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Amphibians: Possible Effects of Insect-Resistant Intacta RR2 Pro® Soybean Diets on Leptodactylus gracilis Tadpoles.

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The toxic or nutritionally deleterious effects resulting from the consumption of genetically modified (GM) foods are still a matter of debate worldwide. In amphibians, the environmental impact of the Bacillus thuringiensis (Bt) toxin (e.g., Cry proteins) engineered into GM Bt crops is barely known. Thus, the aim of the present study was to evaluate the possible effects of a diet based on GM Bt-soybean in contrast with a common vegetable diet (lettuce) on Leptodactylus gracilis tadpoles. We evaluated their growth performance, histological changes in the intestine, and some hematological parameters as indicators of physiological stress. In the laboratory, tadpoles were either fed with green Bt-soybean leaves for 15 days or with lettuce leaves (controls). Both treatments resulted in a low mortality rate (less than 3%) during the experimental period. No external anomalies were detected and development Gosner stages were similar (36-37). However, larvae fed on lettuce grew faster and reached a larger size and greater weight than Btsoybean- fed tadpoles. Thus, the Bt-soybean diet may have induced histopathological changes in the tadpole intestine (greater thickness of the intestinal wall) and some cytotoxic effects on erythrocytes (lower mitotic index and anisocytosis). Our preliminary results highlight the potential effects of a diet based on Bt-soybean leaves on L. gracilis tadpoles. Further research is needed to evaluate the ecotoxicological risk of transgenic insecticidal proteins on non-target herbivores due to the massive use of Bt crops.
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PESTICIDES, BACTERIA AND MICROORGANISMS
BACILLUS THURINGIENSIS
BIOLOGICAL CHARACTERISTICS,
TOXICOLOGICAL EFFECTS
AND ENVIRONMENTAL IMPLICATIONS
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PESTICIDES, BACTERIA AND MICROORGANISMS
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PESTICIDES, BACTERIA AND MICROORGANISMS
BACILLUS THURINGIENSIS
BIOLOGICAL CHARACTERISTICS,
TOXICOLOGICAL EFFECTS
AND ENVIRONMENTAL IMPLICATIONS
ROBERT BÉLANGER
EDITOR
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CONTENTS
Preface vii
Chapter 1 The Development Behind the
Multi-Purpose Microorganism 1
André L. de A. Melo
Chapter 2 Bacillus thuringiensis Based Biopesticides:
Status and Potential Prospects 47
Karim Ennouri
Chapter 3 The Ecological Risks of Bt Crops in Amphibians:
Possible Effects of Insect-Resistant
Intacta RR2 Pro® Soybean Diets on
Leptodactylus Gracilis Tadpoles 67
Rafael C. Lajmanovich, Candela S. Martinuzzi,
Carlina L. Colussi, Paola M. Peltzer,
Agustín Bassó, Andrés M. Attademo
and Lucila M. Curi
Index 97
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PREFACE
In Chapter One, André L. de A. Melo, PhD discusses the spore-
forming bacterium Bacillus thuringiensis and its uses as a bio-insecticide,
also touching on ways to combat insect resistance. In Chapter Two, Karim
Ennouri proposes the bacterium Bacillus thuringiensis as an important
biopesticide because of the entomopathogenic effect of delta-endotoxins as
well as its efficiency against insects resistant to chemical insecticides. In
conclusion, Rafael C. Lajmanovich, Candela S. Martinuzzi, Carlina
Colussi, Paola M. Peltzer, Agustín Bassó, Andrés M. Attademo, and Lucila
M. Curi present a study exploring the impact of a GM Bt-soybean-based
diet, as opposed to a lettuce diet, on tadpoles.
Chapter 1 - Bacillus thuringiensis (Bt) is a gram-positive, spore-
forming bacterium and aerobic growth. It was first studied in the early
twentieth century and caused disease in Bombyx mori, which resulted in
financial losses in the Japanese silk industry. At that time, they did not
imagine that the microorganism would become an important tool in the
next century, especially within the subject of biological control and
production of pest-resistant crops. Despite the isolation of Bt strains and
reiteration of its toxicity, the first formulation of the bio-insecticide only
appeared three decades later, when it was used for the control of
Lepidoptera that caused damage to crops in France. In a short time, the
results stimulated the creation of new formulations with Bt to control moth
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Robert Bélanger
viii
and butterfly larvae. From the 1970s, studies with new strains expanded
the spectrum of action of bacterial toxins, which included species of
Coleoptera, Hemiptera, Simuliidae, Diptera and even Nematoda. The
mechanism of action involves the molecular affinity between Cry toxins
and receptors, which triggers the appearance of membrane pores or
activation mechanisms of apoptosis. Finally, the colonization of
microorganisms and sepsis ends up killing the larva. In spite of the good
results, the protective effect by biological insecticides presents some
limitations, including low efficiency in organisms that feed on internal
parts of the plant and low stability in the external environment. Such
problems were resolved with the development of Bt cultivars, which
received the bacterial genes and produced Cry toxins in their tissues. This
strategy soon presented a high productivity gain, stimulating the fast
adhesion to genetically modified (GM) cultivars, like Bt crops. However,
the emerging resistance of insects against biological insecticides and Bt
cultivars was gradually observed. For this, strategies such as refuge,
pyramidal application and modification of the Cry toxin can be used to
delay the appearance of resistant organisms. From an environmental point
of view, the use of Bt-derived products has made considerable progress.
Estimates indicate that worldwide, between 1996 and 2015, more than
550,000 tons of pesticides were no longer applied to the environment. In
addition, recent studies have verified the bioremediation capability of
heavy metals, chemical pesticides and petroleum and by-products, opening
up a new bias in the use of Bt for the benefit of the environment.
Chapter 2 - Bacillus thuringiensis is a bacterium producing a large
range of insecticidal toxins against several types of insects. Bacillus
thuringiensis, an aerobic, gram-positive, spore-forming bacterium,
continues the focus of the majority of studies and investigations on micro-
organisms and biopesticides. It is characterized from other Bacillus
member genus by a high potential due to the entomopathogenic effect of
delta-endotoxins and the advantages of genetic modifications in
agriculture. The most important toxins are delta-endotoxins or protein
“cry” that interact with specific receptors on the epithelial digestive cells of
the insect. The gut becomes paralyzed and the insect stops feeding.
