Research Strategies to Improve Honeybee Health in Europe Robin F.A

Research strategies to improve honeybee health in Europe Robin F.A. Moritz, Joachim de Miranda, Ingemar Fries, Yves Le Conte, Peter Neumann, Robert J. Paxton

To cite this version:

Robin F.A. Moritz, Joachim de Miranda, Ingemar Fries, Yves Le Conte, Peter Neumann, et al.. Research strategies to improve honeybee health in Europe. Apidologie, Springer Verlag, 2010, 41 (3), pp.227-242. 10.1051/apido/2010010. hal-00892092

HAL Id: hal-00892092 https://hal.archives-ouvertes.fr/hal-00892092 Submitted on 1 Jan 2010

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Apidologie 41 (2010) 227–242 Available online at: c INRA/DIB-AGIB/EDP Sciences, 2010 www.apidologie.org DOI: 10.1051/apido/2010010 Review article

Research strategies to improve honeybee health in Europe*

Robin F.A. Moritz1, Joachim de Miranda2, Ingemar Fries2,YvesLe Conte3, Peter Neumann4, Robert J. Paxton5

1 Institut für Biologie, Martin-Luther Universität Halle-Wittenberg, Hoher Weg 4, 06099 Halle a.d. Salle, Germany 2 Department of Ecology, Sveriges Lantsbruks Universitet Uppsala, Sweden 3 UAPV Abeilles et Environnement, Institut National de la Recherche Agronomique, Avignon, France 4 Swiss Bee Research Centre, Agroscope Liebefeld-Posieux, Switzerland 5 School of Biological Sciences, Queens University, Belfast, UK

Received 5 October 2009 – Revised 26 January 2010 – Accepted 28 January 2010

Abstract – Understanding the fundaments of colony losses and improving the status of colony health will require cross-cutting research initiatives including honeybee pathology, chemistry, genetics and apicultural extension. The 7th framework of the European Union requested research to empirically and experimen- tally fill knowledge gaps on honeybee pests and diseases, including ’Colony Collapse Disorder’ and the impact of parasites, pathogens and pesticides on honeybee mortality. The interactions among these drivers of colony loss will be studied in different European regions, using experimental model systems including selected parasites (e.g. Nosema and Varroa mites), viruses (Deformed Wing Virus, Black Queen Cell Virus, Israeli Acute Paralysis Virus) and model pesticides (thiacloprid, τ-fluvalinate). Transcriptome analyses will be used to explore host-pathogen-pesticide interactions and identify novel genes for disease resistance. Spe- cial attention will be given to sublethal and chronic exposure to pesticides and will screen how apicultural practices affect colony health. Novel diagnostic screening methods and sustainable concepts for disease prevention will be developed resulting in new treatments and selection tools for resistant stock. Research initiatives will be linked to various national and international ongoing European, North- and South-American colony health monitoring and research programs, to ensure a global transfer of results to apicultural practice in the world community of beekeepers.

Apis mellifera / pathology / diagnosis / disease resistance

1. INTRODUCTION products. More importantly from a strictly economic perspective, honeybees are key pol- 1.1. The value of honeybees linators native to Europe and are crucial for and the costs of colony losses many agricultural crops and the conservation of natural plant biodiversity. Indeed, honey- The management of honeybees, Apis mel- bees are the most economically valued pol- lifera, is deeply rooted in human society, and linators and it is estimated that ∼35% of apiculture provides full or additional fam- human food consumption depends directly ily income. There is a considerable market or indirectly on insect mediated pollination for bee products that are used as food and (Delaplane and Mayer, 2000), a vital ecosys- as additives for pharmaceutical and medical tem service contributing to human health and Corresponding author: R. Moritz, well-being. Although the direct value of the honey produced by the bee industry in the EU [email protected] e * Manuscript editor: Marla Spivak is about 140 Mio, the total added value to

Article published by EDP Sciences 228 R.F.A. Moritz et al.

