Introduction

The Varroa mite, Varroa destructor, is one of the important pests of honey bee colonies which cause serious effects on honey bees (Le Conte et al. 2010; Rosenkranz et al. 2010; Nazzi and Le Conte 2016). Mites feed on honey bees which results in deformation of adult bees, reduce bee populations, and decrease colony products. In addition, Varroa mite can transfer and spread other pathogens (mostly viruses) throughout the colonies (Martin 2001; Martin et al. 2012; Nazzi et al. 2012). This causes extra negative effects (e.g., wing deformity and death) for honey bee colonies. Varroa mite was first described as an ectoparasite of the Asian honey bee species Apis cerana by Oudemans (1904). Since then, the mite has been spread widely across the world infecting other honey bee species; such as, Western honey bee, A. mellifera (Anderson and Trueman 2000).

Considering Varroa mite life cycle, there are mainly two ways by which mites can enter the honey bee colonies: (1) through transportation by adult bees (phoresy); (2) via larval stages of honey bees (Fig. 1). Therefore, knowing the life cycle of bee species and subspecies and their exact behavioral and ecological characteristics may help to reduce mite infestation. Especially, the exact duration of capping period is very important step in which Varroa mites spend their reproductive stage in these capped bee brood cells. The duration of brood cell capping (as one of behavioral mechanism of resistant bees to Varroa) has been widely studied in the literature (e.g., Büchler and Drescher 1990; Ardestani 2015; Oddie et al. 2018; see more studies in Table 1). It has been reported that honey bees with shorter capping period have lower Varroa mite infestation. This was shown in A. m. capensis with approximately 10 days of post-capping period compared to the other A. mellifera subspecies which had an approximately 12-day capping period (Moritz 1994). Büchler and Drescher (1990) also suggested a positive correlation between capping period and mite infestation levels.

Fig. 1
figure 1

A schematic overview of concurrent honey bee and Varroa mite life cycles. The Varroa mites switch between a phoretic phase on adult bees and a reproductive phase within the sealed honey bee brood cells. A female mite enters the brood cell shortly before capping. Depending on the post-capping duration in different honey bee species and subspecies, a number of mature mites will leave the brood cell when honey bees emerge. Each color represents one honey bee life stage (i.e. egg, larval, pupal, and adult stages). The outer and the inner layers show the overall Varroa mite and honey bee life cycles, respectively

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Table 1 A summary of post-capping period of the worker bee brood cells in different honey bees
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Honey bee life cycle contains 4 stages: egg, larva, pupa, and adult. A queen bee may lay thousands of eggs per day in the brood cells. After 72 h, the eggs hatch and the first instar larvae come out. Total larval stage may take up to 7–8 days and after that honey bee larva goes to the pupal stage during which the brood cells are sealed. Depending on honey bee species and subspecies, the capping period may vary from 10 to 12 days. A complete life cycle of a worker honey bee, from egg to emerging adult, will be approximately 21 days (Fig. 1).

Generally, Varroa mites need about 10.4 days to complete their life cycles inside the brood cells and come out of the cells when adult bees emerge (Ifantidis et al. 1988). Therefore, this time interval becomes a limiting factor for the mites to complete their reproduction cycle. This can be observed in some honey bees; such as, A. m. capensis with a shorter post-capping duration. Slightly different duration of capping period has been seen in different honey bee species which can be considered as a resistance mechanism against Varroa mites (Table 1). Other resistance mechanisms are grooming and hygienic behaviors which have been comprehensively studied in the literature (Peng et al. 1987; Boecking 1992; Ardestani et al. 2002; Mondragón et al. 2005; Büchler et al. 2010; Ardestani et al. 2011; Kirrane et al. 2012; Pritchard 2016; Invernizzi et al. 2016; Nganso et al. 2017).

The main aim of this work was to collate the information available on the life cycle of Varroa mites. This study also investigated the effects of neonicotinoids on the bee colonies as these chemicals are one of the most frequently used insecticides in the crop fields. The present study also tried to emphasize the eco-physiological effect that these chemicals pose to honey bee colonies and how other honey bee pests specifically Varroa mite are exposed to and affected by neonicotinoid insecticides.

Neonicotinoid Insecticides: Their Application, Toxicity, and Mode of Action

Neonicotinoid insecticides are among the most widespread chemical compounds in crop protection (Elbert et al. 2008; Goulson 2013; Simon-Delso et al. 2015) and widely used in seed dressing (Sur and Stork 2003; Krupke and Long 2015). They are usually sprayed against sucking insects in agricultural fields; however, they can also affect non-target beneficial insects; such as, honeybees (Lundin et al. 2015). Their usage and application have been re-considered due to the link between neonicotinoids and colony collapse disorder in bees (Simon-Delso et al. 2015). The most important neonicotinoid insecticides are: Imidacloprid, Acetamiprid, Clothianidin, Dinotefuran, Nitenpyram, Thiacloprid, and Thiamethoxam (Nauen et al. 2001; Suchail et al. 2001; Iwasa et al. 2004; Decourtye and Devillers 2010; Simon-Delso et al. 2015).

