Diseases of Honeybee (Apis mellifera)

Abstract

Honeybees, important pollinators crucial for ecosystem health, are susceptible to a range of bacterial, fungal, and viral diseases that pose significant threats to their colonies. Bacterial diseases include American Foulbrood (AFB) caused by Paenibacillus larvae and European Foulbrood (EFB) caused by Melissococcus plutonius. AFB results in the death of honeybee larvae and the production of spores that contaminate the hive, while EFB primarily affects young larvae. Fungal diseases like chalkbrood are caused by Ascosphaera apis, Chalkbrood transforms larvae into chalk-like mummies Nosemosis is caused by two pathogenic spores Nosema apis, and Nosema ceranae, which infects the midgut of adult honeybees and viral diseases such as Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), and Chronic Bee Paralysis Virus (CBPV) further weaken honeybee colonies, DWV and IAPV lead to deformed wings and premature death, and CBPV causes shivering hair loss, and paralysis. To manage these diseases, beekeepers employ various strategies including Integrated Pest Management (IPM) techniques, genetic selection for resistance, antibiotic treatments, and maintaining healthy hive conditions. Continued research, monitoring, and education are crucial for effective disease prevention and control, as well as the preservation of honeybee populations and the essential ecosystem services they provide.

1. Introduction

1.1 Bacterial diseases in honeybee

1.1.1 European foul brood

European foulbrood is a disease of bee larvae caused by the Gram-positive bacterium Melissococcus plutonius and worldwide distributed [1], which mainly affects the capped brood, and causes death [2]. The transmission of the pathogen in the hive depends on the survival, the bacteria remain viable in these deposits for long periods [3], and although the cells are cleaned, some bacteria may remain infecting new individuals [4]. Adult bees and honey can act as vectors and transport bacteria between hives and apiaries [5].

The infection process initiates when bees ingest food that is contaminated with the bacterium. Upon reaching the bee’s midgut, the bacterium multiplies and induces physiological damage to the epithelial cells and the peritrophic matrix [6]. Additionally, malnutrition in larvae has been associated with this infection as the bacteria compete for nutrients [6].

Infected larvae within the bee colonies exhibit abnormal distribution and undergo a progressive color change, transitioning from white to yellow and eventually turning brown as they enter the final decomposition phase [7]. The mortality of larvae caused by M. plutonius infection holds significant importance, yet there remains limited knowledge regarding the pathophysiological mechanisms and virulence factors of this pathogen [8]. It has been suggested that variations in the genotypic structure of the bacteria contribute to the differences in virulence factors among different strains, which play a critical role in the development and severity of the disease. Therefore, understanding the molecular epidemiology of the bacterium and its association with the severity of pathophysiological processes is crucial for timely disease diagnosis and effective control in apiaries [9].

1.1.2 Symptoms

This disease can be identified through various characteristic symptoms. Infected larvae exhibit abnormal color changes as the disease progresses, initially appearing pearly white but later turning yellow, brown, or even dark brown [10]. Additionally, affected larvae become sunken and eventually disintegrate within the cells, leading to their decomposition. Twisted or contorted larvae are also observed, which serves as a distinctive symptom of EFB [10, 11]. Scale-like remains of disintegrated larvae can be found adhering to cell walls or the bottom of cells [9]. Infected colonies emit a foul odor resembling rotten or decaying broods [12], further indicating the presence of EFB. The disease also causes a spotty brood pattern within the colony, disrupting regular brood development and resulting in mortality [5]. Chalk-like scales left behind by disintegrated larvae may be visible on cell walls or debris within the hive [12]. EFB weakens honeybee colonies, leading to reduced population size, decreased honey production, and an overall decline in colony strength [11]. Severe EFB infections can increase adult bee mortality, further contributing to colony weakness [10]. Unlike American foulbrood (AFB), EFB does not exhibit rope-like structures when the brood is stretched with a toothpick or matchstick [5]. Early and accurate diagnosis of EFB is vital for effective disease management. Beekeepers and inspectors should closely observe these symptoms and, if necessary, confirm the presence of the bacterium M. plutonius through laboratory analysis (Figure 1) [10, 11, 12].

Figure 1.

A melted larva in its cell from a hive infected with European foulbrood and larvae curled upwards, flaccid, and brown or yellowish dead larva in its cell, [9, 10, 11].