2020 Varroa in Europe

crops due to pollination services has recently ) e been estimated at 14.2 billion in 2005 for the 1515 million then 15 members of the EU. Worldwide, the ( total economic value of pollination by honey- 1010 bee colonies amounted to e153 billion in 2005 55

(Gallai et al., 2008), while the value of bee pol- number of colonies 00 lination to biodiversity is simply inestimable; 196111 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 1011121314151617181920212223242526272829303132333435363738394041424344454647 10111213141516171819202122232425262728293031323334353637383940414243444546471970 1980 1990 2000 it is life itself. In light of the constant decline of year wild non-honeybee pollinators, the importance Figure 1. The annual number of hives reported to of beekeepers and managed bees is greater to- the FAO in western European countries (the former day than ever. 15 EU member states, black circles) and the former Unfortunately, beekeeping is a declining Warsaw Pact countries in Europe (including the for- industry and the past decades have seen a in- mer USSR, open circles). The dramatic decline in crease in colony losses in managed honey- Europe coincides with the political system changes bee colonies (Potts et al., 2010) and an over- in the in eastern Europe, whereas the introduction all decrease of beekeeping activities in Europe of Varroa destructor had no perceptible impact on (van Engelsdorp and Meixner, 2009). In addi- the number of hives reported (data from FAOSTAT, tion and independent of the managed bee pop- 2009). ulations, wild or feral honey bee colonies are also in decline (Kraus and Page, 1995; Moritz et al., 2007;Jafféetal.,2009) most likely due where the lack of state support forced beekeep- to intensification of land-use, pesticide poison- ers to not replace dead colonies, and instead ing, diseases and many parasites (in particu- abandon their apiaries altogether. Moderate lar the ubiquitous ectoparasitic mite Varroa de- and predictable losses can be accommodated structor). and planned for. However, extensive and un- The marked colony losses in Europe at the controllable losses make beekeeping as a pro- continental scale are unlikely to have been fession, with heavy investment in material and driven by pests, pathogens and pesticides. For equipment, an enterprise at permanent risk of example, the losses due to the appearance of bankruptcy (van Engelsdorp et al., 2007). This Varroa destructor in the 1970s and 80s were financial uncertainty also limits recruitment of largely compensated by beekeepers replacing a new generation of beekeepers, especially to their lost colonies with new ones, resulting in the professional ranks. a constant rise in the number of managed hon- Periodic, extensive honeybee colony losses eybee colonies (Fig. 1). The most dramatic are not just a recent phenomenon. For ex- decline in managed colonies in Europe oc- ample, Ireland suffered “The great mortal- curred in the 1990’s coinciding with the so- ity of bees” in 950, and nationwide bee ciocultural changes in eastern Europe whereas losses occurred again in 992 and 1443 colony numbers remained stable in western (Flemming, 1871). Similar repeated honey- Europe. With the lack of state support in the bee mortalities have been recorded in other former socialistic countries many beekeepers countries throughout history. From the late in eastern Europe abandoned their operations 19th century onwards there are references to causing a reduction in colony numbers by such extensive colony losses (Underwood and about 50%. One of the principal reasons for van Engelsdorp, 2007), including the famous the decline in managed honeybee colonies, and “Isle of Wight disease” of the early 1900’s in of beekeepers, is extensive and unpredictable England (Rennie et al., 1921; Bailey and Ball, colony death. While this can be discouraging 1991), the disappearing disease in the 1960– enough for small-scale hobbyist beekeepers to 1970’s in the USA (Wilson and Menapace, drive them to abandon the hobby, for (semi)- 1979), Varroa destructor and the “Bee Para- professional operators this is a crucial limita- sitic Mite Syndrome” of the 1980’s–1990’s (de tion to business planning and expansion. This Jong et al., 1982;BallandAllen,1988; Hung has become most obvious in Eastern Europe et al., 1995) and the mysterious bee losses in Research strategies to improve honeybee health in Europe 229