Besides exposure to the residues through drift from foliar applications, there are two major ways from which honey bees may be exposed to neonicotinoids. The first is through nectar or pollen of crops grown from the seeds treated with neonicotinoids (Codling et al. 2016). These systemic insecticides can be present at ‘trace’ levels (defined here as a range of 1–10 µg insecticide kg−1) in the pollen and nectar of plants; e.g., of the sunflower Helianthus annuus L. (Bonmatin et al. 2003; Desneux et al. 2007; Sánchez-Hernández et al. 2016). Blacquière et al. (2012) also reviewed literature data on neonicotinoid residues in bee-collected pollen, honey, and bee wax and showed that Imidacloprid levels were higher in pollen samples than in honey and beewax samples, ranging between 0.9 and 3.1 µg kg−1 (in pollen). Higher loss of worker bees was observed when bees consumed contaminated pollen with Imidacloprid (40 µg kg−1) (Decourtye et al. 2003). However, other studies have revealed that bees that forage on nectar and pollen from crops grown from the seeds treated with neonicotinoids (Clothianidin) suffer no lethal effects (Cutler and Scott-Dupree 2007). This indicates that the route of exposure may be an important factor influencing the fate of neonicotinoids in honey bee colonies. In addition, the best measure of bioavailability of these compounds is the concentrations measured in the body of worker honey bees. Beside this, the exposure concentration is another important factor for observing effects of these chemicals on honey bees. It should be noted that compounds that are metabolized quickly (i.e. Imidacloprid) may not allow accurate estimation of effective residues in the body of bees. In these cases, actual toxic effects have been estimated from the concentration of neonicotinoids in the ingested food (using LD50s). Another important issue which needs to be considered in the latter studies (Decourtye et al. 2003; Cutler and Scott-Dupree 2007) is that two different neonicotinoids were used in the tests. Therefore, any comparison about observing effects of these two chemicals on honey bees should be taken with caution.

The second potential route of exposure for honey bees to neonicotinoids is through contaminated exhaust dust produced during pneumatic planting of treated seed (Marzaro et al. 2011; Nuyttens et al. 2013). Moreover, the accumulation of neonicotinoids in the contaminated food is another route of exposure to honey bees. In this case, the trace concentrations of neonicotinoids in pollen and nectar will be increased by their cumulative concentrations inside bee brood cells. This may lead to their transport within the colony and contaminate larval food. As a result, further influence on honey bee colonies might be occurred. However, the rate of their transfer from the flowers in the field to the colony might be low. Stewart et al. (2014) analyzed the samples from commercial fields in agricultural production areas in the Mid-Southern US to evaluate the potential exposure of pollinators to neonicotinoid insecticides used as seed treatments on corn, cotton, and soybean. In only 5% of foraging honey bees, the presence of neonicotinoid insecticides (Clothianidin, Imidacloprid, Thiamethoxam, and their metabolites) was detected. In addition, the concentration of neonicotinoids in their collected pollen was rather low (< 1 µg kg−1).

Toxicity of neonicotinoids may vary depending on some factors; such as, the age of the bee, the conditions of the colony, and the subspecies used (Nauen et al. 2001; Wu-Smart and Spivak 2016; Beyer et al. 2018). Toxicity also depends on the route of exposure of honey bees to neonicotinoids, the specific compounds used in the field, exposure time, and concentration levels. It was shown that neonicotinoid metabolites have also some effects on honey bees (Suchail et al. 2001; Decourtye and Devillers 2010) which need to be further investigated. Negative effects of neonicotinoids may be also influenced by other factors; such as, weakness of bee colonies due to the occurrence of Varroatosis (e.g., Straub et al. 2019). So, the observed negative effects on honey bees may be the results of two or more factors. These factors may increase or decrease the effect of each other. In this condition, honey bees might be more sensitive to the chemicals used in the field as a secondary exposure parameter.

