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stingless bee Melipona quadrifasciata and the honey bee
Apis mellifera
Mário Sarto, Eugênio Oliveira, Raul Guedes, Lúcio Campos
To cite this version:
Differential insecticide susceptibility of the Neotropical
stingless bee Melipona quadrifasciata and the honey bee
Apis mellifera
Mário César L. Del SARTO1,Eugênio E. OLIVEIRA1,Raul Narciso C. GUEDES1,
Lúcio Antônio O. CAMPOS2
1
Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil
2
Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil Received 28 October 2013– Revised 23 January 2014 – Accepted 18 February 2014
Abstract– The toxicity of three insecticides frequently used in Neotropical tomato cultivation (abamectin, deltamethrin, and methamidophos) was estimated on foragers of the Neotropical stingless bee Melipona quadrifasciata (Lep.) and the honey bee Apis mellifera (L.). Our results showed that the susceptibility varied significantly with the type of exposure (ingestion, topical, or contact), and there were significant differences between species. While M. quadrifasciata was usually more susceptible to insecticides (except for abamectin) in realistic exposures (via ingestion and contact) than A. mellifera, the former was less susceptible than A. mellifera to topically applied insecticides, a less realistic means of insecticide exposure. These findings challenge the common extrapolation of toxicity bioassays with A. mellifera to all (native) bee pollinators. Such equivocated extrapolation may compromise the significant services provided by native bees in Neotropical ecosystems.
insecticide exposure / acute toxicity / buzz pollinators / wild bees
1. INTRODUCTION
The reported decline of bee populations and the potential impairment of the sustainability of pollination services performed by these insects are the target of current worldwide concerns (Potts et al. 2010; Cameron et al. 2011; Lautenbach et al. 2012). Habitat destruction, climate change, pathogens, and pesticides are thought to be the main contributors to colony decline of the honey bee Apis mellifera (L.) (Biesmeijer et al. 2006; Potts et al. 2010; vanEngelsdorp and Meixner 2010), and recent
findings suggest that multifactor interactions between honey bee diet, parasites, diseases, and pesticides potentiate the decline in managed honey bee colonies (vanEngelsdorp and Meixner
2010; Cornman et al.2012; Becher et al. 2013; Pettis et al.2013).
The significant decline of managed honey bee colonies that has been reported mainly in the USA and Europe drew further attention to wild pollinator communities and their potential importance for pollination services in certain landscapes and crop systems (Winfree et al.
2007; Garibaldi et al. 2013; Jha and Kremen
2013). Native pollinator bees can perform equal to or better than the honey bee as pollinators for some crops (Maeta and Kitamura 1981; Freitas and Paxton 1998), significantly contributing to crop production even when honey bees are
Corresponding author: E.E. Oliveira, eugenio@ufv.br
Manuscript editor: Monique Gauthier
present (Winfree et al. 2007; Garibaldi et al.
2013). The stingless bees of the genus Melipona have been recognized as important pollinators of native plants in subtropical and tropical areas (Antonini et al. 2006) and have been recently recognized as promising agents for commercial pollination in crop systems, such as those of tomatoes (Del Sarto et al. 2005; Bispo dos Santos et al. 2009), eggplants (Nunes-Silva et al.2013), and sweet peppers (Cruz et al.2005). However, considering that pesticide application is a common agricultural practice in tropical areas (e.g., 500 pesticide active ingredients are registered in Brazil (Silveira and Antoniosi-Filho 2013)), and that over 150 pesticides are currently used and known to be toxic to honey bees (Devillers et al.2003; Johnson et al.2010; Mullin et al.2010), it is likely that stingless bee species that provide pollination services may also be the non-intended target of harmful insecticide effects.
Despite the recognized ecological and agri-cultural importance of stingless bees in tropical areas, the majority of studies assessing insecti-cide impacts on pollinators have focused on honey bees with few studies assessing the insecticide susceptibility of stingless bee species (Moraes et al. 2000; Valdovinos-Núñez et al.
