**5. The gut microbiota as an environmental factor for honeybee health**

Honeybees have a beneficial anaerobic and micro-aerobic natural microbiota acquired and installed in their body. This includes Gram-negative groups like species *Gilliamella apicola*, *Snodgrassella alvi*, and *Frischella perrara* and Gram-positive groups like species of *Lactobacillus* and *Bifidobacterium* [49, 50]. That is, *Acetobacteraceae*, *Parasaccharibacter apium* confers resistant to *Nosema* [51] and *Bartonella apis*, a honey bee gut symbiont of the class *Alphaproteobacteria* [52, 53]. So it is natural for bees to acquire these microorganisms through feeding [49]. This honeybee normal microbiota comes from food, pollen, and honey consumption or through contact with other worker honeybees.

The microbiota associated to the honeybee *A*. *mellifera* is complex, and it has been described as being mainly composed of yeasts, Gram-positive bacteria (such as *Lactobacillus rigidus apis*, *S*. *constellatus*, *Bacillus* spp., *Streptococcus*, and *Clostridium*), and Gram-negative or Gram-variable bacteria (*Achromobacter*, *Citrobacter*, *Enterobacter*, *Erwinia*, *Escherichia coli*, *Flavobacterium*, *Klebsiella*, *Proteus*, and *Pseudomonas*) [54–58].

There are several bacterial species negatively affecting honeybee health—*Paenibacillus larvae*, *Melissococcus plutonius*, *Spiroplasma apis*, and *Spiroplasma melliferum* [59–61]. Besides bacteria, there are many fungi, viruses [62], and protozoa, i.e., *Apicystis bombi*, *Crithidia mellificae*, and *Lotmaria passim* (**Figure 1**) [63–65]. *P*. *larvae* is a sporulated Gram-positive *Bacillus* that causes the American foulbrood disease in larvae.

Gilliam reported that these bacteria could be endemic of the digestive tract of adult honeybees and independent of seasons and nutritional factors [11]. They are different depending on the sources of nectar and the presence of other bacterial genera in the stomach of the honeybee. It seems that bees and lactic acid bacteria developed mutualism. Lactic acid bacteria prepare the environment to make nutrients available for honeybees; on the other hand, intestinal tract

**Figure 1.** The pathogens and beneficial microorganisms in honeybee: one pathway of bee food contamination comes from environmental nectar, pollen (on flowers), and water collected by worker honeybees. The food is stored in beehive and can be transferred by trophallaxis among workers and brood. Another pathway is the consumption by honeybees of contaminated honey and/or pollen from other beehives. Common viruses: black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), Sacbrood virus (SV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), Israel acute paralysis virus (IAPV), and slow paralysis virus (SPV). Niche of beneficial microbiota on alimentary tract (gut). The arrows indicate the transfer of microorganisms by food among individuals (larva and adults) in the beehive. For detail, see in the text.

of honeybees is protected from harmful microorganisms. The honeybee regurgitates the nectar stocked in the crop in the hive honeycomb that has an optimum temperature of 35°C [66] for the development of lactic acid bacteria.

The honeybee larvae probably are sterile initially, but as feed on honey from nurse workers, honeybees gain over time this intestinal flora before completing their life cycle [67]. Honeybees harbor a number of commensal or beneficial bacteria distributed throughout the different compartments of their gastrointestinal tract. Each compartment of the honeybee gastrointestinal tract has a distinct environment favoring specific microorganisms [68]. Several findings have indicated that the honeybee gut is colonized by a distinctive set of bacterial species designated as the core gut microbiota [69]. Because the community composition changes through the life cycle of honeybee, the colonization of the gut is believed to be influenced by the age [68]. During the course of their life span, worker honeybee performs many different tasks that can contribute to these variations. Newly emerged worker honeybees nurse larvae within the hive, whereas older worker honeybees build and maintain the wax combs, defend the colony, and receive and process food that is collected by foragers. In addition to the microbiota in the gut, a novel lactic acid bacterial flora composed of 13 taxonomically well-defined *Lactobacillus* and *Bifidobacterium* species were discovered in the crop of honeybees [70, 71]. The crop functions as an inflatable bag that can transport the nectar back to the hive for storage and honey production. It is hypothesized that lactic acid bacteria play a key role in the conversion of both nectar to honey and pollen to beebread (stored food rich in protein) due to their fermentation properties [70, 72]. The lactic acid bacterial microbiota is of great importance to the honeybee health, protecting them against bee pathogens [73, 74] and contributing to the antimicrobial properties of honey [71].

