Viral Infection Affects Sucrose Responsiveness and Homing Ability of Forager Honey Bees,Apis mellifera L.

Honey bee health is mainly affected by Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 10⁷ copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.     

The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV) are part of a complex of closely related viruses from the Family Dicistroviridae. These viruses have a widespread prevalence in honey bee (Apis mellifera) colonies and a predominantly sub-clinical etiology that contrasts sharply with the extremely virulent pathology encountered at elevated titres, either artificially induced or encountered naturally. These viruses are frequently implicated in honey bee colony losses, especially when the colonies are infested with the parasitic mite Varroa destructor. Here we review the historical and recent literature of this virus complex, covering history and origins; the geographic, host and tissue distribution; pathology and transmission; genetics and variation; diagnostics, and discuss these within the context of the molecular and biological similarities and differences between the viruses. We also briefly discuss three recent developments relating specifically to IAPV, concerning its association with Colony Collapse Disorder, treatment of IAPV infection with siRNA and possible honey bee resistance to IAPV.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

蜜蜂病毒学研究进展

蜜蜂是自然界最重要的授粉昆虫,对维护自然生态系统的生物多样性和保持农业生态系统的增产效应发挥着巨大的作用。作为世界第一养蜂大国,中国养蜂业健康发展的意义不仅在于获取大量高品质的蜂产品,更重要的是发挥蜜蜂授粉的农业增产效应,保证我国的粮食安全。和其他动物一样,蜜蜂健康也受到多种病害的威胁,近年来蜜蜂病毒病在世界范围内的广泛流行与传播,是导致世界蜂群持续下降的一个重要原因。蜜蜂病毒长期广泛的以无明显发病症状的低浓度隐性感染方式存在于蜜蜂蜂群中,但多数蜜蜂病毒在特定环境条件下可被激活,在寄主体细胞内快速复制,表现出强烈的致病性,引发致死性蜜蜂病毒病的流行与爆发。蜜蜂病毒病知识的缺乏,以及复杂的蜜蜂病毒鉴定技术使得蜜蜂病毒病难以及时确诊和防治,因此每年在养蜂生产上造成的巨大损失已严重阻碍了我国养蜂业的健康发展。本文将综述这一领域的研究成果和学科发展趋势,为在我国开展蜜蜂病毒学研究提供参考,并介绍国外的一些蜜蜂病毒病诊断方法与防治经验服务于我国养蜂生产实践。     

Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee’s susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.     

In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris

Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an “RNAi-of-RNAi” experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17–18) and also not of SBPV (n = 11–12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections.     

Viral impacts on honey bee populations: A review

Honey bee is vital for pollination and ecological services, boosting crops productivity in terms of quality and quantity and production of colony products: wax, royal jelly, bee venom, honey, pollen and propolis. Honey bees are most important plant pollinators and almost one third of diet depends on bee’s pollination, worth billions of dollars. Hence the role that honey bees have in environment and their economic importance in food production, their health is of dominant significance. Honey bees can be infected by various pathogens like: viruses, bacteria, fungi, or infested by parasitic mites. At least more than 20 viruses have been identified to infect honey bees worldwide, generally from Dicistroviridae as well as Iflaviridae families, like ABPV (Acute Bee Paralysis Virus), BQCV (Black Queen Cell Virus), KBV (Kashmir Bee Virus), SBV (Sacbrood Virus), CBPV (Chronic bee paralysis virus), SBPV (Slow Bee Paralysis Virus) along with IAPV (Israeli acute paralysis virus), and DWV (Deformed Wing Virus) are prominent and cause infections harmful for honey bee colonies health. This issue about honey bee viruses demonstrates remarkably how diverse this field is, and considerable work has to be done to get a comprehensive interpretation of the bee virology.     

Viral transmission in honey bees and native bees,supported by a global black queen cell virus phylogeny

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.     

Genetic evidence for coinfection of honey bees by acute bee paralysis and Kashmir bee viruses

Nucleotide sequence analyses were used to identify acute bee paralysis virus (ABPV) and Kashmir bee virus (KBV) isolated from a single honey bee colony. Most of the bees in this colony carried KBV. Some individual bees also carried ABPV, a coexistence not yet seen between these two viruses. Implications of coinfection on viral efficacy are discussed, along with a restriction enzyme assay that can be used to discriminate between these two widespread viruses.     

Non-Specific dsRNA-Mediated Antiviral Response in the Honey Bee

Honey bees are essential pollinators of numerous agricultural crops. Since 2006, honey bee populations have suffered considerable annual losses that are partially attributed to Colony Collapse Disorder (CCD). CCD is an unexplained phenomenon that correlates with elevated incidence of pathogens, including RNA viruses. Honey bees are eusocial insects that live in colonies of genetically related individuals that work in concert to gather and store nutrients. Their social organization provides numerous benefits, but also facilitates pathogen transmission between individuals. To investigate honey bee antiviral defense mechanisms, we developed an RNA virus infection model and discovered that administration of dsRNA, regardless of sequence, reduced virus infection. Our results suggest that dsRNA, a viral pathogen associated molecular pattern (PAMP), triggers an antiviral response that controls virus infection in honey bees.     

Iridovirus and microsporidian linked to honey bee colony decline

Background

In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.

Methodology/Principal Findings

We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006–2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.

Conclusions/Significance

These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.     

Molecular and phylogenetic characterization of honey bee viruses,Nosema microsporidia,protozoan parasites,and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana‐infecting SBV, and relatively homogenous populations of IAPV and A. meliifera‐infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast‐encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.     

Temporal analysis of the honey bee microbiome reveals four novel viruses and seasonal prevalence of known viruses, Nosema, and Crithidia

Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (～10(11) viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January.     

Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.     

Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae)

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.     

First come,first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.Subject terms: Viral genetics, Microbial ecology, Virus-host interactions, Small RNAs     

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21?days post inoculation (21?days?pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6?days?pi. At 7?days?pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14?days?pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14?days?pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.     

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.