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Preface
ix
Moreover, the advantage of the bacterium is associated with a particular
activity, which allows a selective biocontrol against specific insects while
preserving the non-target entomological fauna (bees and other biological
control aids). Formulation attempts can be added to these findings and may
obtain great products in order to increase pesticide efficiency. Bacillus
thuringiensis is also a promising choice for controlling insects resistant to
certain chemical insecticides.
Chapter 3 - The toxic or nutritionally deleterious effects resulting from
the consumption of genetically modified (GM) foods are still a matter of
debate worldwide. In amphibians, the environmental impact of the Bacillus
thuringiensis (Bt) toxin (e.g., Cry proteins) engineered into GM Bt crops is
barely known. Thus, the aim of the present study was to evaluate the
possible effects of a diet based on GM Bt-soybean in contrast with a
common vegetable diet (lettuce) on Leptodactylus gracilis tadpoles. The
authors evaluated their growth performance, histological changes in the
intestine, and some hematological parameters as indicators of
physiological stress. In the laboratory, tadpoles were either fed with green
Bt-soybean leaves for 15 days or with lettuce leaves (controls). Both
treatments resulted in a low mortality rate (less than 3%) during the
experimental period. No external anomalies were detected and
development Gosner stages were similar (36-37). However, larvae fed on
lettuce grew faster and reached a larger size and greater weight than Bt-
soybean-fed tadpoles. Thus, the Bt-soybean diet may have induced
histopathological changes in the tadpole intestine (greater thickness of the
intestinal wall) and some cytotoxic effects on erythrocytes (lower mitotic
index and anisocytosis). The authors’ preliminary results highlight the
potential effects of a diet based on Bt-soybean leaves on L. gracilis
tadpoles. Further research is needed to evaluate the ecotoxicological risk of
transgenic insecticidal proteins on non-target herbivores due to the massive
use of Bt crops.
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In: Bacillus thuringiensis ISBN: 978-1-53612-724-9
Editor: Robert Bélanger © 2017 Nova Science Publishers, Inc.
Chapter 3
THE ECOLOGICAL RISKS OF BT CROPS IN
AMPHIBIANS: POSSIBLE EFFECTS OF
INSECT-RESISTANT INTACTA RR2 PRO®
SOYBEAN DIETS ON LEPTODACTYLUS
GRACILIS TADPOLES
Rafael C. Lajmanovich1,2,*, Candela S. Martinuzzi1,2,
Carlina L. Colussi1, Paola M. Peltzer1,2, Agustín Bassó1,
Andrés M. Attademo1,2 and Lucila M. Curi1,2
1Laboratorio de Ecotoxicología, Facultad de Bioquímica y Ciencias
Biológicas, Universidad Nacional del Litoral (FBCB-UNL),
Santa Fe, Argentina
2Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Argentina
* Corresponding Author E-mail: lajmanovich@hotmail.com.
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
ABSTRACT
The toxic or nutritionally deleterious effects resulting from the
consumption of genetically modified (GM) foods are still a matter of
debate worldwide. In amphibians, the environmental impact of the
Bacillus thuringiensis (Bt) toxin (e.g., Cry proteins) engineered into GM
Bt crops is barely known. Thus, the aim of the present study was to
evaluate the possible effects of a diet based on GM Bt-soybean in contrast
with a common vegetable diet (lettuce) on Leptodactylus gracilis
tadpoles. We evaluated their growth performance, histological changes in
the intestine, and some hematological parameters as indicators of
physiological stress. In the laboratory, tadpoles were either fed with green
Bt-soybean leaves for 15 days or with lettuce leaves (controls). Both
treatments resulted in a low mortality rate (less than 3%) during the
experimental period. No external anomalies were detected and
development Gosner stages were similar (36-37). However, larvae fed on
lettuce grew faster and reached a larger size and greater weight than Bt-
soybean-fed tadpoles. Thus, the Bt-soybean diet may have induced
histopathological changes in the tadpole intestine (greater thickness of the
intestinal wall) and some cytotoxic effects on erythrocytes (lower mitotic
index and anisocytosis). Our preliminary results highlight the potential
effects of a diet based on Bt-soybean leaves on L. gracilis tadpoles.
Further research is needed to evaluate the ecotoxicological risk of
transgenic insecticidal proteins on non-target herbivores due to the
massive use of Bt crops.
Keywords: amphibian tadpoles, growth, histopathology, cytotoxicity, Bt-
soybean
1. INTRODUCTION
Bacillus thuringiensis (Bt) is a rod-shaped Gram-positive
entomopathogenic bacterium abundant in the soil and plants (Marzban,
2012). During the sporulation phase (resting stages), this bacterium
produces intracellular crystal proteins (Cry), which are effective only when
ingested by susceptible insects (Bora et al., 1993). These crystals are
principally formed by one or more proteins (Cry and cytolytic toxins), also
called δ-endotoxins (Bravo et al., 2007). These toxins are responsible for
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The Ecological Risks of Bt Crops in Amphibians
the selective Bt insecticide activity, since they activate upon eating and
then bind to receptors in the intestinal cells of insects, leading to the loss of
homeostasis and septicemia, and ultimately death (Schnepf et al., 1998;
Whalon and Wingerd, 2003).
Through the use of recombinant DNA (rDNA) technology, genes from
Bt coding for insecticidal δ-endotoxins have been transferred into some of
the most important crops, including maize (Zea mays L.) and soybean
(Glycine max (L.) (Yu et al., 2013; CERA, 2015). Around 64% of the soy
produced today is genetically modified (GM), and Roundup Ready®
soybean is the most common variety of soy worldwide (James, 2007).
Roundup Ready® soybean has been transformed to express the 5-
enolpyruvylshikimate 3-phosphate synthase gene, which provides
resistance to glyphosate, the active ingredient of the herbicide Roundup®
(Padgette et al., 1995).