France in the late 1990’s (Faucon et al., 2002). cessful at the colony level in the short term, The most recent addition to this list is the has not eradicated the pathogen at the popu- Colony Collapse Disorder (CCD) in the USA lation level, particularly if the pathogen has that began in 2006–2007 (van Engelsdorp a high transmission rate and a high infectiv- et al., 2007). In Europe similar phenomena ity. As illustrated by present apicultural real- have not been observed in the past decades ity, any chemotherapy of honeybee colonies and large number colony deaths are mostly immediately leads to unavoidable contami- locally confined (e.g. Genersch et al., 2010). nation of honey (Stanimirovic et al., 2005) For example the “Rhine valley bee poison- and, ultimately more worrisome, to resis- ing” case of 2008 (Benjamin and McCallum, tant pathogens. Moreover, the dramatic colony 2008) caused large scale colony collapses. The losses of the past decade suggest that treat- causes of regionally confined colony deaths ments aiming at a single pathogen may in prin- usually become rapidly clear (e.g. for the ciple fall short in curing colonies altogether if Rhine valley poisoning and similar cases in the interactions between various pathogens are France and Italy that were due to misuse of the main drivers of colony death. neonicotinoid pesticides) and appropriate actions can be taken to prevent future accidents. 1.2. The parasite-virus-pesticide In contrast, the causes of most large scale meltdown colony losses at a national or even broader scale are still ambiguous or inconclusive The most thorough search for a pathogenic (see Underwood and van Engelsdorp, 2007; cause of extensive, unexplained colony losses Cox-Foster et al., 2007; Johnson et al., 2009; is the case of Colony Collapse Disorder (CCD; van Engelsdorp and Meixner, 2010) and have Cox-Foster et al., 2007; Johnson et al., 2009). not yet been reported to reach a continen- However, despite the enormous research ef- tal scale. Nevertheless, this uncertainty pre- forts invested, no single agent or factor has vents the development of rational approaches emerged as the definitive cause of the phe- to managing colony losses and encourages nomenon (Stokstad, 2007; Anderson and East, ad-hoc remedies and blanket prophylactic 2008). Instead, the best hypothesis to emerge application of chemical treatments against from the data is that particular virulent combi- pathogens or parasites, whether present or not. nations of parasites and pathogens rather than Such practices are also encouraged by the in- a classical monocausal disease, is the most adequate diagnostic tools and procedures for likely explanation (Chen and Evans, 2007; disease treatment. Typically, the apiculturist Johnson et al., 2009). Moreover, chronic ex- identifies symptoms at the colony level, and posures to pesticides that cause no problems then initiates diagnostic procedures to identify for healthy colonies are suspected to inter- the disease and initiate a treatment. Yet, when act with pathogens to produce lethal conse- clinical symptoms appear at the colony level, quences for colonies already weakened by dis- diagnosis often comes too late to save or cure ease (Thompson, 2003). Both the European the colony. Consequently, there is a clear need Commission (2007) and the European Par- for fast, reliable, sensitive and cheap diagnos- liament (2008) became aware of the prob- tic tools that alert the beekeeper to potential lem for beekeeping and formulated research problems before colony level symptoms ap- policies to address the issue. The aim was pear. to prevent honeybee colony losses in Europe Treatments typically rely on chemicals, caused by the CCD syndrome and to bet- which are administered into the colony to ter understand host/parasite/pesticide interac- target pathogens before colony death is in- tions as a potential driver of colony collapses. escapable. The development of such treat- Hence in contrast to US research initiatives ments is based on searching for chemicals which aim at understanding past colony losses, that are toxic to the pathogen, but harmless to European attempts have a clear preventive per- the honeybee. However, so far, any chemical spective aiming to understand potential prin- treatment of a honeybee disease, even if suc- ciples of colony death. The classic example 230 R.F.A. Moritz et al. of such interactions among pathogens is the 2. RESEARCH CONCEPTS case of the ectoparasite Varroa destructor, whose lethal effect on colonies is in large 2.1. Strategy for studying interactions part due to its ability to activate and transmit affecting colony health a number of viral diseases (Ball and Allen, 1988; Bailey and Ball, 1991; Bowen-Walker et al., 1999; Sumpter and Martin, 2004; Honeybee pathology has identified a suite Chen et al., 2005, 2006; Tentcheva et al., 2006; of detrimental factors that impact colony Todd et al., 2007). Recent evidence suggests health, including parasites, pathogens and pes- that this may also be the case for other honey- ticides. A major problem is the combination bee ectoparasites (Dainat et al., 2008; Forsgren of factors. A single infection may cause no et al., 2009). The combination of pests, par- harm to a colony, however, if exposed to a pes- asites and pesticides results in an inadvertent ticide at the same time the colony might die. “meltdown” with one negative factor enhanc- Interactions among sublethal factors affecting ing the negative impacts on honeybee health of colony health therefore stand in the centre the others. of European research in the years to come. Unfortunately, the large number of pests, Appreciation of the fact that colonies suf- pathogens and pesticides affecting honeybee fer multiple infections and understanding the health (Bailey and Ball, 1991; Thompson, resultant interactions among pathogens, pes- 2003; Ellis and Munn, 2005) makes it im- ticides and management, will be central ele- possible to experiment with all possible com- ments if we want to comprehend colony losses binations of these in a rigorous, controlled and develop sustainable strategies for promot- manner. The “BEE DOC” (Bees in EuropE ing colony health. It will be essential to exam- and