Lactic acid bacteria are found in two distinct phyla: *Firmicutes* and *Actinobacteria*. The most important genera of lactic acid bacteria within the *Firmicutes* are *Enterococcus*, *Lactobacillus*, *Lactococcus*, *Leuconostoc*, *Pediococcus*, *Streptococcus*, and *Weissella*, which all have a low G+C content. Lactic acid bacteria in the *Actinobacteria* phylum only include species of the *Bifidobacterium* genus that in contrast to the *Firmicutes* members have a high G+C content [75, 76].

Lactic acid bacteria are important inhabitants of the intestinal tract of man and other mammalian and vertebrate animals. *Lactobacillus* and *Enterococcus* are members of this family and are also present in food and fermentation processes [77]. These microorganisms disclose interesting properties not only for the food industry but also for health. The antimicrobial potential of these bacteria includes, among others, the synthesis of compounds such as lactic acid, short-chain volatile fatty acids, and bacteriocin-like molecules [78, 79]. Antagonistic studies are generally directed toward food spoilage and/or pathogenic microorganisms related to the host or product from which the lactic acid bacteria were isolated. Fructophilic lactic acid bacteria are a special group of lactic acid bacteria, which prefer fructose over glucose as growth substrate [80]. They are found in fructose-rich niches, e.g., flowers and fruits. Moreover, the microorganisms can be found in fermented foods made from specific fruits, including wine, fermented cocoa beans, and fermented durian-based condiments [81–83]. *Fructobacillus* spp. and *L*. *kunkeei* are representatives of these microorganisms, and a few novel species have recently been classified as members of this interesting group [84, 85].

Quite recently fructophilic lactic acid bacteria were found in the gastrointestinal tract of several flower- or fructose-related insects, including honeybees, tropical fruit flies, and giant ants [86–88], whose diets are fructose rich. Of these insects, honeybees are economically and agriculturally important for honey production and especially for crop pollination, which links to human food production. However, despite the importance of these insects in nature and in our lives, populations of honeybees are reported to have decreased considerably during the last decade and to be still decreasing worldwide, mainly by colony collapse disorder [89]. To understand and to prevent the disorder, microbial interactions, both symbiotic and pathogenic, have recently been studied [90, 91], and findings have indicated that honeybees carry specific microbiota dissimilar to other animals, including humans. Fructophilic lactic acid bacteria, especially *L*. *kunkeei*, have been found to be one of the dominating bacterial species in several honeybees kept or captured in different regions [73, 90]. Lactic acid bacteria have been successfully applied as probiotics to contribute to health in humans and various companion and farm animals [92, 93]. As lactic acid bacteria are important components in their gastrointestinal tract,

of honeybees is protected from harmful microorganisms. The honeybee regurgitates the nectar stocked in the crop in the hive honeycomb that has an optimum temperature of 35°C [66]

**Figure 1.** The pathogens and beneficial microorganisms in honeybee: one pathway of bee food contamination comes from environmental nectar, pollen (on flowers), and water collected by worker honeybees. The food is stored in beehive and can be transferred by trophallaxis among workers and brood. Another pathway is the consumption by honeybees of contaminated honey and/or pollen from other beehives. Common viruses: black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), Sacbrood virus (SV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), Israel acute paralysis virus (IAPV), and slow paralysis virus (SPV). Niche of beneficial microbiota on alimentary tract (gut). The arrows indicate the transfer of microorganisms by food among individuals (larva and adults)

The honeybee larvae probably are sterile initially, but as feed on honey from nurse workers, honeybees gain over time this intestinal flora before completing their life cycle [67]. Honeybees harbor a number of commensal or beneficial bacteria distributed throughout the different compartments of their gastrointestinal tract. Each compartment of the honeybee gastrointestinal tract has a distinct environment favoring specific microorganisms [68]. Several findings have indicated that the honeybee gut is colonized by a distinctive set of bacterial species designated as the core gut microbiota [69]. Because the community composition changes through the life cycle of honeybee, the colonization of the gut is believed to be influenced by the age [68]. During the course of their life span, worker honeybee performs many different tasks that can contribute to these variations. Newly emerged worker honeybees nurse larvae within the hive, whereas

for the development of lactic acid bacteria.

in the beehive. For detail, see in the text.

240 Honey Analysis

with a reported impact on the intestinal barrier mechanism [94], it is not surprising that lactic acid bacteria, especially fructophilic lactic acid bacteria, may be involved with honeybee health.

Symbiosis is common in nature, in which symbionts as commensals or mutualists evolved to benefit each other. Culture-independent studies of the human microbiota identified recently a complex symbiotic environment with more than 1000 bacterial phylotypes representing more than 7000 strains [95]. The composition of this microbiota has been suggested to be a result of a highly coevolved symbiosis and commensalism influenced by nutrition, physiology, and immunological factors. It varied with the sources of nectar and the presence of other bacterial genera within the honeybee and ended up eventually in the honey (**Figure 1**).
