In recent decades, many modern agricultural technologies developed to control crop pests base their efficacy on pesticide use (Schreinemachers and Tipraqsa, 2012). This, in addition to other factors like diseases and changes in land use, could contribute to the rapid decline of honey bee populations (Hawthorne and Dively, 2011; Wu et al., 2012; Johnson et al., 2013). But not only acute exposure to pesticides can cause the decline of honey bees, also minute, sublethal amounts of pesticides cause fluctuations in populations that might be less noticeable than direct, acute exposure (Thompson, 2003; Pettis et al., 2004; Yang et al., 2008; Wu et al., 2011; Henry et al., 2012; Hopwood et al., 2012; Nabti et al., 2014); studies have shown that sublethal exposure to pesticides could result in (1) adaptation of the colonies to contaminated environments or (2) death in the short-medium term, by slow poisoning (Moser, 1995; Moye and Pritsos, 2010). A similar scenario might be occurring in other highly social bee species. Actually, just few studies have investigated how stingless bees respond, adapt or perish to xenobiotics, even though they play an important role as pollinators in neotropical environments (Valdovinos-Núñez et al., 2009; Stanley et al., 2010; Osborne, 2012; Sánchez et al., 2012; Tome et al., 2012; Barbosa et al., 2015).
Bees provide a critical service to ecosystems and human wellbeing by pollinating natural vegetation and crops; yet, the implementation of strategies aiming to keep them from unfavorable circumstances such as fragmentation, diseases and agricultural insecticides, may not seem enough to ensure survival (Klein et al., 2007; Winfree et al., 2009; Brittain et al., 2010; Johnson et al., 2010; Henry et al., 2012; Whitehorn et al., 2012). Highly social bees, like the honey bee, Apis mellifera, and the bumble bees, Bombus spp, are highly sensitive to a wide variety of insecticides used to regulate insect pest species; actually, given the intricate networks in nature, exposure to these substances cause several unpredictable detrimental effects, from individual to community levels, often disrupting key ecological processes which ultimately threats biodiversity conservation and human food safety (Devillers and Pham-Delegue, 2002; Stefanidou et al., 2003; Decourtye et al., 2004; Choudhary et al., 2009; Alaux et al., 2010; Brittain et al., 2010; Hardstone and Scott, 2010; Johnson et al., 2010). Agricultural landscapes are of special concern, since the risk of contact to insecticides is higher, not only through consumption of pollen and nectar, but also through exposure to contaminated soil, water and foliage, not mentioning spray drift and pesticide runoff (Krupke et al., 2012). Studies have found up to 121 pesticides, including pyrethroids, organophosphates, organochlorines, carbamates, fungicides and herbicides, in pollen, wax and honey from honey bee colonies in agricultural landscapes (Mullin et al., 2010; Kujawski and Namie, 2011; Krupke et al., 2012; Rodríguez et al., 2014). Such scenario in which bees occur near crop fields should raise their importance not only as providers of pollination service and drivers of higher crop yield and quality, but also as biological models for ecotoxicological studies that investigate the pressure that pest management schemes inflict upon non-target organisms like bees.
Exposure to insecticides produces biochemical alterations at cellular levels, which can be used as markers of intoxication (Lagadic et al., 1997). Several biochemical markers of xenobiotic exposure have been investigated in bees (Badiou and Belzunces, 2008; Badiou et al., 2008; Badiou-Bénéteau et al., 2012; Carvalho et al., 2013), and enzymes have attributes that make them suitable as indicators of pesticide contamination (Carvalho et al., 2013). Enzymes such as carboxylesterases (CAE), glutathione S-transferases (GST) and cytochromes P450 have been investigated in bees in search of any evidence of pesticide exposure since they play a role in the detoxification of a large number of toxic compounds and in insecticide resistance (Thompson, 1999; Johnson et al., 2006; Badiou et al., 2008; Karatolos et al., 2012; Badiou-bénéteau et al., 2013). Carboxylesterases hydrolyze carboxylic esters and render organophosphate insecticides useless (Montella et al., 2012). Glutathione S-transferases catalyze the conjugation of glutathione to a variety compounds; likewise CAE, they are linked to resistance based on the metabolism of organophosphates, carbamates and pyrethroids (Thompson, 1999; Li et al., 2007). Cytochromes P450 are detoxifying enzymes that use iron to oxidize a wide array of substances that participate in the normal metabolism; they also catalyze xenobiotics from natural or synthetic origin (Wilce and Parker, 1994; Feyereisen, 1999; Armstrong, 2018). Overexpression of cytochromes P450 is associated with resistance based on the metabolism of insecticides (Agosin, 1985; Hodgson, 1985), and their inhibition can block important metabolic pathways in insects. Thus, a more precise analysis of xenobiotic exposure is achieved by examining all these markers simultaneously (Badiou-Bénéteau et al., 2012). Of special interest is the cholinergic enzyme acetylcholinesterase (AChE), which despite not being a detoxifying enzyme, has been widely used as a marker due to its high sensitivity to pesticides, particularly to organophosphates and carbamates (Walker, 2001; Key and Fulton, 2002; Narbonne et al., 2005; Badiou and Belzunces, 2008; Badiou et al., 2008; Tu et al., 2009; Boily et al., 2013), though it is also sensitive to metals, detergents and complex contaminant mixtures (Gill et al., 1990; Payne et al., 1996; Guilhermino et al., 1998, 2000). The biological role of AChE consists of catalyzing the hydrolysis of the neurotransmitter acetylcholine at the synapsis of the insect’s central nervous system (Casida and Durkin, 2013; Johnson, 2014). The inhibition of AChE results in the accumulation of acetylcholine in the synapse and a consequent sustained excitation of the nerves, which eventually causes tremors, paralysis and dead (Fukuto, 1990; Pohanka, 2011). As a result of alterations in biochemical pathways, several deviations in physiology (fecundity, fertility and longevity) and behavior (home-flight orientation, proboscis extension reflex and choice behavior impairment) have been documented, which can also be used as indicators of insecticide exposure (Decourtye et al., 2005; Han et al., 2010; Cresswell and Laycock, 2012; Henry et al., 2012).
