5.2.1. Climate Change

In order to assess the potential impact of a future climate change on populations of cultured bees and other pollinators in the long term, the effects of Earth's climate changes over the last millennia should be analyzed. There have been three cooling periods and three warming periods over the last 3000 years [56,57]. For example, the first global cooling took place in the Early Middle Ages; the so-called "Dark Ages" (Procopius, History of the Wars) may have resulted from a powerful volcanic eruption, probably in Indonesia, which saturated the atmosphere with dust and toxic gases. It was followed by a global warming between the 10th and the 14th centuries, and consequently a new cold period, known as the "Little Ice Age", which continued until the mid 19th century [58,59]. During these alternating periods, which had somewhat contrasting effects on agriculture and crop zoning as well as on the flora and fauna of the moderate latitude, bees did not disappear completely, even though they had undergone some population fluctuations, as can be seen from the historical documents cited above. The current warming, which has been claimed to have been around since 1850, should not have had an adverse effect on insects, including those involved in plant pollination. Climate change can impact on honey bees at different levels. It can have a direct influence on honey bee behavior and physiology [60]. It can alter the quality of the floral environment and increase or reduce colony harvesting capacity and development. It can define new honey bee distribution ranges and give rise to new competitive relationships among species and races, as well as among their parasites and pathogens [60]. Beekeepers will also be obliged to change their apiculture methods. They will favor moving their hives to new foraging areas and importing foreign races to test their value in the new environments.

Another study has investigated climate's influence on honey bee winter mortality rates across Austria [61]. The results have shown statistical correlations between monthly climate variables and winter mortality rates indicated that warmer and drier weather conditions in the preceding year were accompanied by increased winter mortality.

#### 5.2.2. Unfavorable Weather Conditions

In addition to historical data, a number of contemporary authors [62–64] also attach grea<sup>t</sup> importance to unfavorable weather conditions and dramatic changes in weather as a factor in the reduction of bee colonies. Long periods of cold and rainy or hot and dry weather are associated with the sudden and unexplained disappearance of bees and the emptying of hives [28]. A probable cause is the prolonged lack of nectar and pollen (foraging), which inevitably leads to the collapse of bee colonies. The cold spring of 2013 in the USA led to a later development of hives, thus shortening the period for development of *V. destructor*. The smaller number of developed generations of the parasite, as well as the subsequent warm period suitable for active foraging time, contributed to a significantly lower loss of bee colonies in 2013 compared to the following 2014 [65,66]. In the same connection, but in the opposite direction, there also is the suggestion that chronic hive intoxication due to the treatment with thiomethoxan (a neonicotinoid), may delay the development of the larvae and endangering the existence of the bee colony [67].

Between 2006 and 2011, about 32% of hives in the USA kept dying every year due to unsuccessful wintering [3]. The same was valid for the winter of 2012–2013, when 31% of the hives perished. According to beekeepers, a symptomatic pattern similar to the "empty hive" can be observed in years when the temperature rises early in the spring, often before the snow melts. During this period of the year, bees need more water, including for breeding o ffspring. On the other hand, the e ffects of low temperatures and wind can also hinder the return to the hive, resulting in mass losses. To avoid this particular mechanism of emptying the hive, beekeepers recommend providing warmed liquids with added sugar or honey.

#### **6. Pesticides as a Factor in the Deterioration of the Health Status of Bees and Bee Colonies**

Bees are exposed to pesticides—through the chemical means used by beekeepers to control diseases and pests in or around hives. The contrasting results with clothianidin treated seeds were reported because this pesticide primarily influenced arthropod communities during the four weeks following planting, with disruptions to major natural enemy taxa, but communities showed trends toward recovery at the later corn stages. [68,69]. Rundlof, et al.'s [68] study took place in spring and reported negative e ffects on bees, whereas Sterk, et al.'s [69] study took place in autumn and reported no significant e ffects. Bees come in contact with pesticides when drifting (driven by air currents) through spray drift or dusting from an applied product, for example, in proximity to recently sprayed growing plants, when soil is treated or when treated seed is used for sowing [70,71]. Oral intoxication is also possible when visiting flowering weed vegetation recently treated with pesticide or when ingesting contaminated food and water—pollen, nectar, guttation drops, and honeydew on treated plants, etc. [72–74].