1.1.3 Diagnosis

Accurate diagnosis of European foulbrood (EFB) relies on a combination of clinical examination and molecular identification methods.

Clinical examination involves carefully observing the characteristic symptoms exhibited by infected larvae, such as larval discoloration, sunken and disintegrated larvae, twisted larvae, foul odor, and reduced brood pattern [5, 10, 12], and microscopic analysis, including Gram staining and observation of bacterial cells, can provide preliminary insights into the presence of the causative agent, Melissococcus plutonius [12, 13, 14].

To confirm the presence of M. plutonius and identify specific strains, various molecular techniques are used. Bacterial culture on selective media, such as MYPGP agar, enables the isolation and cultivation of M. plutonius from infected larvae [15]. Polymerase Chain Reaction (PCR) assays target specific regions of the bacterial genome, such as the 16S rRNA gene or the pMA plasmid, providing sensitive and specific detection of M. plutonius [12, 13]. Real-time PCR, or quantitative PCR, offers a rapid and quantitative assessment of M. plutonius presence in bee samples, contributing to accurate diagnosis [14, 15].

Molecular techniques like DNA sequencing allow for precise identification and strain characterization of M. plutonius by comparing obtained sequences with known references [13]. Multiplex PCR assays have been developed to simultaneously detect and differentiate between various honeybee pathogens, including M. plutonius and other brood diseases [16]. Fluorescent In Situ Hybridization (FISH) utilizes fluorescently labeled probes to directly visualize and identify pathogen spores in bee tissue samples [17]. Next-Generation Sequencing (NGS) technologies provide a comprehensive analysis of the microbial community, facilitating the identification of multiple pathogens, including M. plutonius [18]. Additionally, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has shown promise for the rapid identification of M. plutonius through the analysis of protein profiles [19].

Accurate diagnosis and molecular identification of M. plutonius using these diagnostic techniques are crucial for implementing appropriate control measures and preventing the spread of EFB in honeybee colonies. These methods serve as valuable tools for beekeepers and researchers in monitoring and managing EFB outbreaks [10, 12, 15, 17, 20, 21].

1.1.4 Control

Controlling European foulbrood (EFB) disease in honeybee colonies is crucial for maintaining colony health and productivity. Various strategies have been developed to manage and mitigate the impact of this disease. One approach is to encourage hygienic behavior in honeybees, as bees with high levels of hygienic behavior can detect and remove infected larvae, reducing the spread of the disease [22]. Timely removal and destruction of infected brood frames also play a significant role in preventing the further spread of EFB [22].

Another important strategy is to provide honeybees with a balanced and nutritious diet. This helps boost their immune system and makes them more resistant to EFB infection. Supplementing their diet with pollen supplements and ensuring adequate forage can contribute to improved colony health and resilience [23]. Additionally, genetic selection through breeding programs can be a long-term approach to combat EFB, selecting and propagating honeybee colonies that display resistance to the disease can significantly reduce its impact within the population [24].

Proper hive ventilation is crucial as it creates an environment that is less favorable for EFB development, good airflow helps reduce moisture levels and limits the growth of bacterial pathogens [22]. Regularly cleaning and disinfecting equipment and hive components, known as apiary sanitation, is another essential practice to prevent the buildup and transmission of EFB spores. Proper sanitation practices significantly reduce the risk of reinfection [25].

Educating beekeepers about EFB, its identification, and management strategies is crucial for effective control. Recognizing the signs of infection and implementing appropriate control measures contribute to disease prevention and control [26]. Antibiotics such as oxytetracycline may be used in severe EFB outbreaks but should be used cautiously and in accordance with local regulations [12].

Implementing an integrated pest management (IPM) approach that combines multiple control strategies can effectively manage EFB. By integrating various methods, beekeepers can achieve a comprehensive and sustainable disease management plan [22]. Regular inspection of colonies for EFB infection allows for early detection and intervention. Isolating infected colonies and implementing quarantine measures prevent the spread of the disease to healthy colonies [26]. Continued research, monitoring, collaboration among beekeepers, researchers, and regulatory authorities, and adherence to local regulations are also crucial for effective EFB control [12, 26].