Cry toxins are considered non-toxic to higher animals such as
mammals (Zhang et al., 2005; Shimada et al., 2006) and Bt crops have
been suggested to be as safe and nutritious as their
conventional/commercial counterparts for various animals (e.g., Sissener et
al., 2010, 2011; Sanden et al., 2013). Nevertheless, not all studies have
proved this. Indeed, Cry toxins have been reported to bind to the
mammalian intestinal mucosal surfaces (Shimada et al., 2006; Vázquez-
Padrón et al., 2000), and suggested to thereby elicit humoral and mucosal
immune responses in mice (Vázquez-Padrón et al., 1999; Finamore et al.,
2008). Along with the biological responses to Bt maize, worries have been
raised regarding possible allergenicity of GM plant crops, in particular
transgenic proteins (Prescott et al., 2005; Aris and Leblanc, 2011).
It is very important that transgenic crops and other genetically
modified organisms (GMOs) are assessed for their safety (e.g., López et
al., 2012; Snell et al., 2012; Herman and Price, 2013) before they are
allowed to enter the market. To fulfill this goal, the applicant, which is in
most cases a company that has developed a GMO, gives a dossier to the
local authorities, which, among other issues, contains safety data (Kleter et
al., 2005). At present, the possible horizontal gene transfer by GMOs is a
great controversy (e.g., Řehout et al., 2008; Ran et al., 2009; Hanusova et
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al., 2011; Oraby et al., 2014). Indeed, most GM contamination incidents
occur through cross-pollination, contamination of seed stocks, or failure to
segregate GM from non-GM crops after harvest (Price and Cotter, 2014).
Bt toxins produced by transgenic crops can enter the soil in root
exudates (Saxena et al., 1999), plant residues (Zwahlen et al., 2003), or
feces of animals that have fed on Bt plant material (Weber and Nentwig,
2006). Once in the soil, Cry toxins can bind to clay and humus particles
(Tapp and Stotzky, 1998), which safeguard them from biodegradation and
insecticidal effects (Koskella and Stotzky, 1997).
Tank et al. (2010) demonstrated the occurrence of maize detritus and a
transgenic insecticidal protein (Cry1Ab) within the stream network of an
agricultural landscape, whereas Wang et al. (2013) demonstrated that Bt
rice releases detectable amounts of Bt protein into the irrigation water.
Besides, some studies have indicated adverse effects of Bt crops on aquatic
organisms, including Daphnia magna (Bøhn et al., 2008, 2010, 2016;
Raybould and Vlachos, 2011; Holderbaum et al., 2015), larvae of
Trichoptera (Rosi-Marshall et al., 2007; Chambers et al., 2010), larvae of a
crane fly and an isopod (Jensen et al., 2010), and fishes (Gu et al., 2014).
Significant effects of MON810 maize have been recently noted in
salmonid fish (Salmo salar) (Gu et al., 2013, 2014). Also, non-target
effects of some Bt plants (e.g., Populus spp.) have also been reported for
aquatic biota (Axelsson et al., 2011).
It should be underlined that amphibian tadpoles are primarily
herbivorous (Altig et al., 2007). The digestive system of tadpoles is poorly
differentiated, has neutral pH, and serves primarily as a storage site for
food, with some degree of digestive activity (McDiarmid and Altig, 1999),
whereas the Bt target intestine, as in the insect midgut, lacks acid pH
(Whalon and Wingerd, 2003). It is known that, in insects, Cry proteins
bind to specific sites in the midgut epithelial cells, whereas in amphibian
larvae, receptors for these proteins remain unknown. Moreover, according
to oral mammalian toxicology and in vitro digestibility studies, δ-
endotoxins do not show toxicity to these animals and are quickly degraded
in simulated gastric fluid (EPA, 1998). Nevertheless, Fares and El-Sayed
(1998) showed changes in the ileum of a group of mice fed on transgenic
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potatoes which carried the CryI gene of Bt var. kurstaki (strain HD1). In
addition, in a previous study, we demonstrated intestine damage and blood
genotoxicity in Leptodactylus latrans tadpoles exposed to Bt toxins
(Lajmanovich et al., 2015).
Argentina is the third largest producer of biotech crops after the United
States and Brazil. In 2013, Argentina granted the authorization for the use
of Monsanto’s herbicide-tolerant and insect-resistant Intacta RR2 Pro®
(MON87701 x MON89788) soybeans (Bt-soybean). The MON 87701
event has the Cry1Ac gene derived from Bt, whereas the MON 89788
event has the cp4 epsps gene derived from Agrobacterium sp. (Justiniano
et al., 2014). Taking into account all the information available as well as
the results of a previous work where we demonstrated the ecotoxicological
risk for amphibian tadpoles from spraying with Bt toxins (Lajmanovich et
al., 2015), the objective of this work was to analyze the possible effects of
a diet based on GM Bt-soybean on the development and growth, intestine
histology, and hematological parameters (as indicators of physiological
stress) of L. gracilis tadpoles in comparison to a common vegetable diet
(lettuce).
2. MATERIAL AND METHODS
2.1. Test Species
Larvae of the Dumeril´s Striped Frog (L. gracilis; Anura:
Leptodactylidae) were selected as model test organism. In Argentina, this
anuran is categorized as “not threatened” (Vaira et al., 2012). This species
is distributed from Southern Brazil through Uruguay to Paraguay (lower
Chaco and the south), inter-Andean dry valleys in Bolivia, and northern
Argentina (Lavilla and Cei, 2001) and is commonly present in the
agricultural landscape of central-eastern Argentina (Sánchez et al., 2013).
L. gracilis belongs to the L. fuscus group, whose species deposit eggs in
holes in flooded soils, releasing the tadpoles to complete their development
in ponds (Heyer, 1978). This reproductive mode gives higher prevention
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against dehydration and predation, and possibility to a large versatility in
habitat use. Larvae are gregarious, occur in high densities and have a
herbivorous diet (Lajmanovich, 2000).
