**1. Introduction**

The European honey bee, *Apis mellifera* L. (Hymenoptera: Apidae) has been managed for millennia [1]. The species was domesticated by the ancient Egyptians around 2600 BC [2]. Since then, bees have been used as a source of unique, natural, multifunctional products (basically honey and wax). However, the more significant ecological function of the species is related to the pollination of a wide range of agricultural and wild plant species. The honey bee is one of about 200,000 existing species of plant pollinators [3]. Approximately 84% of the crops grown in Europe are dependent on insect pollination [4]. The only major exception is grain crops that are wind-pollinated. Bees, both wild and managed, provide about 80% of the insect pollination of flowering plants [5]. Among them, the honey bee plays the most important role in the process, especially in the pollination of apple, cherry, small grapes, kiwi, broccoli, carrots, cauliflower, celery, cucumber, onion, pumpkin, legumes, sunflower, and many others.

The role of *A. mellifera* as a pollinator in natural habitats is important for several reasons. First, animal-mediated pollination represents vital ecosystem maintenance, i.e., 87.5% of flowering plant species are pollinated by animals [6]. It is well known that the honey bee is considered as a super generalist, which provides a vital role in the functioning of many terrestrial ecosystems [7]. Unlike other pollinators, which have declined significantly in recent years, mainly as a result of habitat loss, *A. mellifera* populations can overcome these disorders [8]. The recent increases in the mortality of managed *A. mellifera* colonies in some parts of the world (USA) may increase the feral population, which is more susceptible mainly to Varroa mite [9]. In some countries, the introduction of populations *A. mellifera* has led to competition with other pollinators [10,11]. A typical example is Australia, where beekeepers are restricted to use agricultural land but not national parks for this very reason [12]. Given the importance of honey bees for the nourishment of humankind, the reducing of the number of bee colonies in Europe and America has been met with increasing concern in recent decades. Some of the published materials describe the unusual and somewhat tragic consequences of a future crisis in honey bee populations and other pollinator species [13,14]. Other studies have linked the problem with the degradation of natural ecosystems and the persistent tendency to lose ever-increasing amounts of the planet's biodiversity [15,16].

The purpose of this report is to summarize and analyze data published in the scientific literature on the causes, drivers, and conditions associated with the poor health status and death of bees and bee colonies.

#### **2. Historical Occurrences of Honey Bee Decline**

#### *2.1. Prehistoric Extinction Event*

There have been known cases of complete bee extinction or population crises. It has been established that prehistoric mass extinction of bee species from the Xylocopinae colony took place during the transition between the Cretaceous and Paleogene geological periods, about 65.5 million years ago [17]. The period has left a characteristic imprint on geological layers—a thin clay layer known as the K–Pg (Cretaceous-Paleogene) or K–T boundary. On the base of sequence and phylogenetic analysis of two mitochondrial COI (cytochrome oxidase subunit 1) and cytb (cytochrome b) and two nuclear genes, F1 and F2 Elongation Factor-1 α (EF-1 α) of Xylocopinae tribe [17], the authors suggested the disruption of many plant-insect relationships at that time, which had a negative e ffect on the bees. The Cretaceous–Paleogene mass extinction event is believed to have been caused by a devastating change in the global climate.

#### *2.2. Medieval Bee Mortalities*

In medieval Europe, there was a mention of "bee mortality" in Ireland in 951, according to the Annals of Ulster. Citing the same source, Fleming [18] noted "high mortality among humans, cattle and bees" again in Ireland in 992. In the same year, the extinction of bees was preceded by a long and severe winter, and drought in the summer, combined with an attack by fungal "diseases or turf" (namely, ergot, *Claviceps purpurea* (Fr.) Tul. 1853) on cereals. The ergo<sup>t</sup> sclerotium contains high concentrations (up to 2% of dry mass) of the alkaloid ergotamine, which accumulates in the so-called sugary honeydew, which attracts bees. The result was a mass famine and an epidemic of ergotism in France in the same year. The harsh winter and dry summer preceded the "unprecedented pestilence and devastation of the bees" a ffecting all of Bavaria in 1035. The unusual cold in the summer of that year eliminated the production of corn and fruit (Annals of Ulster).

Even these historical records from medieval Europe point to a clear causality: bad weather resulting in decreased nectar production, which, in turn, had a negative impact on colony productivity.

The earliest recorded shipment of bees to the New World (North America) took place from England in 1621. By 1650, nearly all farms in New England are reported to have had one or two colonies of bees, but there is evidence that the number of bees managed by American colonists declined after 1670, 50 years after the honey bee was introduced from Europe to North America. According to Pellett [19], the most likely cause was associated with an unknown infectious disease.