Most of the research about pesticide effects on pollinators has been carried out with A. mellifera, which is often considered an acceptable surrogate for other highly social bees, such as stingless bees (USEPA, 2014; Thompson and Pamminger, 2019). Stingless bees are highly social bee species living in the tropical fringe around the world; they are considered important native pollinators, linked to cultural and economic aspects in the areas in which they occur (Kremen et al., 2007; Van-der-Valk and Koomen, 2013; Barbosa et al., 2015; Giannini et al., 2015). They share many ecological features with honey bees, but there are also many biological differences that hamper the use of knowledge obtained with honey bees to understand stingless bees (Van Veen, 2014). Available information indicates that there can be substantial dissimilarities among bee species in their response to xenobiotic exposure. In their seminal review on pesticide sensitivity on bees, Arena and Sgolastra (2014), showed that some bee species have a similar LD50 to several pesticides, but others do not. In the specific case of stingless bees, these authors show that Melipona scutellaris and Scaptotrigona postica are more sensitive to pesticides; on the other hand, Nannotrigona perilampoides, Melipona beecheii, Trigona iridipennis, T. nigra and T. spinipes have a similar sensitivity to that of A. mellifera. Such differential susceptibility to pesticides may be caused to particular life history traits, such as nesting biology and foraging behavior, and even intrinsic physiological traits that have been evolutionarily developed (Cham et al., 2019). Moreover, 65.9%, 31.8% and 2.3% of 869 research items dealing with pesticides and bees, was carried out in honey bees, bumble bees / solitary bees, and stingless bees, respectively (Lima et al., 2016). Therefore, there is a clear lack in our knowledge on stingless bees’ ecotoxicology, which needs to be circumvented if we are to keep ecologically functional ecosystems.
In our study region, el Soconusco, Chiapas, Mexico, the use of insecticides is a common practice for the control of fruit flies (Flores and Montoya, 2010), thrips (Infante et al., 2014), human disease vectors (Fernández-Bremauntz et al., 2004), and other pests. A recent work revealed the presence of 15 organochlorine compounds and glyphosate in water and stingless bee colonies (Ruiz-Toledo et al., 2014, 2018). Such practices and the relentless expansion of urban areas inevitably jeopardize populations of stingless bees (Brown and Albrecht, 2001; Cairns et al., 2005; Brosi et al., 2007; Brosi, 2009; Valdovinos-Núñez et al., 2009; Brown and Oliveira, 2014; Hakrabarti et al., 2014). Still, very little is known about stingless bees susceptibility to insecticides and consequent alterations in enzymatic markers (Osborne, 2012). Understanding their biochemical response to xenobiotic exposure could give more information about their adaptation to anthropogenic changes and the degree of stress they are subjected to in agricultural landscapes (Souza et al., 2015).
In this study, we investigated the expression of the AChE, esterases, GST and cytochromes P450 in foragers of the stingless bee S. mexicana in two sites within an agricultural landscape in El Soconusco, Chiapas, Mexico, with the objective of knowing the changes that occur in these markers along a year. We chose S. mexicana as our biological model because it is commonly used in meliponaries and has been extensively investigated (Guzmán-Díaz et al., 2004; Sánchez et al., 2016), it is relatively common in the study area (Ayala Barajas, 1999), and it has a cultural and economic role in local communities. We also carried the same analyses in A. mellifera to provide evidence about its use as a surrogate for this stingless bee species.