1.2 American foul brood

American foulbrood (AFB) is one of the serious bacterial larval diseases of honeybees Apis mellifera [27, 28], caused by spore-forming, Gram-positive bacteria (Paenibacillus larvae) [29, 30]. The bacterium exhibits highly aggressive pathogenic behavior, resulting in the devastating collapse of bee colonies [30]. The spores of bacteria are ingested through food, specifically honey, pollen, and royal jelly [31] food exchange or feeding between larvae by nurse bees [32].

1.2.1 Pathogenesis

The infective stage pathogen is larval while clinical signs appear in the pupal stage, [27], and collapse the entire colony [28], after the hatching, the larvae are highly susceptible to pathogen infection [33]. Spores only infect the honeybee larvae midgut and multiply rapidly [34], vegetative cells infect the haemolymph and penetrate across the protective membrane [35], and the vegetative cells sporulate and transform into dry flakes which contain millions of spores [36].

1.2.2 Symptom

Capped brood that is sunken, perforated, or discolored, with a dark, coffee-like appearance” [13], and “Dead larvae that exhibit a rope-like consistency when probed with a matchstick or toothpick” [20]. The presence of foul-smelling, decaying larvae that may emit a sour or putrid odor” [28, 32]. Presence of spore-contaminated debris at the bottom of cells and on the hive floor” [20].

Following the initial diagnosis, a bacteriological examination is necessary to isolate and identify the bacteria responsible for the infection [37]. Molecular biology techniques like real-time PCR have become commonly used for identifying the specific bacterial strains causing the infection (Figure 2) [37, 38].

Figure 2.

The earlier stage infection and later stage infection of American foulbrood [20, 28].

1.2.3 Diagnosis

In the field, as a method of confirming the disease, the beekeeper can drill several alveoli, swallowing the larvae and observing the liquid that is formed in this procedure. If the liquid obtained has a brown color and a pasty consistency, forming a filament is a sign of foulbrood [2].

The bacteriological diagnosis must be made, which includes the isolation and identification of the bacteria in the material from the infected colonies. Nowadays, molecular biology techniques such as real-time PCR are used to identify the infecting bacterial strains [37, 38].

The first PCR assay for the identification of P. larvae was developed in 1999 and was based on the 16S rRNA gene, of bacteria and is used for phylogenetic studies and detection of bacteria [38]. Nowadays, real-time PCR techniques are used for direct analysis [38].

1.2.4 Control

Rapid detection and destruction of infected colonies and equipment are essential to prevent the spread of AFB [20]. Strengthening honeybee colonies through proper nutrition and regular requeening can help minimize the impact of AFB [39].

One effective approach is selecting and breeding honeybee colonies with increased hygienic behavior, which involves the removal of infected broods to limit AFB spread [40]. Antibiotics, such as oxytetracycline or tylosin, can be used to control AFB at the individual hive level, suppressing bacterial infection [21]. Other trisulfas (sulfacetamide, sulfadiazine, sulfathiazole), sodium sulfathiazole, and potentiated sulfonamides such as sulfamethoxazole + trimethoprim. Sulfathiazole sodium effectively suppresses AFB and further, antibiotics can be alternated using sulfonamides 1 year and oxytetracycline the next [41].

Another control method involves shaking bees off frames, destroying infected broods, and requeening with healthy queens to reduce the prevalence [40]. Proper disposal of infected material, including burning infected combs and equipment, is essential to prevent spore dissemination [39]. Implementing good apiary management practices, such as maintaining proper hive spacing, promoting ventilation, and reducing stressors, can help minimize the incidence of pathogens [39]. Beekeepers should also conduct regular surveillance for AFB symptoms and promptly report suspected cases to authorities to prevent further disease spread [21]. Adhering to biosecurity measures, including hive hygiene and equipment sterilization, is crucial for preventing AFB transmission [42]. Education and training programs for beekeepers on AFB prevention and control play a significant role in disease management [40]. Furthermore, the establishment of legislation and regulations pertaining to AFB control can support coordinated efforts at a national level [21].

2. Fungal diseases in honeybees

2.1 Chalk brood

Among the main pathogenic agents that affect bee colonies is the Ascosphaera apis fungus, which has spread in several countries worldwide causing ascospherosis, also known as chalk disease [43]. A. apis infection is an invasive mycosis that affects developing larvae [44]. It is generated when the larvae ingest the spores of the fungus with their food. The hyphae grow in the midgut, reaching the surface after sealing the cells, generating a microaerophilic condition, which allows the growth of fungi, later producing mummified corpses with the appearance of chalk [44, 45].