the Decline Of Honeybee Colonies, http:// ine the nature of such relationships to identify www.bee-doc.eu/) research network in the the traits in the host and the parasite that en- seventh EU Commission’s Framework Pro- able increased tolerance and/or reduced viru- gramme (FP7) will therefore adopt a dual- lence, respectively. This makes identifying the track approach to identifying significant inter- genes for host resistance or the management actions. First, an experimental approach will practices that reduce parasite virulence a re- focus on major, pan-European parasites, pesti- alistic option. These ambitions will be greatly cides and pathogens with known or suspected facilitated by the recent progress in the molec- interactions among them, either detrimental or ular characterization of bee viruses and other beneficial, including those factors thought to pathogens, to quantify their transmission effi- be associated with CCD in the United States. cacy and replication in relation to mite infes- Second, a dynamic surveillance approach will tation levels and developmental stage of the be aimed at identifying significant associations bee. The discovery that parasites are not just among as many factors as possible, using both virus vectors, but may also function as an al- existing national monitoring surveys and com- ternative replicative host for certain viruses prehensive additional assays of the samples (Yue and Genersch, 2005;Gisderetal.,2009; produced by the experimental approach. This Dainat et al., 2009;Eyeretal.,2009), sig- strategy combines a detailed investigation of nificantly enhances the epidemiological po- known interactions with the greatest current tential and lethality of the virus infection for significance for colony health throughout Eu- the honeybee, especially at colony level. The rope, with a comprehensive screen for possible examination of the parameters determining interactions of future significance. the virulence of viruses (see de Miranda and Genersch, 2010), the tolerance of individual bees and colonies to virus infection, and the quantification of the interactions between par- 2.2. Experimental test systems asite, virus, and pesticide at the various developmental stages of the honeybee will therefore Following the strategy outlined above, we need to be in the centre of interest. will focus research on selected test systems Research strategies to improve honeybee health in Europe 231 that can be used for detailed experimentation. as residues in honey and other bee products These include: (Bogdanov, 2006). Even so, several tiny pop- Two major parasite-pathogen interactions ulations of European honeybees survive sus- tainably with Varroa mites without chemical ◦ Varroa destructor mites and deformed control (Nordström et al., 1999; Fries et al., wing virus 2006a; Fries and Bommarco, 2007; Le Conte ◦ Nosema spp. Microsporidia, black queen et al., 2007; Büchler et al., 2010; Le Conte cell virus and Israeli acute paralysis virus et al., 2010). There are also several Euro- Two widespread pesticides pean honeybee populations that have never been infested by mites, such as on the Island ◦ the neonicotinoid agro-pesticide thiaclo- of Ouessant in France, large parts of north- prid ern Sweden and the Finnish island Åland in ◦ the pyrethroid beekeeping acaricide τ- the Baltic Sea. Such populations are essen- fluvalinate tial for understanding the genetic mechanisms Two categories of interactions between benefi- driving mite infestation tolerance (for the Var- cial organism with pathogens roa-tolerant feral colonies) and the epidemiological and evolutionary mechanisms underly- ◦ Probiotic honeybee gut microflora and ing the lethal interactions between Varroa and bacterial pathogens viral pathogens (for the Varroa-free popula- ◦ Propolis and plant secondary metabolites tions). There are similar populations elsewhere and parasites & pathogens. (Australian Varroa-free populations; African These must be studied at both individual bee and South American Varroa-tolerant popula- and colony levels, since pathogen virulence at tions) for comparison with European popula- the individual level is often inversely related to tions. virulence at colony level (Sumpter and Martin, 2004; Genersch et al., 2005), and also will include higher order interactions between the 2.3.2. Nosema spp. three categories; for example, between pesticides and parasite-pathogens, or between ben- Nosema fungi are wide-spread mi- eficial interactions and pesticides. crosporidian gut parasites of adult honeybees. They infect host mid-gut epithelial cells and deteriorate the metabolic processes of infected 2.3. Parasites bees (Fries, 1988, 1993). Currently, two different Nosema species have been reported in A. 2.3.1. Varroa destructor mellifera: N. apis, a well established pathogen of A. mellifera with moderate virulence that The parasitic mite V. destructor is with- does not usually cause lethal infections, and out doubt the main obstacle to profitable bee- N. ceranae, thought to be originally a parasite keeping worldwide (Sammataro et al., 2000). of the Asian honeybee A. cerana,which This highly specialized parasite of the hon- has recently been introduced into European eybee feeds on both the brood and the adult honeybee populations (Fries et al., 1996, bee, and reproduces in the brood cell. Al- 2006b; Paxton et al., 2007;Chenetal.,2008) though the mite causes little damage to its and distributed by apicultural trade across the original host, the Asian honeybee Apis cerana, globe (Klee et al., 2007). It is now present in it is lethal for European A. mellifera colonies all continents except Antarctica (Klee et al., to which it transferred more than 30 years 2007; Higes et al., 2009) and honeybees can ago (Matheson, 1995). Therefore, adequate be co-infected with both Nosema species. N. and timely mite control is essential for api- ceranae infestations appear to be more severe culture in Europe and most other regions of in southern than in northern parts of Europe the world. Varroa mite control is overwhelm- (Fries, 2010). Moreover, N. ceranae seems ingly based on chemicals that typically end up to replace N. apis worldwide (Klee et al., 232 R.F.A. Moritz et al.

