Factors Affecting Immune Responses in Honey Bees: An Insight

Bees suffer from a wide range of parasitic mites. More than hundred mite species are associated with honey bees, but Varroa spp. and Tropilaelaps spp. (both ectoparasites) and Acarapis spp. (endoparasite) most threaten bee survival (Sammataro et al., 2000).

Ectoparasitic mite, Varroa destructor, a major threat to honey bees throughout the world, infests and kills a European honey bee colony within one to three years if no control measures are taken (Strauss et al., 2015). Additionally, Varroa further act as a vector to various bacteria (Gliński & Jarosz, 1992), viruses (Allen & Ball, 1996) and fungi (Liu, 1996). Immature and adult stages of this mite feed on the haemolymph of developing and adult honey bees through a single puncture hole. During feeding, mite's salivary secretions damage haemocytes and suppresses the wound healing responses, keeping wound open for days to ensure continuous feeding of its immature stages (Richards et al., 2011). Recent research has shown that Varroa feed primarily on fat bodies rather than haemolymph and these damaged fat tissues may cause immune suppression by altering the production of vitellogenins and antimicrobial peptides (Ramsey et al., 2019). Varroa infestation interferes with both cellular and humoral immunity and suppresses the cellular immune system by reducing total haemocyte count (THC), differential haemocyte count (DHC), surface area of haemocytes and clotting (Salem et al., 2006; Richards et al., 2011). Whereas, the altered expression of immune-responsive antibacterial peptides suppresses humoral immunity in Varroa-infested bees (Gregorc et al., 2012). Tropilaelaps spp. in Asia, another major ectoparasitic mite infesting European honey bees, has similar lifecycle as Varroa. They disturb the regulations of phagocytosis receptor gene and antimicrobial peptide encoding genes in infested honey bees (Khongphinitbunjong et al., 2015). On the other hand, endoparasitic mite, Acarapis woodi, enters the trachea of young bees and punctures the tracheal wall to suck haemolymph causing nutrient loss and damaged wing muscles. They also affect air flow and allow secondary infections through punctured wounds (Eischen, 1987). Feeding on haemolymph may reduce the number of haemocytes as reported in Varroa infested bees (Salem et al., 2006). Colonies infested with this mite have reduced social immunity as the bees become unable to thermoregulate due to damaged wing muscles (McMullan & Brown, 2009).

Like other organisms, honey bees are naturally infected with disease-causing pathogens including bacteria, viruses, fungi and protozoans, which along with other pests are considered as a reason behind the global honey bee decline (Ratnieks & Carreck, 2010). Haemocytic sensitivity of honey bee immature and mature stages to American foul brood (AFB) infection was reported by Zakaria (2007). The bacterial pathogen Paenibacillus larvae, causative agent of AFB disease, suppress larval honey bees’ innate immunity as the antibacterial peptide encoding gene's transcript levels are negatively correlated with the disease levels (Evans & Pettis, 2005). They directly suppress the cellular immunity by damaging haemocytes in larvae (Gregorc & Bowen, 1998).

Expression of genes encoding immunity-related enzymes and antibacterial peptides vary after infection with Nosema apis and Nosema ceranae (Antúnez et al., 2009). N. ceranae suppress both cellular and humoral immunities by degrading or hypertrophying host cells (Higes et al., 2007) and by reducing the expression of immunity related genes (Chaimanee et al., 2012). Nosema also causes energy stress in the host probably by competing for nutrients (Mayack & Naug, 2009). Nosema lacks mitochondria and develop only after direct contact with the cytoplasm of its host cell which fulfils the requirement of an external energy source for reproduction of this microsporidia (Fries et al., 1996; Weidner et al., 1999; Martín-Hernández et al., 2011). Such heavily infested epithelial cells lining the bee gut assure poor nutrient absorption resulting in bees dying early death due to starvation (Liu, 1984). The Nosema-caused energetic stress reduces the trehalose levels in haemolymph (Mayack & Naug, 2010) and affects the flight efficiency and thermoregulatory ability of bees (Campbell et al., 2010). Nosema directly interfere with the innate immune responses by reducing apoptosis in host bees through enhanced transcription of apoptosis protein-(iap)-2 inhibitor gene (Kurze et al., 2015).