A cohort of tadpoles of L. gracilis larvae were collected with dip net
from temporary ponds located in riparian forests of the Espinal and Deltas
e Islas del Río Paraná ecoregions (Burkart et al., 1999), surrounded by
Paraná River floodplain (Cayastá, Garay Department, Santa Fe province,
Argentina) in December 2015. These sites had not been treated with
agrochemicals or biopesticides, as specified by the local laws for wildlife
protection. Average tadpole size (snout-vent length; SVL) was
12.2 ± 0.5 mm and weight was 0.18 ± 0.05 g; Gosner stages were
32-34 (Gosner, 1960). Tadpoles were acclimated in glass aquaria that
contained dechlorinated tap water (DTW; pH 7.42 ± 0.05, conductivity:
165 ± 9.5 µmhos/cm, dissolved oxygen concentration: 6.2 ± 1.5 mg/L and
hardness: 54.8 mg/L of CaCO3), under laboratory conditions (12/12-hr
light/dark cycle and at 22 ± 2ºC). During the previous 24 h before the
experiment, tadpoles were not fed.
2.2. Experimental Design
Glass aquaria containing 10 L of DTW were used in the experiments.
Laboratory experiments were conducted at 22 ± 2ºC with at a 12/12-h
light/dark cycle. Treatment groups were: 1) controls: tadpoles fed on
lettuce, which is the diet recommended by numerous experimental
protocols for herbivorous tadpoles due to its very high nutritional quality
(e.g., McDiarmid and Altig, 1999; Tyler, 2009; Benavides et al., 2005),
and 2) tadpoles fed on green Bt-soybean leaves. Both treatments were
prepared in duplicate with one hundred tadpoles per aquarium (N = 400).
Lettuce (Lactuca sativa) and Bt-soybean leaves were provided by local
farmers. Fresh Bt-soybean leaves were collected at different vegetative
stages before anthesis (V2 to V4 phenological stages) (McWilliams et al.,
1999) from a GM crop in Paraná Department (Entre Ríos Province,
Argentina). No residues of glyphosate and aminomethyl phosphonic acid
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were detected in soybean leaves samples by gas chromatography with
electron capture detector. These analyses were performed by Fares Taie
analytical service laboratories, Mar del Plata, Argentina, using sampling
protocol 135965 (detection limit of 0.01 mg/kg).
Larvae were exposed to the two different diets for 15 days. Both types
of food were prepared crushing the leaves in a ceramic mortar and pestle to
obtain food homogenates. The same ration of each diet was calculated
following the mass specic portions provided by Peltzer et al. (2008). The
water in the aquaria was replaced every five days with freshly prepared
solution of the same characteristics. Larval mortality was monitored and
dead larvae were removed from the aquaria every 24 h. A subsample of
each treatment (N = 20, respectively) was used to test the effects on
development and growth, histological changes in intestines, and
hematological parameters. Larvae were killed according to the criteria of
ASIH et al. (2004) and with the approval from the Animal Ethics
Committee of the Faculty of Biochemistry and Biological Sciences of
Universidad del Litoral (Santa Fe, Argentina). After blood extraction, each
larva was dissected along the mid-ventral line by making a longitudinal
incision, and digestive organs (coiling gut) were removed, washed in
distilled water and placed on filter paper to remove excess of fluids.
2.3. Response Variables
After 15 days of feeding, we independently determined biometric
parameters of each tadpole, including the snout vent length (SVL, digital
calliper 0.01 mm precision), Gosner stages (Gosner 1960), and weight of
each tadpole (digital balance 0.01 g precision).
2.4. Histological Studies
For light microscopy examination, the middle intestine of tadpoles was
fixed in Bouin’s solution for 48 h, dehydrated in ethanol, cleared in xylol,
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
and embedded in paraffin. Fixed tissues were then serially sectioned (5
μm) using a microtome and stained with hemotoxylin-eosin. The
histological sections were examined and photographed under a light
microscope at a magnification of 40 X (Arcano model with camera
attachment Moticam® 10.0). The intestinal wall thickness was measured
based on the methodology proposed by Romero Arauco et al. (2007) for
amphibian larvae, using ImageJ software (National Institutes of Health,
NIH, USA).
2.5. Analysis of Erythrocytes
Approximately 20 µL blood was taken from each tadpole by cardiac
puncture (Lajmanovich et al., 2005) and blood smears were prepared on
clean slides, fixed, and stained by means of the May-Grünwald/Giemsa
method (Barni et al., 2007). It is important to consider that red blood cells
(RBC) in amphibians are nucleated and undergo cell division in the
circulation, particularly during the developmental stages (Duellman and
Trueb, 1986). The mitotic index (MI) frequencies were determined in 1000
erythrocytes from each tadpole, with a microscope under 100 X
magnification (Krauter, 1993). The presence of erythrocyte nuclear
abnormalities (ENAs) was determined according to the procedures of
Guilherme et al. (2008) in mature erythrocytes by determination of the
frequency of micronuclei (MN) and the following nuclear lesions: notched
nuclei (NT), binucleated erythrocyte (BER), erythroplastid (EP), kidney
nucleated (KN), and lobed nuclei (LN) (Lajmanovich et al., 2014). Coded
and randomized slides were examined blind by a single operator.
The blood smears were photographed with a Moticam® 10.0 camera
and using ImageJ software (National Institutes of Health, NIH, USA).
Subsequently, four parameters were measured from erythrocytes (without
mitosis or ENAs): large cell diameter (D-cell), small cell diameter (d-cell),
large nuclear diameter (D-nucleus) and small nuclear diameter (d-nucleus).
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2.6. Data Analyses
Data of MI and ENAs were analyzed using the binomial proportion
test (Margolin et al., 1983). All other data were compared using a
Student’s t test with BioStat software 5.0 (Ayres et al., 2008). Differences
were considered significant at p < 0.05.
3. RESULTS
3.1. Tadpole Growth
Both treatments resulted in a low mortality rate (less than 3%)
throughout the experimental period (15 days). Throughout the experiment,
larvae of both dietary treatments fed actively and presented no abnormal
trophic behavior (for example immobility, starvation). At the end of the
experiment, all larvae reached Gosner stages 3637. Tadpoles fed on
lettuce had a mean SVL of 15.20 mm 0.38) and a mean weight of 0.40 g
0.04), whereas tadpoles fed on Bt-soybean were shorter (SVL 13.37 ±
0.24 mm) and lower in weight (0.25 ± 0.05 g) (p < 0.01) (Figure 1).