2007) although it does not appear to have a 2.4.1. Deformed Wing Virus (DWV) competitive advantage within an individual host bee (Forsgren and Fries, 2010). DWV is by far the most widespread honey- In Spain, N. ceranae has been reported to bee virus (Ribière et al., 2008; de Miranda and be linked to the sudden collapse of A. mellif- Genersch, 2010) due to its close association era colonies (Higes et al., 2008) and increased with V. destructor (Bowen-Walker et al., 1999; risk of colony death if not actively controlled Tentcheva et al., 2006; Gauthier et al., 2007). (Martín-Hernández et al., 2007). This high DWV became almost ubiquitous throughout colony level virulence of N. ceranae in Spain Europe with the spread of Varroa (Allen and may be a regional phenomenon, as high colony Ball, 1996). Of itself, DWV is one of the least mortality is not always observed (Siede et al., virulent of bee viruses and its damage to indi- 2008; Invernizzi et al., 2009). Moreover, sev- vidual bees and colonies is only due to its rela- eral viruses are associated with Nosema infec- tionship with Varroa mites. It has been shown tions that can signiﬁcantly aﬀect the apparent to be transmitted by, and to replicate inside virulence of Nosema (Bailey and Ball, 1991). V. destructor (Ongus et al., 2004;Yueand Very little study has so far been dedicated to Genersch, 2005). Furthermore, DWV symp- these potentially important interactions, which toms in emerging bees appear to be related European research strategies aim to address. to virus replication in the infesting mites dur- Due to the similarity in life histories of the two ing the pupal phase (Yue and Genersch, 2005; Nosema spp., it is likely that the interactions Gisder et al., 2009). The paradox of the DWV with other factors may be similar for N. apis – Varroa – bee interaction is that it is precisely and N. ceranae.TheN. apis genome is cur- the low virulence of DWV that allows Var- rently being sequenced (J. Evans, unpubl. data; roa infested pupae to complete development Chen and Huang, 2010) which will greatly fa- despite the virus infection, thus liberating the cilitate its study. reproducing mites and sustaining the Varroa - DWV epidemic that ultimately becomes lethal at colony level (Sumpter and Martin, 2004). This contrast between individual and colony- level virulence is also seen with other bee 2.4. Viral pathogens pathogens, emphasizing the need to study such interactions at both levels.