Pathogenic viruses also cause immune suppression. Mites act as a vector or activator to turn the asymptotic presence of viruses into lethal infections (Shen et al., 2005). The occurrence of such pathogenic viruses as deformed-wing virus (DWV) (Shen et al., 2005; Ryabov et al., 2014), Kashmir-bee virus (KBV) (Shen et al., 2005) and acute bee paralysis virus (ABPV) (Ball & Allen, 1988) are correlated with the presence of mites, and together they suppress the immunity of host bees. DWV vectored by Varroa suppresses host immunity by interfering with the nuclear factor-kappa B (NF-KB) protein family and reducing the Toll pathway controlled antiviral response (Nazzi et al., 2012). Presence of Tropilaelaps, another mite, is also linked to infections of DWV (Forsgren et al., 2009). Such immune responses as melanisation and encapsulation correlate negatively with DWV levels in a host (Di Prisco et al., 2016). Threats posed by DWV-Varroa association become more severe in the presence of additional stresses including poor diet and pesticides which further promote virus replication (Nazzi & Pennacchio, 2014; Goulson et al., 2015). The interference of neonicotinoids with the NF-KB immune signalling reduces immune responses and promotes the DWV replication in honey bees bearing covert infections (Di Prisco et al., 2013). DWV replication in several honey bee body tissues including fat bodies (Fievet et al., 2006) regulate various physiological processes including immune defences through vitellogenin (Trenczek & Faye, 1988). The interference of DWV replication with the expression of vitellogenin may be responsible for its lower levels in collapsed colonies in comparison to healthy colonies (Dainat et al., 2012b).

For the well-being of the entire colony including immature and mature stages, the hive temperature is maintained between 32–35°C by the resident bees (Kronenberg & Heller, 1982; Seeley, 2014). They use fanning and evaporative cooling mechanisms to combat higher temperature whereas cluster formation or metabolic heat generation through contraction and relaxation of flight muscles combat lower temperature (Southwick & Heldmaier, 1987).

Elevated temperatures induce in honey bees a heat shock response with an increased expression of heat shock proteins. At the same time, heat shock suppresses the multiple immunity-related genes (McKinstry et al., 2017). Compared to summer bees, winter bees exhibit a more reduced expression of immunity related genes and higher DWV loads (Steinmann et al., 2015), and the occurrence of Varroa and DWV reduces their life span (Dainat et al., 2012a). Temperature also affects the flight muscles of bees, which either by shivering or through heat loss maintain a temperature range for efficient working of muscles. Any temperature below this range is responsible for the failure of wing, leg and body muscles (Esch, 1988) which may affect social immunity.

Relatively high and low temperatures cause oxidative stress in arthropods (Lopez-Martinez et al., 2008), which occurs when there is imbalance of free radical production and antioxidant activity (Sies, 2000). Its interference with such cell components as DNA (increased mutations due to breakdown of single-strand, base deletion or degradation) (Jena, 2012), protein (reduced enzymatic activity due to modification of amino acids and rupturing of peptides) (Davies, 2016) and lipids (destruction of cell membrane by lipid peroxidation) (Cheeseman, 1993) affect the physiology of an organism (Simone-Finstrom et al., 2016). Exposure to pesticides, heat, cold, ultraviolet radiations and hydrogen peroxide induces oxidative stress in honey bees (Li et al., 2016) and bees respond to these stresses through varying expression levels of detoxifying or immunity related genes (Zhu et al., 2016). Modified environmental conditions in greenhouses induce oxidative stress and thus adversely affect the cellular and humoral immune systems of honey bees (Morimoto et al., 2011). Simulated heat waves alter the vitellogenin level which protects against oxidative stress (Bordier et al., 2017). Glutathione S-transferases (GSTs) play an important role in oxidative stress management and insecticide resistance, but variations in temperature change GST-related gene expression in a time dependent manner (Yan et al., 2013). Due to oxidative damage, bees face severe survival (Perry et al., 2015; Chakrabarti et al., 2020) and memory issues (Farooqui, 2008).