Sublethal doses of pesticide products, or even some pesticides considered to be completely safe, can lead to severe losses or endanger the existence of the bee colonies [75]. According to some observations, if during the treatment of apiaries is adjacent to the other apiaries, bees fly through the spray drift of the pesticide, and then they absorb with their bodies an odor di fferent from the one specific to the hive [76,77]. Impacted individuals are, thus, not recognized by the bees guarding the bee colony, and, as a result of which, they are not allowed into the hive or are killed as invaders [78–80]. This circumstance further necessitates the strict application of the requirement to broadcast warnings locally before carrying out all chemical treatments—spraying, sowing of treated seeds, etc. It is obvious that the phenophase of the target culture—flowering or other—is irrelevant in this case.

#### *6.1. E*ff*ect of Pesticides (Other than Neonicotinoids) on Bee Health*

Exposure of bees to sublethal doses of pesticides occurs not only with systemic products (most commonly, aqueous solutions applied to vegetative plants) but also with non-systemics, such as pyrethroids and organophosphates, which can reduce the lifespan of individuals [75,76]. Recent studies have shown that low levels of intoxication—oral or contact, with active substances other than neonicotinoids—can weaken the immune system of exposed individuals, impair their ability to learn by monitoring and communicating with other bees, thus leading to memory loss and a change in their eating behavior and ability to distinguish flavors [72–84].

Increased levels of chlorothalonil fungicide have been detected in bee pollen in hives where high mortality has been observed. However, artificial feeding of larvae and adults with a product containing the same pollen does not cause an increase in mortality among individuals during an experiment [85]. The bees are more susceptible to most insecticides but particularly to fipronil (the most deadly to bees), most neonicotinoids and pyrethroids, and some organophosphates. According to Smith et al. [35], the greatest challenge for researchers is to extrapolate data on the e ffects of pesticide action on bees obtained at the individual experimentation level to the whole bee colony. Studies on their e ffects on bees are approved by OECD (Organisation for Economic Co-operation and Development) toxicity test guidelines. By DNA microarray analysis of transcribed products from the intestinal tract of beehives in decay, Johnson, et al. [86] found the presence of unusual RNA fragments that are thought to result from infection with one or more viruses. The authors did not find increased expression of genes related to the body's response to pesticide intoxication. For at least two decades, individual researchers have focused on exploring the mechanisms of bee detoxification and, in particular, on the role of the P450 gene, which encodes the ability of bees to metabolize toxic compounds that have fallen into the hive [87,88]. It has been suggested that there is a risk of honey bee survival if honey bees collect and drink water from water puddles in crops treated with neonicotinoid insecticides [89]. Also, honey bees and other native pollinators are threatened by cumulative exposure to these insecticides from residues in pollen, nectar, and water [89].

#### *6.2. Neonicotinoid Insecticides*

Neonicotinoids are synthetic alkaloid insecticides, analogs of natural nicotine. In treated plants, they systematically propagate acropetally (ascending xylem). They are placed in the group of neurotoxic insecticides. Neonicotinoids bind to the nicotinic receptors located in the postsynaptic membrane of neurons to cause their activation because they are agonists of the receptor [90,91]. Indeed, they are selective because they tend to bind preferentially to one of the subunits ( α4β2) that make up the receptor, which happens to be more common in insects than in vertebrates. Neonicotinoids are applied in di fferent forms—spraying of aboveground parts of plants, treatment of seeds, and application directly to the soil in a wide range of crops. Two groups of neonicotinoids are known to date: cyano-substituted (acetamiprid and thiacloprid) and nitro-substituted (imidacloprid, thiamethoxam, clothianidin, nitenpyram, and dinotefuran). Furthermore, it has been demonstrated that the three main neonicotinoids used in agriculture the world over, namely imidacloprid, thiamethoxam, and clothianidin, pose the highest risks to bees among all other pesticides [92,93]. The di fference is in the chemical formula determining the di fferent toxicity of the two groups of active substances against bees [92,93]. For example, cyano-substituted neonicotinoids, like thiacloprid and acetamiprid, increase their toxicity to bees by 500 and 100 times in the presence of azole fungicides.

From the dawn of this millennium, a broad debate has started within the scientific community, giving rise to a number of research programs on the negative impact of neonicotinoid pesticides on pollinating insects, in particular, the managed honey bee or otherwise exploited wild bee colonies [3,38,94,95].