#### *2.3. Other Unexplained Evidence for Bee Mortality*

Many of the bee epidemics recorded in detail in the past remain unexplained. For example, in 1903, 2000 colonies were lost to an unknown "disappearing disease" in the Cache Valley of Utah [20]. Significantly, however, the previous winter had been hard and the spring cold. In 1995–1996, beekeepers in Pennsylvania lost 53% of their colonies to no specifically identifiable cause [21]. One of the most famous epidemics occurred on the Isle of Wight, a small island o ff the south coast of England, when three separate epidemics between 1905 and 1919 wiped out 90% of the island's bees [22]. In this case, pathogenic factors, such as the tracheal mite *Acarapis woodi* and the microsporidia *Nosema apis*, were involved. Thereafter, after long and lengthy research over the years it is suggested that the disease had been due to a combination of factors, in particular, infection by Chronic Bee Paralysis Virus (completely unknown at the time), together with unfavorable climatic conditions that restricted the growth of plants and, hence, indirectly a ffected the foraging by bees because of the lack of flowers [23].

Although the number of managed beehives globally has increased by 45% since 1961, the proportion of bee-dependent pollinating crops has increased much more steeply—by 300% [24]. In this regard, many beehives, especially in countries with industrialized, high-intensity agriculture, are raised as mobile units for the purpose of pollination of crops, not for the production of bee honey and other products [25]. Insu fficient attention has been paid to the potential negative e ffect of long-distance transport of beehives and the related stress for bees [26,27].

#### **3. Colony Collapse Disorder Syndrome**

Many studies link the death of bee colonies to the popular Colony Collapse Disorder symptom/syndrome (CCD). As the name suggests, a typical characteristic of this pathological phenomenon, is the total absence or the presence of very few adult (imagined) bees in the hive, in the presence of live brood (eggs, larvae, pre-pupa<sup>е</sup>, and pupaе) and an alive bee queen. A common feature is that worker bees leave the hive and do not return [28,29]. In fact, there is no factual evidence of the death of individuals. No dead bodies are found inside or in front of the a ffected hive, which is a characteristic sign of acute poisoning [30]. In addition, there is no indication of an invasion of the hive by other insects, such as wasps or hornets. Visual inspection shows that live individuals in the hive, both imagined and brood, are usually infested by the ectoparasitic mite—*Varroa destructor* [31]. Other pests found in all bee colonies a ffected by the syndrome are intracellular, intestinal parasites (microsporidia), mainly *Nosema ceranae*, and other representatives of the same genus [32]. Other unspecific signs indicating that the bee colonies are in crisis but not necessarily dictated by CCD include the shortened period (from approximately 25 to 5 days) during which young bees take care of larvae and pupae before becoming workers that produce wax; significantly delayed attacks by hive pests, colony includes mostly young adult bees, etc. In its classic form, the syndrome appears to be localized only in the United States and, in some cases, in Europe [33].

#### **4. What Actually Leads to Honey Bee Decline?**

Widespread notions, including such among the academic community, relate the main problem—the decrease in the number of bee colonies and any other form of bee losses—mainly or exclusively, to the aforementioned pathological phenomenon of CCD. However, scientists and experts involved in researching the problem consider two forms of honey bee losses:

1. Annual (most frequent)—as a result of unsuccessful wintering caused by biotic factors (such as infections and parasites), acute intoxication, and a number of other causes, which are subject of the discussion in this review [34]