2009; Lourenço et al.2012; Tomé et al. 2012). Here, we compared the susceptibility between foragers of the honey bee A. mellifera and of the Neotropical stingless bee Melipona quadrifasciata (Lepetelier) to three insecticides (deltamethrin, methamidophos, and abamectin) that are commonly used against insect pest species in Brazilian tomato fields (MAPA2013). Concern regarding the stingless bee M. quadrifasciata is due to its wide distribution in Brazil, especially where field and protected tomato systems are cultivated (Del Sarto et al.2005; Bispo dos Santos et al. 2009). Furthermore, M. quadrifasciata belongs to the same genus as the bee species Melipona capixaba (Moure and Camargo), which is recognized as an endangered species not only by the Brazilian Ministry of Environment (Normative Instructions no. 3, May 27, 2003 (Resende et al.2008; Luz et al.2011)) but also by the International Union for the Conservation of
Nature and Natural Resources (IUCN 2013), reinforcing the importance of assessing insecti-cide impacts on M. quadrifasciata. Insectiinsecti-cide bioassays were also performed with the honey bee because of its broadly recognized role as the bee pollinator model in ecotoxicology protocols for assessing the toxic effect of pesticides on pollinators (Felton et al. 1986).
Insecticide toxicity assessments using dif-ferent means of exposing the stingless bee M. quadrifasciata (in addition to honey bees) pro-vide basic toxicological information to guide insecticide use and minimize their potential non-target impact on native pollinators. The findings of this study also assist in assessing the validity of the honey bee as an indicator of insecticidal impact on native stingless bees.
2. MATERIAL AND METHODS
2.1. Insects
All insects used in this investigation were obtained from seven colonies of the Neotropical stingless bee M. quadrifasciata or from seven colonies of the Africanized honey bee A. mellifera maintained under field conditions at the Experimental Apiary of the Federal University of Viçosa (UFV, Viçosa, MG, Brazil, 20°45′ S, 42°52′ W). To ensure genetic variability between the colonies and to obtain more reliable toxicological estimates, sets of foragers from different colonies of either bee species were considered as replicates.
2.2. Insecticides
The concentration–mortality bioassays followed directive numbers 213 and 214 of the Organization for Economic Cooperation and Development (OECD
São Paulo, Brazil; avermectin abamectin: Vertimec 18 EC, Syngenta Proteção de Cultivos LTDA, São Paulo, Brazil). For the assays in which the bees were topically exposed or contact-exposed, technical grade insecticides (purity≥90 %) were used and directly obtained from the manufacturers (deltamethrin and methamidophos: Bayer CropScience, São Paulo, Brazil; abamectin: Syngenta Proteção de Cultivos LTDA, São Paulo, Brazil). Whenever suitable, the insecticides were classified according to their toxicity to bees as described by Felton et al. (1986) in the following categories: highly toxic (median lethal dose (LD50) values <1.0 μg a.i./bee),
moderately toxic (1.1<LD50<10μg a.i./bee), slightly
toxic (10.1<LD50≤100 μg a.i./bee), and virtually
non-toxic (LD50values >100μg a.i./bee).
2.3. Insecticide susceptibility bioassays
The insecticide susceptibility of bee foragers was evaluated using three means of exposure: ingestion (in honey syrup 50 % v/v), topical, and contact with dry residues. In all cases, preliminary concentration– mortality bioassays were performed to determine the concentration range to be used in the bioassays (i.e., the interval between the highest concentration for which no mortality was observed and the smallest concentration for which 100 % of the tested bees were killed). Once the concentration range was recognized, six to eight concentrations were established for the definitive concentration–mortality bioassays. In the control treatments, the bees received only honey syrup (oral exposure), were topically exposed to the solvent (acetone), or were exposed to a solvent-treated surface (free of insecticide residue).
2.3.1. Ingestion bioassays
For these bioassays, M. quadrifasciata bees were collected using a 10-cm-long plastic tube (diameter of 20 mm) connecting the colony entrance to a wooden cage (9×9×3 cm). The bees were arrested on the wooden cage as they left the colony. To collect the workers of A. mellifera, these wooden cages were placed at the hive entrance. In each wooden cage, a 1-cm-diameter hole was drilled in one of the cage walls, and a 2-mL Eppendorf tube was inserted. At the bottom of this tube, a small hole (~0.5 mm) was
drilled to allow the bees (group of seven) access to the honey syrup diet. Insecticide solutions were mixed in the diet and never exceeded 10 % of the total diet volume. The bees were fasted for 1 h prior to the experiments before allowed access to the insecticide-treated diet for the subsequent 5 h. The food consumption during this 5-h period was calcu-lated by subtracting the amount of food left in the Eppendorf tube, which allowed us to estimate the dose of insecticide to which the insects were exposed. After this 5-h period, the bees were allowed access to an insecticide-free diet, and mortality was recorded after 24 h. The bees were considered dead if unable to walk when prodded with a fine hair brush. Seven replicates (i.e., a wood cage containing seven bees from different colonies of the same species) were used for each insecticide concentration. During the experimental period, the cages were maintained at 28±2 °C and 65±5 % relative humidity (r.h.).