3.2. Intestine Histology
No anomalies were observed in the intestine of tadpoles in the control
treatment (lettuce diet). For this group, light micrographs showed that, at
Gosner stages 36-37, the intestine is a long simple tube with a single layer
of cuboidal epithelial cells, the primary epithelia are surrounded by fine
layers of muscles with little intervening connective tissue, the submucosa
is further enclosed by a thin serosa, and the mean thickness of the intestinal
wall is 25.12 ± 3.35 µm (Figure 2 A). At the same Gosner stages, the
intestines of tadpoles fed Bt-soybean presented a great thickness of the
intestinal wall (36.45 ± 6.32 µm; Figure 2 B). The thickness of the
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
intestinal wall was significantly different between treatments (p < 0.01)
(Figure 3).
Figure 1. Comparative values of snout-vent length (SVL) and weight in Leptodactylus
gracilis tadpoles for 15 days on two types of leaf diets; Gosner stages (36-37). Data are
expressed as mean ± SD. Asterisks indicate statistically significant differences between
lettuce and Bt-soybean diets treatment, according to Student’s t test (** p < 0.01, n =
20).
Figure 2. Light micrographs of intestinal walls (IW) of Leptodactylus gracilis tadpoles;
Gosner stages (36-37). (A) Control diet (lettuce leaf); (B) 15 days to Bt-soybean leaf
diet. L: lumen; MS: muscular and serosa; SM: submucosa; CE: columnar epithelium;
BB: brush border. See: greater thickness of the intestinal wall in tadpoles treated with
Bt-soybean diets. Haemotoxylin-Eosin, 40 x, bar =10 µm.
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Figure 3. Comparative values of intestinal walls thickness in Leptodactylus gracilis
tadpoles for 15 days on two types of leaf diets; Gosner stages (36-37). Data are
expressed as mean ± SD. Asterisks indicate statistically significant differences
between lettuce and Bt-soybean diets treatment, according to Student’s t test
(** p < 0.01, n = 20).
3.3. Red Blood Cell Profile
The mature erythrocytes of L. gracilis tadpoles are oblong/oval-shaped
with a central nucleus. The nucleus was visibly structured and had a well-
defined boundary, which facilitated the recognition of fragments in their
cytoplasm. The mitotic erythrocytes were used to determine the rate of cell
division or MI (Figure 4). The MI frequency was 3.57 0.46) in the
lettuce diet treatment and was significantly different (p < 0.05) from that in
the Bt-soybean treatment (MI = 1.83 ± 0.4). Although the MI decreased
with the Bt-soybean treatment, the frequency of all other ENAs also
decreased. The difference between food treatments regarding BER and LN
(Figure 5) was statistically significant (p < 0.05 and 0.01, respectively).
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
Figure 4. Example of mitotic erythrocytes (arrow) found in blood samples of
Leptodactylus gracilis tadpoles (lettuce leaf diets); Gosner stages (36-37). May
Gründwald-Giemsa, 100 x, bar = 2 µm.
Figure 5. Frequency of cells in mitosis (mitotic index; MI) and erythrocyte nuclear
abnormalities (ENAs): micronuclei (MN), notched nuclei (NT), binucleated
erythrocyte (BER), erythroplastid (EP), kidney nucleated (KN), and lobed nuclei (LN)
in Leptodactylus gracilis tadpoles for 15 days on two types of leaf diet treatment
(Gosner stages 36-37). Asterisks indicate statistically significant differences between
lettuce and Bt-soybean leaf diets treatments, according to Binomial Proportion´s test
(*, p < 0.05; ** p < 0.01, n = 13).
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Table 1. Results from the comparative variation-statistical analysis of
metric erythrocyte parameters (µm) (big cell diameter (D-cell), small
cell diameter (d-cell), big nuclear diameter (D-nucleus) and small
nuclear diameter (d-nucleus) of the Leptodactylus gracilis tadpoles fed
for 15 days on two types of leaf diets; Gosner stages (36-37)
Diets treatment
Parameters
Lettuce
Bt-soybean
D-cell
11.69 (0.18)
13.60 (0.36)**
d-cell
9.45 (0.14)
9.95 (0.30)
D-nucleus
5.68 (0.09)
5.58 (0.13)
d-nucleus
4.40 (0.08)
4.20 (0.12)
Data are the means ± SD; ** p < 0.01; significant (Student's t-test) difference from the
Bt-soybean leaf treatment.
Table 1 shows the final results from the measuring and statistical
processing of 620 erythrocytes of L. gracilis larvae under the two food
treatments evaluated. The major diameter of the cells (anisocytosis) from
tadpoles fed Bt-soybean was significantly different (p < 0.01) from that of
those from tadpoles fed lettuce.
4. DISCUSSION
There is a strong international debate in relation to the consumption
and cultivation of the new generation of GM plants (e.g., Giovannetti,
2003; Séralini et al., 2012, 2014; Grunewald and Bury, 2013; Horak, et al.
2015), particularly on their mutagenicity, teratogenicity and
carcinogenicity (Domingo, 2016). For years, Bt-based formulations, which
hold high concentrations of Cry proteins, have been used as an example of
totally safe insecticides. Indeed, this gene coding for these proteins has
been widely cloned in different crops, and consequently high quantities of
Cry toxins are released into the environment. However, their harmful
effects on non-target organisms are poorly understood and analyzed
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
(Ramiro and Faria, 2006). The reduction observed in this study in the
growth, SVL and weight of the L. gracilis tadpoles fed on the Bt soyben
diet could be linked to toxic elements (e.g., Cry proteins) in this soybean.