Hive transportation provides pollination services to different crops, and transported bees underwent transport stress due variable temperatures, humidity, pressures and vibrations (Melicher et al., 2019). Migratory bees have more oxidative damage than stationary bees (Simone-Finstorm et al., 2016). Transport stress affects both, individual immunity by downregulating the genes associated with immunity as well as social immunity by decreased thermoregulation (Melicher et al., 2019). Migratory bees have a short life span compared to stationary bees (Simone-Finstorm et al., 2016). Transportation also reduces the hypopharyngeal gland size in nurse bees which is essential for brood food production (Ahn et al., 2012).

Apart from direct effects, varying temperature in different seasons create conditions suitable for infestation by various parasites and pathogens (Le Conte & Navajas, 2008). The death of honey bee colonies in winters is closely associated with mite attack (van Dooremalen et al., 2012). DWV titres increase between summer and fall when mite incidences increase as well (Gauthier et al., 2007; Dainat et al., 2012b). The expression of immunity related gene (eater) decreases from summer to fall and then increases from fall to winter (Dainat et al., 2012b). High relative humidity combined with low temperatures enhances the chalkbrood development (Flores et al., 1996). Suboptimal brood temperatures increase the susceptibility of adults to pesticides (Medrzycki et al., 2010).

Pesticides especially insecticides as well as fungicides and herbicides are reported to harm honey bees (Belzunces et al., 2012) either individually or synergistically with other stresses (Vadame & Belzunces, 1998). Honey bees may be directly exposed to pesticides when visiting crop flowers during pesticide application or indirectly when coming in contact with a crop after pesticide application (May et al., 2015). They also become exposed when pesticides are directly applied to hives for the control of various bee enemies or diseases (Martel et al., 2007). Short-term effects of pesticides include instant bee death and long-term effects include morphological deformities, compromised foraging, impaired learning, weakened thermoregulation, exploitation of nutritional reserves and reduced immunities (Alaux et al., 2010a; Henry et al., 2012; Sánchez-Bayo et al., 2016; Meikle et al., 2016). Neonicotinoid (imidacloprid, thiacloprid, clothianidin etc.) and pyrethroid (ethofenprox and bifenthrin) insecticides interfere with haemocyte densities and affect cellular immunity (Brandt et al., 2016; Perveen & Ahmad, 2017). They reduce antimicrobial activity of haemolymph and responses including wound healing, melanisation and encapsulation (Brandt et al., 2016; Brandt et al., 2017) and induce such cell deformities as de-nucleation, nuclei side displacement, ruptured cell wall, distorted cell shape and agglutination (Perveen & Ahmad, 2017). Fungicides increase the transcript levels of enzyme related to melanisation, while insecticides and herbicides decrease them (Gregorc et al., 2012). Thymol and coumaphos acaricides downregulate genes involved in detoxification pathways and those genes whose products are involved in humoral (Basket) and cellular (Dscam) defence responses (Boncristiani et al., 2012), whereas flumethrin upregulate the expression of genes that encode antimicrobial peptides and immunity-related proteins (Garrido et al., 2013). Also, direct interferences of azole fungicides and glyphosate herbicide with Cytochrome P450 enzyme system, known to play a role in pesticide resistance and detoxification (Claudianos et al., 2006), affects the innate immunity of honey bees (Yoshida, 1988; Gregorc et al., 2012). Glyphosate also alters midgut microbial communities which defend honey bees against various pathogens (Dai et al., 2018; Motta et al., 2018). Apart from directly affecting cellular immunity (Cousin et al., 2013), herbicides cause indirectly food shortages and nutritional stress by removing alternative food source for bees (Albrecht, 2005; May et al., 2015).