Neonicotinoid insecticides enter the body of honey bees, bumble bees, or other pollinators when insects feed on nectar and pollen from treated plants [96]. Krupke, et al. [97] considered di fferent routes of contamination of beehives with neonicotinoids but focused primarily on the use of coated seeds and granules, which contaminate the soil. Yang, et al. [98] found a negative e ffect of sublethal doses of imidacloprid on honey bee behavior. Feeding individuals with sugar syrup containing 50 μg/<sup>L</sup> has shown to prolong the time interval between two visits to the feeding site [99,100]. The use of 1250 μg/<sup>L</sup> imidacloprid syrup for food results in significant behavioral changes, with some individuals failing to return to their normal foraging habits, while for others, the time required to return to the hive is extended significantly [99,100]. Increased mortality as a result of disorientation and inability to find the way back to the hive has been observed in bees intoxicated with thiamethoxam [101,102]. Bee colonies exposed to chronic e ffects of clothianidin and thiamethoxam experience significant, detrimental short and long-term impacts on colony performance and queen fate, which suggests that neonicotinoids may contribute to colony weakening in a complex manner [46]. The same authors have found that losses in individual bee colonies are determined by di fferent levels of genetically determined resistance of bees to intoxication [46].

Some comparisons are needed with regard to the usage of neonicotinoides in di fferent countries. In Australia, 80%–90% of beekeepers avoid agricultural fields and place their hives in forests to ge<sup>t</sup> the best harvest of honey. Therefore, their exposure to pesticides is minimal, unlike what occurs in the USA, Europe, Japan, or China. Australian beekeepers also know that whenever they take their hives for pollination of almonds, they lose hives, and when their bees forage on neonicotinoid-treated canola fields, they ge<sup>t</sup> sick and lose more hives the following winter. Moreover, Australia has been fortunate, to date, to avoid any incursion of Varroa, which presents a major threat to the health of honey bees [103]. For this reason, Australia exports bee products, bee queens, and whole hives for pollinating crops. On the contrary, imports are strictly prohibited, and quarantine measures are mandatory [104]. Because the use of neonicotinoid insecticides have proven to have particularly harmful e ffects on the environment (especially pollinators, and that includes bees above all). France becomes the first country in Europa that banned the use of five neonicotinoids completely.

#### **7. Interactive and Cumulative E** ff**ects: Action of Biotic and Abiotic Stressors**

With regard to the causes of death of bee colonies, opinions are most often polarized, claiming that one or another individual stress factor is the main, if not the sole, cause of the phenomenon. Recently, it has become increasingly accepted that the combined action of two or more adverse factors of di fferent nature increases the risk of colony collapse. It has been hypothesized that the poor health status of bees is the result of individual or combined action of various factors such as stress due to poor nutrition, fasting and "monocultural" diet, abrupt meteorological changes, reduced genetic diversity in honey bee populations, etc., not excluding additional, chronic pesticide intoxication [30,39]. Too little is known about the immune response in bees at the individual and colony level. However, as social insects, bees can rely on a collective immune response to protect the colony as a whole [105]. Recently, balanced feeding of pollen and propolis has been found to be able to activate detoxifying enzymes in the individual bee [106]. It is also considered that experimental data on the e ffects on the protective capabilities of an individual cannot be automatically extrapolated to the entire colony in actual field experiments [35].

Recent studies have shown that interactions between pesticides and pathogens lead to deterioration in the health status of bee colonies [74,107,108]. Exposure to neonicotinoid pesticides increases the sensitivity of bees to the intestinal parasite *N. ceranae* [109]. Imidacloprid is able to synergistically increase the level of infection with Nosema spp. [73], as well as mortality [107], when both stressors are present simultaneously in the hive. Similarly, Aufauvre, et al. [74] found higher mortality from fipronil intoxication and infection with *N. ceranae* combined than when the two agents acted in isolation.

The interaction between stressors is not limited to pesticides and pathogens. Very often, the malnutrition of beehives is highlighted as a factor increasing the losses caused by bee parasites. For example, the parasite Crithidia spp. causes less mortality if wild bees have a complete food source [110]. Goulson, et al. [30] sugges<sup>t</sup> that nutritional stress influences LD50 values (lethal dose, 50%) for individual bee toxic compounds. This is evidenced by the varying values of LD50 for pesticides in separate, independent studies [75].