At least 18 viruses have been identified that affect brood and/or adult honeybees (Bailey 2.4.2. Black Queen Cell Virus (BQCV) and Ball, 1991; Ribière et al., 2008). Even healthy colonies are usually covertly infected BQCV itself is not particularly damag- by several viruses (Tentcheva et al., 2004). V. ing. However, it has a synergistic effect with destructor has been shown to be an impor- Nosema infections, making the latter more tant vector for several of these viruses. Like- harmful (Bailey et al., 1981, 1983). Recent wise, a number of viruses seem to be closely surveys show it to be present in about 30% linked to Nosema infections. Since the large of colonies in France and central Europe number of viruses affecting bees renders it (Tentcheva et al., 2004; Berényi et al., 2006; impossible to run full factorial experimen- Gauthier et al., 2007) making it a significant tal designs on all possible interactions, it is virus. Although BQCV is named for its effect will be more meaningful to focus on the best on queen pupae, it is primarily distributed in established and most dramatic virus–parasite adult bees (Tentcheva et al., 2004)whichis interactions. Once these interactions are un- also the only stage known to be affected by derstood, other viruses can perhaps also be Nosema (Fries, 1988, 1993). BEE DOC will assessed, though currently only from correla- focus on this virus–endoparasite combination tional evidence from large scale field data to to test for interaction mechanisms occurring at understand their impact on colony health. the adult stages of the bee. Research strategies to improve honeybee health in Europe 233