The intestinal damage observed in L. gracilis fed Bt-soybean may be due
to poor absorption of nutrients, and may then reflect a process of
physiological stress (e.g., lower MI and blood anisocytosis). In fact, in
carnivorous fish (e.g., Salmo salar L.), soybean meal induces enteritis and
other pathological alterations on the digestive tract (Urán et al., 2008,
2009). In contrast, for herbivorous frog larvae, non-GM soybean is
commonly recommended as a staple diet with good nutritional
requirements for amphibians (McCallum and Trauth, 2002). Besides, Gu et
al. (2014) suggested that the Cry protein in the GM Bt maize causes
alterations in the intestine of S. salar, but without affecting its overall
survival, growth, development or health. In a previous study, we observed
that tadpoles exposed for 48 h to Bt commercial insecticide containing Cry
toxins (Introban®) showed intestinal histopathologies such as
inammatory lesions in the connective tissue subjacent to the epithelium
and dilation of blood vessels (Lajmanovich et al., 2015). However, it is
also important to consider that, before anthesis (growth stages at which we
collected our samples), the Cry content in Bt-soybean leaves is highest and
ranges from 25.50 to 37.50 μg g1 (Yu et al., 2014).
A significant difference in the thickness of the intestinal wall between
the two diets was observed. In addition, we found a significant difference
in growth (weight and length) of soybean-fed tadpoles compared to
controls. The data obtained show a possible interaction in vivo between Bt-
soybean leaves and the animal’s intestine which could induce changes in
the physiological status of the intestinal wall. In this sense, and in
agreement with that found by Chen et al. (2015), Wang et al. (2014)
concluded that Cry proteins do not affect amphibians. Their conclusions
are principally based on the detection of Bt proteins in roots, stems and rice
leaves and on their bioactivity (mortality) in their target insects. However,
it is not clear whether tadpoles or froglets had been in contact with these
toxins or whether they had ingested these toxins and for how long, taking
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into account that the Cry protein concentration in Bt rice fields decreases to
50% during the first month and to 21% after 7 months (Li et al., 2007).
According to oral mammalian toxicology and in vitro digestibility
studies, δ-endotoxins show no toxicity to this vertebrate and are rapidly
degraded in simulated gastric fluid (EPA, 1998). However, Fares and El-
Sayed (1998) showed changes in the ileum of a group of mice fed on
transgenic potatoes that carried the Cry gene of Bt var. kurstaki (strain
HD1). The results obtained by us indicate that Bt-soybean leaves could
induce some histomorphometric changes in the thickness of the intestinal
wall of tadpoles (in comparison to similar wild diet) perhaps due to the
existence of a mechanism that eliminates or inhibits the toxic effect.
Likewise, Vázquez-Padrón et al. (2000) noted intestinal histopathologies in
mice (the mean area of intestinal absorptive cells of mice increased in δ-
endotoxin and transgenic-treated mouse groups). In addition, Ibrahim and
Okasha (2016) concluded that consumption of GM corn containing Bt
genes alters the intestinal histological structure.
Histopathological investigations on insect larvae (e.g., mosquitoes)
affected by the Cry toxin have shown morphological lesions in the midgut
epithelium, which exhibited swollen cells, degenerated brush borders,
disorganized nuclei, enlargement of intercellular spaces and cell lysis (Rey
et al., 1998). However, the lack of harmful effects of δ-endotoxins on non-
target species (e.g., mammals) has been explained by stomach acidification
and by the nonexistence of specific binding sites for Cry toxins in the
intestine (McClintock et al., 1995). Like mosquito larvae, amphibian
tadpoles are mainly herbivorous (Altig et al., 2007). The gastrointestinal
tract of tadpoles is poorly differentiated, has neutral pH, and serves
primarily as a storage site for food, with limited digestive activity
(McDiarmid and Altig, 1999).
On the other hand, cell division is an essential condition for the
formation of ENAs (Heddle et al., 1991). Hence, the MI is critical in
determining the rate of cell division (Moore et al., 2011). Little is known
concerning the impacts of experimental stress on rates of cell division or
on mitotic activity in amphibian tadpoles (Lajmanovich et al., 2014).
However, we observed a significant decrease in the MI in the group fed the
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
Bt-soybean diet as compared to tadpoles fed on lettuce. However, it is clear
that if the rate of cell division had decreased, the number of ENAs may
have diminished in response to experimental stress. This decrease in the
MI could lead to a false reduction in MN and other clastogenic endpoints
(e.g., nuclear alterations), because the RBC must be on division. In this
sense, Mezzomo et al. (2016) showed that the Bt spore crystals genetically
modified to express individually Cry1Aa, Cry1Ab, Cry1Ac, or Cry2A can
cause some hematological risks to vertebrates and increase their toxic
effects with long-term exposure. Reduction in the MI could be due to the
inhibition of DNA synthesis or the blocking of the cell from entering
mitosis (Tülay and Ozlem, 2010). Also, Vyuyan (2002) explained that the
decrease in the MI because of the increased number of interphase or dead
cells and the accumulation of interphase cells may be due to the inhibition
of DNA synthesis. According to Sazada et al. (2010), this inhibition shows
that some plant extracts might interact with DNA subsequent mitotic
inhabitation. Similar results were observed by Sobita and Bhagirath
(2005), Mondal et al. (2006) and Sazada et al. (2010) in evaluations of
toxic effects of leaf extracts on cell division and chromosome morphology.
Numerous studies have suggested that the mechanical properties of red
blood cells are altered by oxidative damage (e.g., Mohanty et al., 2014).
Furthermore, Atatür et al. (1998, 1999) determined differences in the
metric parameters of RBC from some anuran species and looked for the
reasons in the different environmental conditions of the biotopes. Other
authors (e.g., Arnaudov et al., 2008; Zhelev et al., 2006, 2013) also argue
that various environmental factors (e.g., anthropogenic pollution) could
affect the size of erythrocytes. Our hematological results allow us to point
out some peculiarities related to some changes in the metric features of
erythrocytes of L. gracilis tadpoles under the two different diets studied. If
we consider tadpoles fed lettuce as relatively normal (they reached further
development), the Bt-soybean diet caused anisocytosis. There are few
similar studies that concern the metric values of tadpole erythrocytes under
the effects of diet toxicants. Even so, this degenerative form of
erythrocytes (change in size) was also studied in amphibians in response to
chemical stressors in the aquatic environment (Barni et al., 2007).
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On the other hand, in human blood, the erythrocyte size variation is
frequently found in leukemias and in most forms of anemia. Anisocytosis
results from anomalous cell development, and typically results from a
deficit in the raw materials (e.g., iron, vitamin B12, folic acid) needed to
synthesize them or by a congenital disorder in the cell structure (Bain,
2005). The Cry protein contained in Bt-soybean has been found to induce
microcytic hypochromic anemia in mice (Mezzomo et al., 2016).