2.3.2. Topical bioassays
In these bioassays, the bees were ice-chilled prior to the application of the technical grade insecticides, which was performed by applying a 1-μL insecticide solution to each adult forager bee with an automatic microapplicator (Burkard, Rickmansworth, UK). Acetone was the solvent used in the application of the technical grade insecticides. The insecticide was applied on the ventral side of the region located between the second and third pair of legs. The bees were subsequently placed in a 9-cm-diameter Petri dish with the bottom covered with filter paper. Each Petri dish received seven foragers of the same bee species. The bees were kept on the Petri dishes for 24 h with a honey syrup diet offered ad libitum. Seven replicates were used for each insecticide concentration. Bee mortality was recorded 24 h after topical insecticide application. The bees were considered dead if unable to walk when prodded with a fine hair brush. During the experimental period, the Petri dishes were maintained at 28±2 °C and 65±5 % relative humidity (r.h.).
2.3.3. Contact bioassays
papers (Whatman no. 1), which were left to dry for 30 min. These filter papers were used to cover the bottom of Petri dishes (9.0-cm diameter) that had their inner walls coated with Teflon® PTFE (DuPont, Wilmington, DE, USA) to prevent the bees from escaping. Each Petri dish received a group of 10 adult forager bees from different colonies of the same species. Insect mortality was recorded after 24 h of exposure, and the bees were considered dead if unable to walk when prodded with a fine hair brush. An insecticide-free honey syrup diet was provided ad libitum to the bees during the exposure. The bioassays were performed at 28±2 °C and 65±5 % relative humidity (r.h.).
2.4. Statistical analysis
The concentration–mortality data were subjected to probit analysis (PROC PROBIT; SAS Institute2008). The differential insecticide susceptibility between M. quadrifasciata and A. mellifera was calculated for each insecticide based on the estimated LD50 (or median
lethal concentration (LC50)) for each insecticide and bee
species and dividing the LD50(or LC50) value obtained
for M. quadrifasciata by the LD50 (or LC50) value
obtained for A. mellifera (Robertson and Preisler1992). The 95 % confidence limits of these toxicity rate estimates were considered to be significantly different (P<0.05) if they did not include the value 1 (Robertson and Preisler1992).
3. RESULTS
Insecticide susceptibility was assessed in for-ager bees of M. quadrifasciata and A. mellifera (the model pollinator species). The insecticide susceptibility varied significantly with the type of exposure (ingestion, topical, or contact), and there were significant differences between species for some means of insecticide exposure.
3.1. Susceptibility to ingested insecticides
Based on the LD50 obtained in the concen-tration–mortality bioassays, M. quadrifasciata forager bees were very susceptible (LD50values ≤1 μg a.i./bee) to ingested deltamethrin, methamidophos, and abamectin. These LD50values
fell within the highly toxic range of insecticide toxicity suggested by Felton et al. (1986). Deltamethrin and abamectin were also highly toxic to foragers of A. mellifera¸ unlike methamidophos (LD50= 3.7 μg a.i./bee; Table I). For M. quadrifasciata, the scale of insecticide toxicity was abamectin>methamidophos>deltamethrin; for A. mellifera, the scale was abamectin>deltamethrin> methamidophos.
3.2. Susceptibility to topically applied insecticides
Our results showed that M. quadrifasciata and A. mellifera were very tolerant to topically applied deltamethrin and methamidophos (LD50 values ≥100 μg a.i./bee; Table II); therefore, these compounds are virtually non-toxic to these bee species. Topically applied abamectin was also virtually non-toxic to M. quadrifasciata (LD50=136.7 μg a.i./bee) but was moderately toxic to A. mellifera (LD50=7.8μg a.i./bee). The scale of insecticide toxicity for M. quadrifasciata was deltamethrin >abamectin>methamidophos; for A. mellifera, the scale was abamectin > deltamethrin>methamidophos.
3.3. Contact susceptibility to dry insecticide residues
4. DISCUSSION
The present study assessed the insecticide susceptibility of the Neotropical stingless bees M. quadrifasciata and the honey bee A. mellifera. The insecticides exhibited low overall toxicity when topically applied to adult forager bees, while the ingested insecticides were usually very harmful to both bee species. Furthermore, contact exposure to dry residues of deltamethrin and abamectin was moderately harmful, while methamidophos was virtually non-toxic to both bee species. Our results revealed that M. quadrifasciata is usually more susceptible to insecticides in realistic exposures via ingestion and contact than A. mellifera, with the exception of abamectin. In contrast, M. quadrifasciata is usually less susceptible to topically applied insecticides, which is a less realistic means of insecticide exposure. The findings reinforce the importance of testing ingestion and contact exposure rather than only topical exposure and challenge the notion that the honey bee is a suitable pollinator model (or bioindicator) for insecticide toxicity and risk assessment.