The fungus A. apis exclusively infect brood larvae and not adult bees [46]. Unlike other entomopathogenic fungi that can enter insects through their cuticles, the spores of A. apis cannot germinate on the larval cuticle and instead rely on the larvae ingesting the spores along with their food [44, 47]. Once the spores of A. apis are ingested, they require high concentrations of CO₂ to initiate germination, which occurs in the anaerobic environment of the larval intestine where CO₂ is produced by the larval tissues [48]. The optimal temperature for germination is 35°C [49]. Upon activation, the spores swell and develop germ tubes that grow into dichotomous hyphae, these hyphae penetrate the peritrophic membrane of the larvae and extend into the body cavity, eventually reaching the posterior end and breaching the barrier. As a result, spore cysts are formed. The infectious units that cause chalk disease are the ascospores, which form within spore balls localized in resistant cysts. The spores consist of two nuclei, with the larger one located at the center and the smaller one near the tip of the spore [49]. The spore wall is composed of three layers and primarily contains chitin, which contributes to the long-term viability of ascospores [46, 49].

2.1.1 Symptoms

One of the initial clinical signs of chalk disease is the presence of dead larvae that are covered in white fungal growth and usually appear swollen [49]. Over time, these larvae shrink and change color to black, gray, or white, depending on the presence of reproductive structures [50]. As the fungal development cycle progresses, the infected larvae become mummified [50]. Chalk disease can be easily identified by observing mummified bee brood, commonly referred to as plaster brood or plaster mummies, on the bottom board of beehives and in exposed cells [49]. However, it is difficult to detect low-level infestations of this disease (less than 12% infection) because worker bees eliminate the brood of infected bees (Figures 3 and 4) [51].

Figure 3.

White fungal growth and usually appear swollen [49]. Over time, these larvae shrink and change color to black, gray, or white, [50].

Figure 4.

Mummified bee brood, commonly referred to as plaster brood or plaster mummies, on the bottom board of beehives [49].

2.1.2 Diagnosis

Various methods have been developed to detect and confirm chalkbrood infection, including both traditional and molecular approaches.

Traditional diagnosis involves visual inspection of infected broods, which appear as mummified larvae covered with a white down of mold [49]. However, visual examination alone may not provide definitive results, necessitating the use of molecular techniques for accurate identification of pathogens.

Polymerase chain reaction (PCR) and DNA sequencing detect and identify the presence of the fungal pathogen A. apis, which causes chalkbrood disease. PCR-based techniques target specific regions of the pathogen’s DNA, such as the internal transcribed spacer (ITS) region, to amplify and detect its presence [52]. Additionally, real-time quantitative PCR (qPCR) has been employed for rapid and quantitative detection of A. apis in honeybee samples [53]. This method allows for the quantification of fungal load in infected colonies [54]. Furthermore, molecular techniques have facilitated the development of specific primers and probes to differentiate A. apis from other related fungal species, ensuring accurate identification [55].

In recent years, next-generation sequencing (NGS) technologies have been utilized for comprehensive analysis of the honeybee microbiome, including the identification of pathogens like A. apis [54].

2.1.3 Control methods

Controlling chalkbrood disease in honeybees is crucial for maintaining colony health and productivity. Various strategies have been employed to manage and mitigate the impact of this disease. These strategies include promoting hygienic behavior among bees to detect and remove infected brood, timely removal and destruction of infected brood frames, providing a balanced [22], and nutritious diet to boost the bees’ immune system, ensuring proper hive ventilation and moisture control [23], breeding honeybee colonies for chalkbrood resistance, cautious use of fungicides such as oxytetracycline [56]. Maintaining optimal hive temperatures around 35°C can help suppress the disease [49], and practicing apiary sanitation to prevent the buildup and spore transmission [25].

Strengthening weak colonies [23], and educating beekeepers about chalkbrood disease [25]. Exploring biological control agents [57], implementing integrated pest management approaches, regular quarantine and hive inspection, and supporting ongoing research and monitoring efforts to enhance disease management strategies [22, 25]. Stimulating the feeding of colonies, with energetic liquid food and protein food, is also an important action, increasing the number of healthy bees and reducing colony stress [43, 58].