2.4.3. Israel Acute Paralysis Virus colony level (Genersch et al., 2005), since the (IAPV) larvae die and are removed by hygienic behaviour before spore production can be max- IAPV is part of a larger species complex imised. It is therefore essential to combine in- (de Miranda et al., 2010) that also includes dividual and colony level studies to determine Kashmir Bee Virus (KBV) and Acute Bee the significance of virulence and transmission Paralysis Virus (ABPV), two viruses that can for the epidemiology of AFB. This informa- be lethal at individual bee and colony lev- tion is essential for developing management els (Todd et al., 2007; Ball and Allen, 1988). and selection programmes for disease control IAPV has been considered an associative fac- (Dieckmann et al., 2000). tor for Colony Collapse Disorder (CCD) in America (Cox-Foster et al., 2007; Johnson et al., 2009). In the past, European colony 2.6. Pesticides losses with symptoms similar to CCD have been associated with ABPV (Berényi et al., 2.6.1. Lethal and sublethal 2006). Although the significance of IAPV for The honeybee is unusually sensitive to a CCD is still unclear (Oldroyd, 2007; Stokstad, range of chemical insecticides (Stefanidou 2007; Anderson and East, 2008), it is sensible et al., 2003; Thompson, 2003; Barnett et al., to include this virus complex at the experimen- 2007), most likely due to a relative deficit tal level to clarify its importance for European of detoxification enzymes (Yu et al., 1984; honeybee populations, including the possible Claudianos et al., 2006). Foraging bees can interchangeability of IAPV, KBV and ABPV encounter lethal pesticide levels when forag- as risk indicators for colony collapse in differ- ing but they can also bring back contaminated ent geographic areas (de Miranda et al., 2010). nectar and pollen to the hive. In addition to the pesticides the bees are exposed to during 2.5. Bacterial pathogens foraging, beekeepers also use various acaricides to control mite infections, particularly V. American foulbrood (AFB) is caused by destructor (Sammataro et al., 2000). Most of the spore-forming bacterium Paenibacillus lar- these are lipophilic and accumulate in the wax, vae and is the most significant bacterial dis- increasingly contaminating the combs where ease in apiculture. Although this disease was the brood develops. More importantly, nothing identified >100 years ago, it still plagues bee- is known about the interactions between agri- keeping in the EU. On a global scale, antibi- cultural pesticides foraged on by bees, the aca- otic treatment for AFB is standard, although ricides applied by the beekeeper, and pests and less common in Europe. Employing selection pathogens. for resistant honeybee strains has had pri- The EU directive 91/414 Section 2.5.3 regu- ority in breeding work for many years, as lates the use of pesticides in the context of api- has the disruption of infection pathways as culture. “. . . no authorization will be granted tools to develop contamination-free remedy if the hazard quotients for oral or contact ex- schemes. Clearly, the prime goal is to make posure of honeybees are greater than 50, un- antibiotic treatments obsolete. Not only do less it is clearly established through appropri- as they inevitably lead to honey contamina- ate risk assessment that under field conditions tion and pathogen resistance (Spivak, 2001), there are no unacceptable effects on honey- but they will most likely also kill the ben- bee larvae, honeybee behaviour,or colony sur- eficial honeybee probiotic microflora that is vival and development after the use of plant part of the natural honeybee defence against protections product according to he proposed AFB (Forsgren et al., 2009; Yoshiyama and conditions of use”. Acute mortality can occur Kimura, 2009). Although there is wide vari- and its diagnosis is usually easily established ation in virulence between P. la rv a e strains, by the presences of many dead bees in front the most virulent strains at the individual lar- of the hive. However, honeybees can also en- val level are the least virulent strains at the counter sub-lethal effects of pesticides that are 234 R.F.A. Moritz et al. much more difficult to detect since they affect 1999; Tremolada et al., 2004). It is one of the longevity or behaviour. Such sub-lethal effects most common pesticides found in honeybee can cause disruptions in social interactions colonies. that are essential for colony function (Weick and Thorn, 2002). Since many pathogens have similar sub-lethal effects on longevity and be- 3. TOOLS AND CONCEPTS haviour (Ball and Bailey, 1991), the cumula- TO IMPROVE COLONY HEALTH tive impact of different sub-lethal effects may be significant at colony level, even when they 