Therefore, more research is needed to evaluate the possible
ecotoxicological risk of GMO products containing the Bt toxin on non-
target wild fauna because some effects are evident.
CONCLUSION
The present study provides the first results concerning the exposure of
non-target tadpoles to transgenic soybean Bt leaves. The tadpoles that were
fed the Bt-soybean diet showed low growth in comparison with the ones
fed similar to the diet eaten in the wild during a short time (15 days).
Moreover, our results provide preliminary evidences of potentially
histopathological changes and cytotoxic effects (lower MI and anisocytosis
in RBCs) on amphibian larvae exposed to the Bt-soybean diet. Taking into
consideration the health risk of humans and wildlife exposed to different
levels of Cry toxins, especially through the diet and environmental
exposure, our results highlight the need of further studies to clarify the
toxic mechanism involved in the blood and digestive tract of amphibians
and other herbivores that potentially forage on Bt leaves.
ACKNOWLEDGMENTS
This work is dedicated to the memory of Dr. Oscar Scremin, Facultad
de Ciencias Médicas, Universidad Nacional de Rosario, Rosario
(Argentina) and David Geffen School of Medicine at UCLA, California
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R. C. Lajmanovich, C. S. Martinuzzi, C. Colussi et al.
(USA) an outstanding scientist who spent his life defending science at the
service of society and not of international corporations.
We thank Ana Canal and Amorina Sánchez for helping in
histopathological studies. We would like to extend a special thanks to
Silvia Seib for providing us with excellent logistic support in the field and
Dr. Maria Victoria Eusevi for English Editing Service. This study was
supported in part by National Agency for Promotion of Science and
Technology (ANPCyT-PICT 470).
COMPLIANCE WITH ETHICAL STANDARDS
Animals used in this research have been treated according to ASIH
(2004) criteria and with approval from the animal ethics committee of the
Faculty of Biochemistry and Biological Sciences. http://www.fbcb.unl.
edu.ar/pages/investigacion/comite-de-etica.php.
CONFLICT OF INTEREST
The authors declare that they have no actual or future conflict of
interest, financial or otherwise.
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Reviewed by:
Hernán R. Hadad, PhD. Química Analítica, Instituto de Química
Aplicada del Litoral (IQAL), Facultad de Ingeniería Química, Universidad
Nacional del Litoral (UNL)-Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET). Santiago del Estero 2829, Santa Fe
(3000), Argentina.
E-mail: hadadhernan@gmail.com.
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... The impact of modifications and alterations caused by human activities as a result of direct (habitat loss) and indirect causes (contamination by chemicals, UV radiation, diseases) on the survival, bioecology, population status, dynamics and diversity of amphibians, began to be referenced in our country with some isolated studies since the mid-70s (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, among others). However, since the 1990s, research on specific threats, as well as, the synergy of several threats, became a more notable beginning in 2005 (some examples: Rengel and Pisanó, 1991;Salibián, 1992;Lavilla and Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich and Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti and Dieguez, 2006;Natale et al., 2006;Barrionuevo and Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). The studies are distributed in different localities, provinces or regions of the country, however, most of the research referring to the contamination by different substances was developed under laboratory conditions, being insufficient to those analyzed under real conditions of field or in situ. ...
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In September 2005, the IUCN Species Survival Commission (SSC) held a summit for the conservation of amphibians where specialists from around the world agreed that, in addition to the need to document the declines and extinctions, we need to promote action strategies designed to respond to the global biodiversity crisis. The outcome of this summit was expressed in the Amphibian Conservation Action Plan (Gascon et al., 2007) with the recommendation that governments, civil society and the academic community adopt it and implement the suggested measures. Since 2007, almost 60 Action Plans have been developed at the national or regional level throughout the world, including six in South American countries (source: http://www.amphibians.org/publications/national-actionplans/). In Argentina, there is a growing number of research groups that develop projects related to the conservation of amphibians with different approaches and objectives. However, a unified national strategy providing an overview of amphibian conservation actions in the country including a declaration of principles, clearly defined priorities, and short, medium and long-term goals for the implementation of conservation actions does not yet exist. It is certainly of great value to have a comprehensive strategy that provides both the national and provincial governments with a framework to promote and support conservation programs and a tool that guides research activities, promotes the creation of information networks, training programs, environmental education, and outreach and community participation, thereby strengthening efforts to protect the biodiversity of our country. The Asociación Herpetológica Argentina (AHA) has long led national actions related to the conservation of amphibians by compiling and publishing the first Categorization of the State of Conservation of the Herpetofauna of Argentina (Lavilla et al., 2000) And the subsequent update (Vaira et al., 2012). In a similar vein, the AHA was actively involved in the first Global Amphibian Assessment (GAA) led by IUCN. Specialists convened during the Argentine Herpetology Congress of Puerto Madryn in 2003 to contribute their knowledge and validate the data compiled by the IUCN regional coordinator (http://oldredlist.iucnredlist.org/initiatives/ amphibians/process/methods). As of a result of these activities, the need to design a national strategy for the conservation of amphibians became evident, not only to evaluate the conservation status of amphibians of Argentina, but also to outline and address the problems negatively affecting the species and the courses of action that should be taken. Based on the input from a local network of specialists on various topics, the development of a base document recommending priority actions was proposed, which could serve as a guideline for the design of conservation initiatives and a national strategy for the conservation of amphibians in our country. The process included the creation of a framework to organize the activities to be developed, the implementation of a standard procedure and the formation of thematic groups of specialists. This initiative was launched in september 2015 during the XVI Argentine Congress of Herpetology held in the city of San Miguel de Tucumán. There, the general objectives of the strategy were outlined, and a tentative work schedule was developed, convening the local specialists who wanted to join this proposal. This was the