2.2 Nosemosis

2.2.1 Microsporidian of honey bee

Nosemosis is an adult honeybee disorder, that infects the epithelial cells of the intestines of adult bees [59], causing the integrity of the intestine to be destroyed and the digestive system to be disturbed [60], this caused a series of abnormal remarkable in worker bees [61]. Accelerate the development of bee behavior polymorphisms, that is, the early appearance of adult bee behavior [62], decreased ability to feed larvae [63], shortening life [64], and the increase in overwintering mortality [65]. Resulting in reduced bee colony productivity, fecundity, and viability [66]. The geographic environment will have an impact on the evolution of the pathogen [67].

2.2.1.1 Nosema apis

Nosemosis A is a disease caused by N. apis [68]. The high incidence of N. apis is from the autumn months, being during the winter as a result of increased colony mortality or collapse [69, 70]. It frequently shows apparent signs of the disease, caused by N. apis such as brown fecal stains with a peculiar acid odor observed on the frames and at the entrance of the hive [71]. As a result of high mortally and weak bees at the entrance of hives or swallow’s abdomens [72].

2.2.1.2 Nosema ceranae

Nosemosis C is a disease caused by N. ceranae [73], it is present throughout the year and in the absence of clinical signs related to infection by N. apis such as fecal deposits or a dilated abdomen [74]. N. ceranae high virulent pathogen as compared to N. apis [75].

N. ceranae effect cellular defense mechanisms, and nutritional metabolism [76]. Expression of the hormone vitellogenin decreases [61], which participates in the synthesis of royal jelly [77], promotes immunity, acts on the response to stress and longevity of the bee [78].

2.2.2 Effects on colonies

Both type A and type C nosemosis have been shown to be extremely damaging to honey bee colonies [79, 80]. The most evident appearances of type A nosemosis are the unusual presence of feces and dead bees around infected hives, the increase in the size of the abdomen of the affected bees, and its seasonality, with infection peaks in spring and autumn [81]. Type C nosemosis is considered asymptomatic, at least during a long initial incubation period of the infection which, together with the imbalance of polytheism that it causes in bees, accelerates the onset of the hive. At early ages, have favored its relationship with the decline of bee hives [79, 80, 82, 83] and in particular with Colony Collapse Disorder (CCD), in different regions of the world [83, 84, 85].

The different pathogenicity of nosemosis has been related to the host response to the spore load administered to bees in experimental infections. Normally, the higher the dose of infection, the greater the effects on fecundity and host survival [83]. This phenomenon has been confirmed in experimental infections by N. apis and N. ceranae, where bees infected with higher doses presented higher mortality and higher sugar consumption [86].

There is clear evidence that N. ceranae affects various aspects of individual physiology (metabolism and immune response), morphology, and behavior of infected bees [61, 87, 88, 89]. For all these reasons, the infection unfavorably affects the viability and survival of the bees, and compromises the health of the hives, decreasing their productivity (Figure 5) [65, 90].

Figure 5.

Dysentery at the entrance of Nosema-infected bee hive.

2.2.3 Diagnosis

For diagnosis, the Adult foraging bees are collected from the entrance of the hive and avoid the internal bee which is probably less infected by spores [71].

2.2.3.1 Microscopic examination

Perform a microscopic examination of the midgut of the bee, generally infected bees have lighter and whitish color midgut [91], while uninfected bees are dark and dull midgut [71].

For the conformation of infected and uninfected colonies under microscopy, at least 30 bees are collected, and macerate of the midgut in 15 ml of water, three drops of the suspension are placed on a slide under a coverslip and examined at 400x magnification under a bright field or phase contrast microscope [71, 92]. Spores are dark outlines and oval-shaped spores and it is hard to differentiate the species under a light microscope [71, 93].

2.2.3.2 Molecular biology techniques

In Different molecular methods the Multiplex PCR as described by Martín-Hernández et al. [94] is used by small subunit 16S rRNA genes, with control of mitotic gene (COI) in a single reaction [95]. Further, quantitative PCR has been used for identification and also to quantify the degree of infected samples by Nosema [96], additionally, RT-PCR has been used for the number of infected cells quantification [71].