Neonicotinoids are generally neurotoxins that act as agonists of insect nicotinic acetylcholine receptors and can be classified as lethal through disruption of insect nervous system (Matsuda et al. 2001; Elbert et al. 2008). They affect the post-synaptic membrane by mimicking the natural neurotransmitter acetylcholine and binding with high affinity. Some of the major metabolites of neonicotinoids are equally neurotoxic, acting on the same receptors (Suchail et al. 2001; Decourtye and Devillers 2010), thereby prolonging the effectiveness of these systemic insecticides. These neurological effects of neonicotinoids may result in the occurrence of adverse effects on the honey bee life stages and behavior (see below). A summary of different studies in which toxicity of several neonicotinoids to honey bees were assessed is shown in Table 2.

Table 2 A summary of neonicotinoid insecticides toxicity to the honey bee Apis mellifera
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Although the toxicity of neonicotinoids to honey bee adults has been reported in the literature and a growing trend of research is focused on this topic (Goulson 2013; van der Sluijs et al. 2013; Fairbrother et al. 2014; Sandrock et al. 2014; Bonmatin et al. 2015; Pisa et al. 2015; Lundin et al. 2015; see also Table 2); however, there is limited information in the literature on their behavioral and ecological effects inside honey bee colonies. This is important when studying different factors; such as, pathogens and parasites (biotic factors), pollutants (abiotic factors), and their interactions influencing bee populations. As described before, honey bee colonies are attacked by other parasites of which Varroa mite is an important pest for the colonies. These mites decrease bee population to a large extent (Rosenkranz et al. 2010). The effect of neonicotinoids on Varroa mites may be beneficial or harmful inside bee colonies. It should be noted that a clear discussion on the factors affecting honey bee health would be useful for current and future research.

Possible Exposure Routes for Varroa Mite to Neonicotinoids

There are several routes of exposure for Varroa mites to neonicotinoids as systemic chemicals:

  1. 1.

    Exposure of mites in adult stage outside honey bee colony

When a mite spends its phoretic stage on honey bees, it is possible for the mite to be exposed to neonicotinoids when these chemicals are sprayed in the field. If the concentrations of these chemicals reach the lethal doses, Varroa mites suffer. This, of course, would be beneficial to the beekeepers by reducing the number of mites in the colony and thereby decreasing damages to honey bee population. If the exposure concentrations are sub-lethal, then the mites may survive and go back to the colony by adult bee workers after foraging. Another potential way of exposing Varroa mites in the adult stage during their phoretic period is via exposure of adult honey bees. When a honey bee drinks nectar from flowers which were previously sprayed with neonicotinoids or, were grown from neonicotinoid-treated seeds, these chemicals enter into the bee’s body and mites may be exposed to these chemicals by sucking honey bee hemolymph. The outcome of this exposure route is also concentration-dependent.

  1. 2.

    Exposure of mites in adult stage inside honey bee colony

There are different ways of exposing to these chemicals when mites are inside honey bee colonies. Mites can be exposed to neonicotinoids when feeding on honey bee larvae. Prior to this step, honey bee larval food is prepared by honey bee workers and it may contain contaminated nectar or pollen collected from the field by foragers (Bonmatin et al. 2003, 2015). The new adult mites emerging from the sealed broods will be exposed to these chemicals by feeding on foraging worker bees. Honey bees inside the colony may take up collected pollen and nectar to transform it to larval and royal food. This may be another exposure route for adult mites in the colony. In all steps, the concentration of neonicotinoids is important. Exposure of the mites to the sub-lethal level of neonicotinoids may be an advantage for the mites (not killing them directly). But, the mites may be suffered when exposing to the lethal concentrations.

  1. 3.

    Exposure of juvenile mites inside the colony

The Varroa mite nymphal stages can only be exposed to neonicotinoids inside brood cells. The main exposure for these stages will be either by feeding on larval food which may be contaminated or by sucking larval hemolymph. In the latter case, larvae have already been fed with the contaminated food by nursing bees and the accumulation of neonicotinoids in larval body (binding to their target receptors in the neurons) would negatively influence the freshly hatched Varroa nymphs.

In summary, toxicity of neonicotinoids to Varroa mites depends on different factors of which exposure concentration may be the most important one. Other factors; such as, the life stage of mites, the type of neonicotinoids used in the field, exposure time, and exposure routes are other important elements in the toxicity of these chemicals to Varroa mite.