Insecticide application is very common in agroecosystems, which may harm many insect species that assist in pollination (Freitas et al.
2009; Johnson et al.2010; Krupke et al.2012). Studies with pyrethroids, fipronil, and espe-cially neonicotinoids have provided accumu-lated evidence that insecticides can cause potential problems for colonies of A. mellifera (Cresswell et al.2012; Gill et al. 2012; Tirado et al. 2013). However, the risk posed by insecticides on other pollinator species has been largely unexplored, although suspicions that these native pollinators are seriously threatened by insecticide applications have begun to arise (Morandin et al.2005; Thompson et al.2007; Osborne2012; Biddinger et al.2013; Decourtye et al.2013).
Bee exposure to insecticide may take place through a variety of means. Although pesticide exposure may occur with compounds applied to control Varroa parasitic mites in beehives, pesticide exposure usually results from the
contact between bee foragers and contaminated plant surfaces, the harvesting of contaminated pollen and nectar, or the ingestion of contami-nated sap from plants originating from insecticide-coated seeds (Johnson et al. 2010; Gill et al. 2012; Mullin et al. 2010). In our study, we assessed insecticide toxicity in polli-nator bees and explored distinct means of exposure (ingestion, topical, and contact expo-sure) by using highly controlled conditions and rigorous experimental design bioassays that enabled comparative toxicity assessments be-tween Neotropical stingless bees and A. mellifera. The results obtained here revealed that ingested insecticides are very harmful for both bee species but are 10-fold more effective in killing native species than the honey bee. This is in accordance with previous studies that have shown that other native bees are more susceptible to insecticides (Cresswell et al.
2012; Lourenço et al. 2012; Hardstone and Scott2010). However, the insecticide abamectin provided contrasting results, showing that the native stingless bee is more tolerant than the honey bee for ingested and topically applied abamectin.
between A. mellifera and M. quadrifasciata observed here may result from differences in the life histories of both bee species. Life histories of different bee species vary con-siderably, and life story traits (e.g., sociality, body size, target-site sensitivity, and capacity for detoxification by enhanced metabolism) have been linked to differential sensitivity to pesticides (Liu et al. 2005; Hardstone and Scott2010; Brittain and Potts2011; Decourtye et al.2013).
The overall higher insecticide susceptibility of the native species compared with the honey bee emphasizes the limited value of the extrap-olation of the results of the toxicity bioassays that compare the latter species to native polli-nator species. Several studies have registered sublethal insecticide effects on other bee species despite the fact that these compounds were considered safe in a risk assessment with honey bees (Decourtye et al.2013); thus, the use of a higher diversity of pollinator species should be considered in such studies.
The Brazilian situation affords an interesting case study due to the economic and ecological importance of the native stingless bee species M. quadrifasciata. This native species is a frequent and important pollinator of native and cultivated species in the country, while the honey bee is an introduced species that does not appear to be a consistent model for assessing pesticide impact in pollinator species (Kevan 1999; Kremen et al.2002; Tomé et al.
2012). The native species M. quadrifasciata is likely a more suitable model for such studies in Brazil because it is a close relative to native endangered species, notably M. capixaba (Moure and Camargo), which is formally recognized as endangered (Resende et al.
2008; Luz et al. 2011; IUCN 2013). Thus, our findings may contribute to improving the overhaul of the insecticide registration process in Brazil by demonstrating that native bees can be more sensitive to insecticide exposure than honey bees. This information may also support the adoption of suitable programs aiming to conserve and use Neotropical stingless bees in agriculture.
ACKNOWLEDGMENTS
This work was supported by grants from the Arthur Bernardes Foundation (FUNARBE), Minas Gerais State Foundation for Research Aid (FAPEMIG), the National Council of Scientific and Technological Development (CNPq), and the CAPES Foundation.
Différence de susceptibilité aux insecticides entre l’abeille sans aiguillon néotropicale Melipona quadrifasciata et l’abeille domestique Apis mellifera
Exposition à un insecticide / toxicité aigüe / pollinisateur / abeille sauvage
Unterschiedliche Empfindlichkeit gegenüber Insektiziden bei der neotropischen stachellosen Biene Melipona quadrifasciata und der Honigbiene Apis mellifera I n s e k t i z i d b e l a s t u n g / a k u t e To x i z i t ä t / Vibrationsbestäubung / wildlebende Bienen
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