Furthermore, Aronstein et al. [97] developed a cost-effective ELISA technique for identifying N. ceranae, reducing the need for PCR. Characterization of monoclonal antibodies (mAc) against N. ceranae and N. apis, enabling the development of a rapid and sensitive indirect immunofluorescence (IFI) technique for the identification of both species in infected bees samples [98]. Ptaszynska et al. [99] introduced a loop-mediated isothermal amplification (LAMP) technique for distinguishing N. apis from N. ceranae. The method utilizes multiple pairs of primers, increasing sensitivity compared to conventional PCR by specifically targeting the DNA sequence of interest.

2.2.4 Control

Fumagillin was the most widely used active product to control nosemosis, supplied in a mixture of sugar and water [71, 100, 101]. In 1949, fumagillin; from (Aspergillus fumigates) has been used to treat N. apis, and was later used to treat N. ceranae [102]. Recent studies show that fumidil-b (antibiotic) is effective temporarily against N. ceranae [103]. Further, fumagillin is toxic to bee-caused alterations in the ultrastructure of the hypopharyngeal glands of bees [102].

As an effective method, the replacement of the queen each year achieves a high survival ability for hives and the production of honey [104]. The application of natural compounds (phenolics and organic acid) is one of the best chemotherapeutics against nosema, further, tannic acid, toltrazuril, and resveratrol reduce the infection of N. ceranae [105], Porphyrins (aromatic compound) used in treatment to control Nosemosis [106].

2.2.4.1 Natural product

Natural substances, such as oxalic acid and formic acids, are mainly used to control varroa mites [107], and also reduce the parasite load of bees infected by N. ceranae [108]. The use of caffeine, gallic acid, and kaempferol supplement diets increased the survival rate of N. ceranae-infected bees [109].

Feeding with thymol a decrease in the parasite load in bees infected with N. ceranae compared to controls [110]. Porrini et al. [111] observed a decrease in spore counts in bees infected with N. ceranae that were administered syrups supplemented with plant extracts of Artemisia absinthium, Allium sativum, and Ilex paraguariensis, obtaining significant results with L. nobilis where one of the major components is thymol. Damiani et al. [112], similarly observed similar results against N. ceranae in the application of extracts of L. nobilis.

2.2.4.2 RNA interference

RNA inferences (RNAi) are post-transcriptional gene silencing techniques that develop for the controlling of N. ceranae and significantly decreased the parasite load percentage of nosema spores [113]. RNAi from Nosema spp. genes that code for ATP/ADP transporter proteins, for the polar tube protein 3 (PTP3), and for a cuticle protein (nkd) of the insect [113, 114]. The microRNAs (miRNA) expressed by bees infected with the microsporidium N. ceranae, by ultra-sequencing, suggest that there is a differential expression of these miRNAs in response to infection [115]. These RNAi reduced the spore load in the host and increased the immune response of the bees [116]. Other works target genes from the bee itself that are essential for the development of the pathogen so that its silencing is capable of effectively reducing its proliferation without affecting the host cell itself [117].

3. Viral disease of honeybee

Single-stand RNA viruses are highly infected pathogens of honeybees [118] about 20 bee-identified viruses, that is, Sac-brood virus (SBV), Deformed-wing virus (DWV), Kashmir-bee virus (KBV), Israeli-acute-paralysis virus (IAPV), Chronic-bee-paralysis virus (CBPV), Acute-bee-paralysis virus (ABPV), and Black-queen cell virus (BQCV) [119, 120, 121, 122], and KBV, IAPV, and ABPV complex viruses reported last year caused several colonies losses and bee mortality [123, 124, 125].

Due to the great diversity of virus species, aspects such as pathogenicity, virulence, and impacts (at a social and individual level) on honey bees are not constant traits, nor are they independent of host conditions. For example, the DWV and VDV-1 viruses are part of the same viral complex with DWV type A and DWV type B, respectively [126], both viruses cause abnormal conditions on the wings and abdomens of adult bees of any caste, leading to early mortality [127, 128]. On the other hand, the BQCV virus can infect any caste of honey bees, but they are only pathogenic for queen larvae, causing sterility [129]. Through these examples, we can visualize how the susceptibility of the different strains to viruses depends directly on the species of the virus.