2. Multi-annual—permanent reduction in the number of bee colonies in separate, specific regions.

For example, it is known that from the mid-1980s until now, the number of bee colonies in Europe has decreased by 25% and in the United States by 50–60% [35]. At the same time, despite sporadic local extinction, the number of bee colonies globally has increased by about 45% over the last 50 years and more specifically since 1961 [24,35]. The latter seems hopeful, but these data must be interpreted with care. Usually, beekeepers compensate for the loss of one hive by splitting it into two. This results in the "weakening" of the two new bee colonies, and the long-term e ffect of repeated application of this practice has not been fully studied. Very often, there is a lack of objectively collected information on the status of the honey bee in individual countries, on population changes over the years, and by region. For example, in Austria and Czechia, colony losses during winter were fluctuating from year to year, with strong regional di fferences. It has been observed that winter losses related to queen problems, di fferences in population dynamics, and treatment against the Varroa mite, di fferences in the number of beekeepers and colonies, etc. [36]. Generally, in the 20th century in Europe, two periods were distinguished according to the prevailing trend: an increase in the total number of bee colonies in 1965–1985, and a decrease in the number of beehives in 1986–2005 [4]. The number of beekeepers, both professionally engaged and those for whom beekeeping is a hobby (holdings with up to 50 bee colonies), has also been decreasing in Europe. The changes recorded between 2000 and 2010 show an increase in the total number of hives in Europe from 15 million in 2000 to 16.4 million in 2007, a decrease in 2008–2009, and again, some increase towards the end of the period—2010, to about 15.8 million hives [37]. In comparison, in 2010, the registered bee colonies in Bulgaria were over 613,000 [35]. While, in 2006, bee colonies in the country amounted to 671,674, in 2007 to 718,822, in the following years, their number kept steadily decreasing, and in 2012 it reached 529,117—a reduction from 21% to 26.4%. During this period, the number of holdings engaged in beekeeping (beekeepers) also decreased from 31,026 in 2008 to 19,238 in 2012—a 40% reduction.

#### **5. Factors A** ff**ecting the Health of Honey Bees**

There are five stressors of global importance that are thought to be relevant to the reducing number of bee colonies in di fferent parts of the world. These are the anthropogenic driven worldwide spread of pathogenic organisms and pest beetles (*Aethina tumida*), climate change and adverse climatic conditions, landscape changes with limitation of natural habitats, intensification of agricultural production (including the use of fertilizers and pesticides), and invasion of new non-native species [38].

## *5.1. Biotic Factors*

A total of 29 diseases and pests are known to be the cause of the annual loss of bee colonies almost everywhere in the world [35]. Among the causative agents and pests in the honey bee colonies are Varro mite (*Varroa destructor*), microsporidia (*Nosem apis*; *N. ceranae*, the more virulent one); fungi such as *Ascophaera apis*; bacteria (*Paenibacillus larvae, Melissococcus plutonius*), amoebae (*Malpighamoeba mellificae*), septicemia and spiroplasm, small hive beetles (*Aethina tumida*), wax moths (Pyralidae), and others [3,38].

The parasitic mite *Varroa destructor* was introduced into Europe (possible in the 1950s) [39], and North America (first detected in 1987s), [40], making it a nearly ubiquitous honey bee pest [41]. An exception is Australia, as well as some isolated islands and probably individual Central African countries where the parasite has not ye<sup>t</sup> been identified [4]. This mite is responsible for significant annual losses of bee colonies in Canada and a number of European countries [35]. In regions where *V. destructor* is not a major problem, beekeeping is negligible or does not occur at all [42]. A number of authors have identified this species as the most probable cause of bee populations extinction in Europe, USA, and Canada [31,35,43–45]. In addition to being direct pests, mites are a vector of various

viral diseases in parasitized bees [28,42]. Examples of the latter include the Deformed Wings Virus, the Acute Bee Paralysis Virus, the Israeli Acute Paralysis Virus, and the Kashmir Bee Virus—all directly related to the empty hive syndrome [46–48].

Microsporidian infections with Nosema spp.—*N. ceranae* and *N. apis*—lead to acute diarrhea in bees during the winter-spring season or to a latent infection [49,50]. There have been cases where infections with *N. ceranae* cause symptoms identical to the decline of bee colonies [51]), but the relationship between the two pathological phenomena are poorly understood [35].

The small hive beetle, *Aethina tumida*, adversely affects all aspects of beekeeping, including queen rearing, honey production and processing, and pollination operations. In honey bee colonies, they feed on pollen, honey, and occasionally brood. The damage associated with an *Aethina tumida* infestation is caused by the beetle larval stage; adults have little negative impact on a colony besides distracting worker bees from their normal hive duties [52].

The Greater wax moth (*Galleria mellonella*) is another opportunistic pest found in honey beehives, and cause significant damage to stored combs [53]. The damage caused by *G. mellonella* larvae is severe in tropical and sub-tropical regions and is believed to be one of the contributing factors to the decline in both feral and wild honeybee populations.

While in Europe, biotic factors, mainly infections and parasites of the hives, are given grea<sup>t</sup> importance; most of the loss of bee colonies in the United States is attributed to adverse weather factors, starvation, loss of the bee queen or stress from transporting hives over long distances, and infection (mainly viruses, bacteria, and fungi) that may contribute to CCD syndrome [27,29,54,55]. However, the studies cited here do not take into account the prior health status of beehives killed in case of transport stress [35].

## *5.2. Abiotic Factors*