Goulson, et al. [30], note that the individual factors that have a negative e ffect on bee health do not act in isolation. Obviously, all types of bees are subjected to di fferent stress factors at the same time and with an accumulating e ffect over time. In doing so, each individual factor reduces the ability of bees to

overcome the negative e ffects of the action of other stressors. The mortality of bees and bee colonies is likely to be lower if the parasite-infested hive is not further exposed to sublethal doses of toxic substances, incl. antibiotics and acaricides used in beekeeping. Moreover, the pesticides in agriculture and/or bees are not starved or subjected to a monotonous diet, often as a result of adverse weather conditions, such as prolonged drought or low temperatures. The conclusion is that complex causes require the search for complex solutions to the problem. The strategy should be aimed at reducing the general and individual stress from the action of various adverse factors by radically changing the environment in which the bees live and in which they perform their functions.

#### **8. Some Examples for Solving the Problem of Honey Bee Population Decline**

#### *8.1. The Hindu Kush Lessons*

The prospect of life without bees and other pollinators was demonstrated in the Maoxian region, Sichuan province, Southwestern China, part of the Hindu Kush Mountain, where bees, both wild and honey-bearing (European and Eastern,) disappeared more than 20 years ago [111]. In the early 1990s, local, until then self-su fficient, farmers, largely catering to their own needs, set out to create market-oriented apple and pear plantations. Both fruits are self-sterile, which requires pollinating the flowers with pollen from other, genetically distant species (varieties) of the species. With the intensification of production and the desire to market better looking fruits, the use of pesticides, and in particular, insecticides, increased. The mistaken perception that the cause of the lower pollination is insect pests attacking the flowers has led to an even more intensive use of insecticides. Natural habitats—alternative sources of nectar for bees—mainly forests and natural vegetation, have been replaced by new, industrial plantations. This e ffectively reduced the bee feeding period to 14 days a year. The second big problem in the region was the lack of managed honey bees and other pollinating insects. As of 1999, the problem covered neighboring Hindu Kush areas—territories of India, Pakistan, and Nepal [111,112]. The case is unique in that, in order to survive, local farmers were forced to switch to manual pollination of fruit tree flowers [111]. In India, Nepal, and Pakistan, the problem was solved with the restoration of native vegetation, providing habitats and food sources for bees, appropriate managemen<sup>t</sup> of the natural pollination process, and training of farming colonies. Thus, bee populations were restored, and after 2011, manual pollination has rarely been practiced.

What conclusions can be drawn from the Hindu Kush experience? The main problem is, however, the use of certain pesticides in agriculture, even as recommended on the labels [113]. Another problem is the approval of certain compounds and formulations for use in some crops because they pose more risks to bees and the environment than those estimated by the regulatory authorities. The latter is due to authorities using insu fficient or inappropriate information and out of date methods [114]. Improper use of pesticides in agriculture is part of the problem, but it is not the only cause of death for honey and other bee species. Refusing to use pesticides, even if possible and as a sole measure, would not be a solution to the problem. Restoring and preserving the natural habitats of pollinators today can ensure the diversity of our table tomorrow [115].

#### *8.2. The Experience of North America*

With the decrease in the number of bee colonies in the United States by more than 50% as a result of the introduction of parasitic mites, heavy pesticide use, and industrialized plantations, crossbreeds between the European, *A. mellifera* and the African honey bee, *Apis mellifera scutellata* Lepeletier have been introduced [116]. New, hybrid forms show increased resistance to parasites, infectious diseases, and some pesticides [117,118]. They also find successful application in the agricultural practices of Latin American countries, such as Brazil, where they have replaced the European bee and have even been rated as better pollinators than the European one [119]. The problems associated with the use of these: the so-called "Africanized" bees or "killer bees" include smaller numbers of workers in one colony, shorter flight and perimeter of feeding around the colonies, and high mortality when moving to

a new foraging site. In addition, Africanized bees are extremely aggressive. They are now considered inapplicable to the USA because of the mobile nature of bee pollination used there for many crops [117]. The measures taken in the country are aimed at the use of less intensive forms of agriculture related to reducing the dependence of agriculture on the use of pesticides. The conservation of weeds and other vegetation around arable land where bees would find additional foraging for a longer period is promoted. Research in this regard shows that structural changes in the landscape distribution of crops with the conservation and/or introduction of wild plant species increase the species diversity of bees and pollinating butterflies [120].