3.1 Symptoms

IAPV infect bees having the following symptoms as paralyzed bee, decrease flying ability, crawling, changing in orientation, and shaking of wings [130].

ABPV caused loss of hair from the bee thorax, and paralysis [123].

KBV declined bee population [131].

CBPV can paralyze bees, No fling ability, loss of hair from the abdomen, trembling, and the bee to black in color [132].

DWV as the name indicates the deformed bee wings, shortened abdomens, and hypoplastic glands (Figures 6 and 7) [133, 134, 135].

Figure 6.

The deformed bee wings, shortened abdomens, and zero fling ability [133, 134, 135].

Figure 7.

IAPV infect bees are paralyzed bee, decrease flying ability, crawling, changing in orientation, and shaking of wings, and CBPV can paralyze bees, loss of hair from the abdomen, trembling, and the bee to black in color [130, 132].

3.2 Transmission of viruses

Viruses in honeybees can be transmitted through various pathways, contributing to their spread within colonies. Vertical transmission occurs when infected queen bees pass on the virus to their offspring through infected eggs or sperm [136]. Horizontal transmission, on the other hand, involves the spread of viruses between individual bees within the colony through direct contacts, such as grooming or feeding activities [136]. Varroa mites (Varroa destructor) play a significant role as vectors in virus transmission. As these mites feed on honeybees’ hemolymph, they can introduce and transmit viruses directly into the bee’s body, facilitating their dissemination within the colony [137].

Contaminated food and water sources within the hive can also serve as vehicles for virus transmission. Bees may consume infected pollen, nectar, or honey, resulting in the ingestion and subsequent spread of viruses [138]. Robbing behavior, where bees from different colonies steal resources from each other, can contribute to the transmission of viruses. Infected bees involved in robbing activities can introduce viruses into previously healthy colonies, thus spreading the infection [137]. Furthermore, certain beekeeping practices can inadvertently facilitate virus transmission. The use of contaminated equipment or the sharing of beekeeping tools between infected and healthy colonies can lead to the transmission of viruses [139].

Bee-to-bee transmission through direct contact is another important pathway for virus dissemination. Bees in close proximity can transmit viruses to each other, such as when Nosema-infected bees pass viruses to healthy bees through mutual grooming behaviors [140]. Pollination activities involving honeybees can contribute to virus transmission between different colonies. Bees may carry viruses on their bodies or in their honey stomachs, transferring them to flowers and subsequently infecting other colonies during pollination [139]. Fecal-oral transmission also plays a role, as viruses present in infected bees’ feces can contaminate hive surfaces and resources. Healthy bees can then come into contact with these contaminated surfaces or ingest the infected feces, leading to virus transmission [137]. Additionally, environmental factors, including temperature and humidity, can influence the survival and transmission of viruses in honeybees. Higher temperatures and increased humidity can enhance viral replication and increase the likelihood of virus transmission [141].

3.3 Diagnosis

Various methods are used to identify and detect viral pathogens in honeybee samples. Molecular techniques such as (PCR), Reverse Transcription PCR (RT-PCR), and Quantitative PCR (qPCR) are widely used for their ability to detect and quantify specific viral genetic material [142, 143]. These methods enable the identification of RNA viruses and provide valuable information on virus levels within honeybee populations [144].

Immunological assays like Enzyme-Linked Immunosorbent Assay (ELISA) are used to detect the presence of viral antigens or antibodies, indicating the presence of viral infection in honeybee samples [145]. Dot blot assays involve the immobilization of viral genetic material or antigens on a membrane, allowing specific probes to detect the presence of viral infection [146]. Western blotting, on the other hand, is a technique that detects and identifies specific viral proteins in honeybee samples, providing valuable information on viral infections [147].

Advanced technologies such as Next-Generation Sequencing (NGS) and metagenomics offer comprehensive approaches to identifying known and novel viral pathogens in honeybee samples [148, 149]. These methods involve high-throughput sequencing of the entire DNA or RNA content, enabling the detection and characterization of viral pathogens present in the sample. Viral microarrays utilize DNA or RNA probes immobilized on a solid support to simultaneously detect multiple viruses, providing a comprehensive viral profile [147].