starting point for the design and consolidation of specific programs and projects whose goals and actions could be measured and evaluated periodically. It was then proposed to generate a list of concrete and realistic goals, with respective actions and performance indicators that would allow their evaluation and monitoring in the short, medium and long term. As a result of the process, we finalized this Action Plan for the Conservation of Amphibians of Argentina,which outlines a set of 47 actions that respond to 18 identified problems grouped into 6 components that can be undertaken in predefined terms. The Plan proposes to execute these actions that experts have considered as priorities or necessary, however this does not imply that it constitutes a plan that has exhaustively evaluated all of the problems and possible actions for the conservation of the amphibians of Argentina. The Plan propose goals and actions that are considered priority to cover the existing information gaps and face the current and future threats to the conservation of amphibians in our country. It aims to provide clear guidance on issues that are considered relevant to conservation by identifying and ordering a set of measurable goals and the respective actions that respond to specific recommendations grouped in the thematic lines proposed and that can be implemented in the short, medium and long term (1, 3 and 5 years). Since the fulfillment of the actions is expected to generate changes in the conservation status of the amphibian species, the Plan includes monitoring of its development, evaluating the progress made in achieving the objectives according to the established deadlines. This will allow for the incorporation of changes and addition of new goals during the periodic reviews. We hope that this Plan will become a starting point for the design and consolidation of inter-institutional and interdisciplinary programs to guarantee the long-term persistence of the amphibian diversity of Argentina. We also hope that this document will be relevant for all actors of civil society and that it will increase awareness of the biodiversity crisis, encouraging them to participate in the proposed action plans. Finnally, we acknowledge the revision of the english translation of the Plan by Kelsey Neam. Marcos Vaira Mauricio S. Akmentins Esteban O. Lavilla
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como el sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
Article
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El público general desconoce que la República Argentina es uno de los países con mayor diversidad de anfibiosen América Latina, presentando unas 175 especies, con una gran proporción de endemismos, ciclos de vidaúnicos y adaptaciones fisiológicas y/o comportamentales a ambientes extremos (entre otras). La visión generales que la diversidad de anfibios se limita solo a sapos, ranas (inclusive, es común que sean consideradas elfemenino de los sapos) y escuerzos. Uno de los aspectos más problemáticos a la hora de generar interés en lacomunidad sobre la conservación de anfibios, es el desconocimiento del rol que cumplen en los ecosistemas, asícomo también los beneficios que este grupo puede proveer. También, abundan en el saber popular leyendas omitos sobre la peligrosidad o características nocivas de los anfibios. En gran parte, esta falta de conocimiento sebasa en la dificultad que enfrenta el público general, educadores y/o interesados en la naturaleza para acceder ainformación de calidad y con un lenguaje simple sobre la fauna de anfibios. Bajo la premisa ?no se conserva loque no se conoce? es que se debería lograr generar información, sobre la diversidad de especies de anfibios de laArgentina y sus problemáticas de conservación, que involucre un lenguaje claro y resulte accesible a todo tipode público.Por otra parte, los museos, privados o estatales (nacionales, provinciales y municipales), son los lugares máscomunes donde el público general concurre a interiorizarse sobre las ciencias naturales. Aunque los anfibiosrara vez ocupan lugares preponderantes dentro de las exposiciones permanentes o temporales, esta tendenciase ha revertido en los últimos años, principalmente por iniciativas como ?La Noche de los Museos?, dondelos investigadores pueden comunicar directamente al público concurrente la información sobre sus líneas deinvestigación y organismos de estudio.
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como del sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., 2003Peltzer et al., , 2004Peltzer et al., , 2006Peltzer et al., , 2008Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Cuello et al., , 2009Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
... El impacto de las modificaciones y alteraciones por actividades humanas por causas directas (pérdida de hábitat) e indirectas (contaminación por químicos, radiación UV, enfermedades) en la supervivencia, bioecología, el estatus poblacional, dinámica y diversidad de anfibios, comenzaron a ser referenciadas en nuestro país con algunos estudios aislados desde mediados de la década del ´70 (Bustoabab et al., 1977;Salibián et al., 1984;Pérez-Coll et al., 1986, entre otros). Sin embargo, a partir de la década de los ´90 se incrementaron las investigaciones y aproximaciones sobre el estudio de una amenaza puntual como el sinergismo de varias de éstas, tendencia que fue más notable desde el año 2005 (algunos ejemplos: Rengel y Pisanó, 1991;Salibián, 1992;Lavilla y Buti, 1999;Lajmanovich et al., 1998Lajmanovich et al., , 2002Lajmanovich et al., , 2003aLajmanovich et al., ,b, 2005Lajmanovich et al., , 2010Lajmanovich et al., , 2011Lajmanovich et al., , 2015Lajmanovich et al., , 2017Úbeda et al., 1999;Natale et al., 2000;Lavilla, 2001;Izaguirre et al., 2000Izaguirre et al., , 2001Peltzer et al., , 2004Peltzer et al., , 2010Peltzer et al., , 2013Peltzer et al., , 2015Peltzer et al., , 2017Lajmanovich y Peltzer, 2001;Ponssa et al., 2001;Vaira, 2002;Attademo et al., 2005Attademo et al., , 2007Attademo et al., , 2011Perotti y Dieguez, 2006;Natale et al., 2006;Barrionuevo y Ponssa, 2008;Agostini et al., 2009Agostini et al., , 2012Cuello et al., 2006Junges et al., 2010;Bionda et al., 2011aBionda et al., ,b, 2013Nori et al., 2013;Sánchez et al., 2014;López et al., 2015;Pollo et al., 2016;Akmentins et al., 2015;Curi et al., 2017;Velasco et al., 2018). Los estudios se encuentran distribuidos en distintas localidades, provincias o regiones del país, sin embargo, la mayoría de las investigaciones referidas a la contaminación por distintas sustancias han sido desarrolladas bajo condiciones de laboratorio, siendo insuficientes las analizadas en condiciones reales de campo o in situ. ...
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El Plan de Acción para la Conservación de los Anfibios de la República Argentina que reseña un conjunto de 47 acciones que responden a 18 problemáticas identificadas, agrupadas en 6 componentes que pueden acometerse en plazos preestablecidos. El Plan propone ejecutar estas acciones que los especialistas convocados han considerado prioritarias o necesarias, aunque ello no implica que constituya un Plan que haya agotado y evaluado todos los problemas y acciones posibles para la conservación de los anfibios de la República Argentina.
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