Further, honey bees respond to pesticide induced oxidative stress (Chakrabarti et al., 2015) by increasing the activity of such antioxidant enzymes glutathione peroxidase and catalase (Balieira et al., 2018). In addition to pesticides, antibiotics cause immune suppression by downregulating the expression of immune-related genes (Li et al., 2019).

Proper nutrition is imperative for the maintenance of an individual's health and disease prevention (Sharma & Prajapati, 2014). Pollen and honey/nectar serve as key components in the honey bee's diet. Pollen, being a source of protein, provides many essential amino acids to bees for peptide synthesis in immune pathways (DeGrandi-Hoffman & Chen, 2015). On the other hand, honey is a source of carbohydrates and provides energy for various metabolic processes associated with cellular and humoral immune responses (DeGrandi-Hoffman & Chen, 2015). Thus, proper nutrition plays a role in the maintenance of the immune system. Short-term nutritional deprivation downregulates the immune system, whereas food access rapidly upregulates the immune system of insects (Siva-Jothy & Thompson, 2002). Both individual and social immunities are modified by protein consumption. Glucose oxidase (GOX) activity, a parameter of social immunity, greatly increases with pollen consumption in bees (Alaux et al., 2010b). GOX act as a catalyst in the oxidation process of β-D-glucose into gluconic acid and hydrogen peroxide. The antiseptic nature of hydrogen peroxide helps in colony food sterilization (White Jr et al., 1963).

Protein deficiency reduces metabolic cell activity but increases total haemocyte count and granulocyte count (Szymaś & Jędruszuk, 2003). This may occur as a compensatory mechanism for protein deficiency or reduced metabolic cell activities. Diet diversity also affects immune competence levels because polyfloral diets have more GOX activity compared to monofloral diets (Alaux et al., 2010b).

The effectiveness of individual and social immunity is directly related to the nutritional status of a colony. Invasion by such outer parasites as Varroa disturbs this relationship and hence supresses the immune system (DeGrandi-Hoffman & Chen, 2015). Nosema feeding patterns on honey bees elevates their hunger level, reduces nutrient absorption and causes energetic stress leading to downregulation of the immune system (Martín-Hernández et al., 2011). This type of energetic stress is further enhanced by pesticides, and adverse effects could be seen in haemolymph sugar levels, foraging and thermoregulatory behaviours (Alaux et al., 2010a; Mayack & Naug, 2010). Starved bees might modulate the vitellogenin levels which induce early foraging in infected bees and these bees died during foraging due to the energetic stress (Amdam & Omholt, 2003; Nelson et al., 2007; Mayack & Naug, 2009). Compared to pollen restriction (nutritional stress), pollen ingestion upregulates the immunity-related genes (Corona et al., 2019). Furthermore, the treatment of nutrient-deficient honey bee colonies with antibiotics had more pronounced adverse effects - reduced lifespan, hypopharyngeal gland development, nutrient and immune gene expression and enhanced viral loads (Li et al., 2019).

Honey bees develop certain mechanisms to react to pollen shortage in their colony. They cannibalize their young brood to maintain the older brood but during a long-term pollen shortage they even stop rearing which affects colony strength and social immunity (Brodschneider & Crailsheim, 2010). Stress related to transportation and habitat change may also occurs due to reduced diversity or nutrition. Polyfloral diets, good source of nutrition, enhance immunity-related enzymes and increase the resistance to such stresses (Huang, 2012). The use of pollen substitutes during dearth periods also reduces the total haemocyte count and metabolic activities of cells but the reductions are still lower than in protein free diets (Szymaś & Jędruszuk, 2003).