Microscopy techniques such as Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) allow for the direct visualization of viral particles, aiding in the identification of viral infections [150, 151]. In situ hybridization utilizes labeled probes to detect and localize viral genetic material within honeybee tissues, providing visual confirmation of viral infections [152]. These techniques enhance the accuracy of viral diagnosis and aid in understanding the spatial distribution of viruses within honeybee samples.

Other diagnostic approaches include Multiplex PCR, which enables the simultaneous detection of multiple viral pathogens, providing a cost-effective and efficient method [153]. High-Resolution Melting Analysis (HRM) differentiates between viral species or strains using PCR and melting curve analysis [144]. Loop-Mediated Isothermal Amplification (LAMP) is an isothermal amplification technique that allows for the rapid detection of viral RNA or DNA in honeybee samples, providing a simple and cost-effective diagnostic approach [153]. Additionally, serology methods detect specific antibodies produced by honeybees in response to viral infections, providing indirect evidence of viral exposure or current infection [154].

3.4 Control

Firstly, maintaining hive hygiene through regular cleaning, sterilization of equipment, and removal of infected brood and contaminated comb can limit the spread of viruses [140]. Secondly, effective varroa mite management, using integrated pest management strategies and mite-resistant honeybee stocks, is essential for reducing viral transmission [155]. Additionally, genetic selection for honeybees with traits associated with resistance to specific viruses has shown promise in disease control [156].

Biosecurity measures, including preventing the introduction of infected bees or equipment, and quarantine protocols for newly acquired colonies or queens, can minimize the risk of viral transmission [137, 156]. Regular monitoring of honeybee colonies for viral pathogens using techniques like PCR or ELISA allows for early detection and timely intervention [157]. Vaccination strategies targeting specific viruses are being researched as a potential means of disease prevention and control [141].

Reducing stress factors on honeybee colonies, such as providing adequate nutrition, minimizing pesticide exposure, and ensuring proper ventilation, can enhance their immune response and resilience to viral infections [137]. Selecting honeybee strains with enhanced hygienic behavior, which remove diseased brood, can reduce the viral load and prevent infection spread [22]. Maintaining high-quality queen bees by regularly replacing aging or diseased queens can improve colony resistance to viral infections [155]. Integrated pest management (IPM) strategies that combine various control methods, such as monitoring, cultural practices, biological controls, and targeted chemical treatments, can also help reduce the impact of viral infections [158].

Providing honeybees with a diverse and nutritious diet through pollen substitutes or access to diverse forage sources boosts their immune system and resistance to viral infections [159]. Educating beekeepers on best management practices, disease identification, and prevention strategies is crucial for effective viral infection control [158]. Selecting suitable apiary locations away from potential sources of viral contamination, ensuring strong and populous colonies, establishing treatment thresholds for varroa mite management, regularly sterilizing beekeeping equipment, replacing colonies with high viral loads, and promoting collaboration and research among beekeepers, researchers, and government agencies are additional measures that contribute to viral infection control [158, 159].

4. Conclusion

In conclusion, honeybees face significant threats from bacterial, fungal, and viral diseases. American Foulbrood (AFB) and European Foulbrood (EFB) are bacterial diseases that result in larval death and hive contamination. Chalkbrood, caused by a fungus, transforms larvae into chalk-like mummies. Nosemosis, caused by two spore-forming parasites, affects the midgut of adult honeybees. Viral diseases like Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), and Chronic Bee Paralysis Virus (CBPV) weaken colonies, causing deformities, premature death, and paralysis. The transmission of these diseases is facilitated by external factors such as Varroa destructor mites, which also act as viral vectors. Beekeepers employ various strategies like Integrated Pest Management (IPM), genetic selection for resistance, antibiotic treatments, and hive maintenance to manage these diseases. However, continued research, monitoring, and education are essential for effective prevention and control, ensuring the preservation of honeybee populations and the critical ecosystem services they provide.

Acknowledgments

The authors are highly grateful to the Department of Entomology, PMAS-Arid Agriculture University for allowing us to get all the available technical support for the write up of this book chapter.

Conflict of interest

The authors declare no conflict of interest.

Notes/thanks/other declarations

The authors are also obliged to Mr. Bee Honey Farms for providing literature in the form of up to date books and published research articles, which helped us to improve the write up of this chapter.