] and pseudocholinesterase [12–14] in the developmental phases of D. discoideum. Diazinon is a non-systemic organophosphate, and its effects observed in our work seem to be related to its specific action as a cholinesterase inhibitor. In fact, the amoebae exposed to diazinon show a decrease in the pseudocholinesterase activity and a reduction in both fission rate rhythm and differentiation cycle. This data is in accord to previous results that showed a correlation between alteration of Dictyostelium pseudocholinesterase and delay in the two phases of developmental cycle [12, 14]. The dicofol is an organochlorine pesticide chemically related to DDT. Our
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results show that it affects the D. discoideum developmental cycle like diazinon but not by cholinesterase alteration. This data is not surprising because it is known that in animals, dicofol acts on the nervous system by inhibition of ATPase [34]. In nature, the amoebae are exposed to different environmental stress factors. Our data show that the effect of pesticides changes drastically by varying the temperature at exposure. Temperatures below 14 °C increase the effect of Cd, Hg, diazinon and dicofol, as well as temperatures below 12, 10 and 8 °C make the amoebae sensitive to Cu, Al, Zn and Ag, respectively. In particular, the transition from 8 to 6 °C determines the death of the entire cell population if exposed to pesticides. This reduced resistance to pesticides may be due to an effect of temperature on detoxification systems, as observed in our data about HSP70 that reduces its presence at low temperatures. Even more evident is the effect of high temperatures, which caused the pesticides stress, prevented the cells to maintain a correct homeostasis and induced their death. In conclusion, our work, despite having the criticality of being a laboratory experiment, highlights how the sensitivity to some pesticides widely used in agriculture changes to 2 °C changing from nothing/low to critical. This data are interesting if we consider that the concentrations used are in the range described in the agricultural areas [35–37] and the same concentrations have an effect on other soil microorganisms [38] (www.pesticidesinfo.org). In addition, The European Directive 2009/128/EC [3] has led to a new approach to agriculture with the transition from an old concept of agriculture based on high use of both pesticides and fossil fuels to an agriculture more and more aware and careful to biodiversity and public health issues. This modern approach cannot fail to consider the role and the importance of soil protozoa and slime mould in particular as well as their sensitivity to pesticides with respect to the prokaryote microbial community [39]. In fact, a “healthy” soil is able, thanks to the role of these microorganisms, to fertilize and to counteract the pathogens that afflict many crops [40]. Our work suggests considering, in future regulations, about the use of pesticides as their toxic effect on non-target organisms is strongly influenced by climate temperatures that vary from season to season and from country to country and are constantly influenced by human activity. In addition, there is need for a new consideration of the protozoa, which takes into account recent researches about the presence in this microorganism of classical neurotransmitters [13, 19, 32, 41, 42] that, similar to those in animals, make protozoa an innocent target of neurotoxic pesticides in the battle against pest crops.
Acknowledgments I am grateful to Prof. Maria Giovanna Chessa and Prof. Carla Falugi, for their scientific support of my research in the fields of protozoa and cholinergic system, and to Dr. Martina Morabito and Prof. Benjamin Wolf for the critical reading of this manuscript. This research has been partly funded by the MInistero dell’Università e della
Ricerca (MIUR), Fondo Integrativo Speciale Ricerca (FISR–Bando 2002).
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