The Interaction Between Exposure of Varroa Mites to Neonicotinoids and Mite Life Cycle

Varroa mites that are transported by adult bees inside the colony (from phoretic stage) prefer middle-aged and older nurse bees compared to younger ones (Kraus et al. 1986). The mites invade the non-sealed brood cells of the 5th instar bee larvae shortly before cell capping, as their most favorable target. The complete life cycle of Varroa mite has been described by previous authors (Ifantidis et al. 1988; Rosenkranz et al. 2010; see also Fig. 1). In short, Varroa mother mite starts laying eggs 70 h (2.92 days) after cell capping (Ifantidis et al. 1999; Steiner et al. 1995). Although the first egg is unfertilized, the next fertilized eggs laid in 30 h (1.25 days) intervals (Rehm and Ritter 1989; Ifantidis 1990; Martin and Kryger 2002). It will take 5.8 to 6.6 days for the mite offsprings (female and male, respectively) to become adult (Rehm and Ritter 1989; Ifantidis 1990; Donze and Guerin 1994; Martin and Kryger 2002). Since, mite reproduction is performed inside brood cells, the number of healthy mated female mites which come out of the cells at the time of adult bee emergence, is very important. Therefore, the duration of capping stage of cells is an important factor for successful reproduction of Varroa mite (Rosenkranz et al. 2010).

In Varroa life cycle, mites may be exposed to neonicotinoids via larval food (oral exposure), or via suction of larval body hemolymph (oral exposure), or via metabolized compounds in the body and bound to the neurons (see previous Section). Exposure to sub-lethal concentrations of neonicotinoids may lead to a reproductive advantage for Varroa mites. For example, Wu et al. (2011) reported that honey bees exposed to pesticide residues in brood comb contaminated with neonicotinoids showed a reduction in adult emergence and an increase in brood mortality. This can be resulted in higher fecundity of Varroa mites due to a delay in development and emergence of adult bees and an increase in susceptibility to other pathogens. As a consequence, a reduction in honey bee colony health may be occurred. Except the latter study (Wu et al. 2011), no other data was available on the effect of neonicotinoids on Varroa mite survival and its reproduction inside bee colonies.

The Advantages and Disadvantages of the Effects and Side-Effects of Neonicotinoids

In the literature, it has been shown that insecticides affect individual honey bees in the colony in different ways. Among other effects documented, some are known to lower the developmental rate of queen honey bees, some of them increase the occurrence of queen rejection and lower queen weight (Pettis et al. 1991, 2004), and finally some of them cause honey bee cardiotoxicity (Papaefthimiou and Theophilidis 2001) and affect foraging bee mobility and communicative capacity (Medrzycki et al. 2003). For example, the neonicotinoids Clothianidin and Thiacloprid have been reported to impair the memory processing in honey bees (Piiroinen and Goulson 2016; Tison et al. 2016, 2019). Their exposure to honey bees may disrupt olfactory learning which will be resulted in a negative effect on their foraging behavior (Farooqui 2013). It interferes with the normal transduction of the neural impulse by binding to the receptor of the neurotransmitter acetylcholine on the post-synaptic membrane (Tomizawa and Casida 2005). In addition, sub-lethal exposure concentrations of neonicotinoids have been linked to immune suppression in bees (Sánchez-Bayo et al. 2016).

Only few studies showed the adverse effects of neonicotinoids on honey bee larval developmental stages (Decourtye et al. 2005; Gregorc and Ellis 2011). For instance, Decourtye et al. (2005) showed a delay in the time needed for honey bee adult to emerge from a sealed brood cell (post-capping period) when fed with contaminated food with Imidacloprid at 5 µg kg−1. This might be related to the effects of neonicotinoids on the secretion of Ecdysone hormone which suppresses honey bee pupa to move to adult stage, or other neurophysiological effects. When a mother mite feeds on the hemolymph of bee pupa, this may cause a reduction in the emergence weight, metabolic reserves, and physical deformities of host bees (Bowen-Walker and Gunn 2001; Amdam et al. 2004; Rosenkranz et al. 2010; Nazzi and Le Conte 2016). These negative effects on bee pupa may cause a delay in the developmental stage and prolonged pupal duration. The negative developmental effects of neonicotinoids on bee larvae may have advantages for the life cycle of the Varroa mites, by helping more juvenile to reach the adult stage, thus increasing mite population in bee colonies. But, these effects need to be further investigated in honey bee colonies.

Conclusions

The present study highlighted the interactions among honey bee health stressors and their negative impacts on honey bee colonies. Although neonicotinoid insecticides are applied to kill pests in the crop field; these chemicals also affect beneficial insects like honey bees. Similarly, the ectoparasitic Varroa mites also pose negative impacts on honey bees at different life stages. The routes of exposure of neonicotinoids to honey bees were investigated with emphasizing on the possible combination of their effects in the presence of Varroa mites. However, few studies could be found in the literature on the influence of these chemicals and their metabolites inside bee colonies due to the difficulties in experimental testing. Together with those effects, side-effects of neonicotinoids on honey bee behavior and its survival need to be further assessed. Inclusion of their negative effects, as confirmed by their involvement in the recent colony collapse of bees, should be comprehensively taken into account in long-term beekeeping programs.