Age-associated immune dysfunction, also known as “immune senescence”, results in increased susceptibility to infections and impaired functioning of other systems (Ponnappan & Ponnappan, 2011; Sharma et al., 2019). Similarly, the expression of immunity-related genes also varies with the age of bees. Brood has a higher haemocyte density than adults with maximum THC in pupal stage. Nurse bees have a higher fat-body mass and foragers a higher phenoloxidase activity, which generally increases with ontogeny (Wilson-Rich et al., 2008). In simple words, the development of an adult from nurse to forager is accompanied by both reduced haemocytes and fat body content and increased phenoloxidase and GOX activity (Schmid et al., 2008; Alaux et al., 2010a). Furthermore, the presence of a dominant haemocyte type varies with the age, from granulocyte dominance in European honey bee larvae to plasmatocyte dominance in adults (Richardson et al., 2018). Expression levels of a cytochrome P450 gene not only vary from highest in the egg stage to lowest in the adult stage but also within a single stage with maximum expression in the first instar larva and brown-eyed pupa (Zhu et al., 2016). Hence, minimum haemocyte densities and reduced cytochrome P450 expression in older bees indicates lower immunity in foragers as compared to younger stages. However, this age-dependent immune suppression is considered beneficial for bees as the foragers already have higher death rates due to foraging exhaustion. Because maintaining the immune system is a costly process, reduced immunities among foragers helps in the redistribution or saving of energy at colony levels (Schmid et al., 2008).

Besides individually, these factors interact to supress the immune system more negatively. Moreover, immune suppression by one factor creates conditions favourable for other factors and thus reduces the survival of infected bees. Interrelationships among colony's nutritional state, immunity, Varroa and viruses have been well elaborated by DeGrandi-Hoffman & Chen (2015). Colonies without Varroa parasitism have optimum brood rearing and colony growth which facilitate sufficient resource collection and proper nutrition, leading to a built--up immunity which reduces viral loads and ensures proper colony growth. However, the presence of Varroa can reverse this (DeGrandi-Hoffman & Chen, 2015). A Varroa-virus association becomes more dangerous in the presence of such additional stresses as pesticides and poor nutrition (Nazzi & Pennacchio, 2014; Goulson et al., 2015). Pesticides especially neonicotinoids promote virus replication by interfering with the expression of the NF-KB gene family (Di Prisco et al., 2013). Workers infested with Varroa have compromised protein levels which cannot be raised even if sufficient pollen is provided (van Dooremalen et al., 2013). Nosema feeding patterns also induce nutritional or energetic stress in bees and interfere with both individual and social immunities (Mayack & Naug 2009; Campbell et al., 2010; Chaimanee et al., 2012). Neonicotinoids and fipronil promote Nosema infections in colony by suppressing immunity-related genes (Aufauvre et al., 2014) or by affecting GOX activity (Alaux et al., 2010a). Inhibition of cytochrome P450 mediated ergosterol synthesis by azole fungicides increases insecticidal toxicity (Johnson et al., 2013).

Similar to insecticides, fungicidal residues also promote Nosema infections in colonies but their mechanisms are not known (Pettis et al., 2013). Unfavourable temperatures reduce the expression of immunity-related genes (Steinmann et al., 2015; McKinstry et al., 2017) and enhance Varroa, viral, fungal infections and susceptibility to pesticides (Flores et al., 1996; Medrzycki et al., 2010; Steinmann et al., 2015). High and low temperatures, pesticides and UV radiations cause oxidative stress in honey bees (Li et al., 2016) and this stress through cellular damages suppresses innate immunity. Natural compounds in the food of honey bees upregulate the immune system by detoxifying certain pesticides, but pollen and honey deficiency downregulates it (Mao et al., 2013; Negri et al., 2015; Corona et al., 2019).

Food collection by honey bees strongly depends on outside weather. Adverse climate conditions-rainfall, high winds and extreme temperature range restrict bee foraging (Riessberger & Crailsheim, 1997) and cause nutritional stress in the colonies. Improper nutrition thus plays a central role in immune suppression by reducing resistance to Varroa, Nosema, viruses and pesticides (Huang, 2012). Interrelationships between the various stress factors are shown in Fig. 2.

Fig. 2