3.1. Probiotic bacteria are not immediately apparent when studied in isolation, and at individual bee level. Bifidobacterium and lactic acid bacteria The huge suite of agro-chemicals currently (LAB) from the genus Lactobacillus are gen- used in agriculture makes it clearly impossible erally beneficial bacterial species. They are to run full factorial design experiments test- part of the natural gastrointestinal flora of ing the effects and interactions of all of those healthy animals and are sold commercially as compounds. If we focus on selected model probiotics, live micro-organisms, that confer compounds we will be able to extract the health benefits to their host. The honeybee principle effects of the pesticide interactions also has a unique honey stomach probiotic with parasites and pathogens on colony health. flora involving Lactobacillus and Bifidobac- For example the BEE DOC research network terium bacteria (Olofsson and Vasquez, 2008; will focus only on two major compounds, one Yoshiyama and Kimura, 2009) that protects neonicotinoid agro-pesticide, thiacloprid, and the bee from harmful micro-organisms in ex- one pyrethroid acaricide, τ-fluvalinate. Thia- change for a nutrient rich niche. LAB pro- cloprid and τ-fluvalinate therefore represent duce such antibacterial compounds as organic the two most important and common pesticide acids, hydrogen peroxide, diacetyl, benzoate, groups (pyrethroids and neonicotinoids) with and bacteriocins, all of which are beneficial for different modes of action on the target organ- humans and animals (Coenye and Vandamme, isms. 2003; Ouwehand et al., 2002) and presum- ably for honeybees as well. Most interestingly, LAB have recently been shown to strongly in- 2.6.2. Thiacloprid hibit P. la rv a e , the bacterium causing Ameri- can Foulbrood (Forsgren et al., 2009). Thiacloprid is a broad-spectrum neonicotinoid insecticide with a fairly low acute bee toxicity, and is used against a wide range of lepidopteran, coleopteran and orthopteran 3.2. Plant and honeybee derived crop pests, including oil seed rape and fruit compounds orchards which are crops intensively used by ffi honey bees. Consequently, the active ingre- Apiary hygiene alone is insu cient for dis- dient is commonly found in the hive and in ease control and prevention, and persistent and pollen pellets of foragers (Chauzat et al., 2006; prophylactic chemical treatments inevitably Chauzat and Faucon, 2007). lead to pathogen resistance (Lodesani, 1995; Milani, 1995, 1999; Elzen, 1998; Thompson, 2002). European research strategies will there- 2.6.3. τ-fluvalinate fore include a focus on the development of novel treatments by using therapies de- τ−fluvalinate is a pyrethroid used to control signed by nature and by the honeybees a broad range of pests including moths, aphids, themselves. Compounds from propolis col- thrips and leafhoppers. It is also used widely lected by bees will be tested for their im- to control the mites in beekeeping, and accu- pact on disease control and prevention. Also mulates in the wax of the comb at high con- honeybee-produced peptides will be produced centrations (Bogdanov et al., 1998; Wallner, by recombinant technology with the goal of Research strategies to improve honeybee health in Europe 235 applying them to the colony for disease treat- 3.3. Disease resistance genetics ment. The honeybee colony is armed against and genomics pathogens with a very effective exogenous defense system based on the multi-functionality In an ideal beekeepers’ world, honey- of nutritive proteins and antimicrobial phyto- bees should not require any treatment against chemicals. These compounds are present in diseases at all, which would prevent the con- nectar, pollen and propolis and have proven tamination of colonies with in-hive chemi- ff to be e ective acaricides and antimicrobial cals used in apicultural management. EU re- agents in many organisms, including humans search therefore focuses on the identification (Bankova et al., 1995; Gil et al., 2000;Tomás- of genes that regulate resistance towards Var- Barberán and Wollenweber, 1990;Tomás- roa and American Foulbrood. In the context Barberan et al., 1989; Popova et al., 2004, of the BEE DOC network, the immune sys- 2007;Bankova,2005). Paradoxically, little in- tem of honeybees lies at the centre of in- ffi formation is available about the e cacy of terest because the responses of the bees to- these compounds against honeybee parasites wards virus infections need to be addressed. and pathogens. In particular propolis is consid- Although A. mellifera lacks about 30% of ered to be the most important chemical defense immune system genes that are known from mechanism of honeybees against microorgan- different dipteran species (e.g. Drosophila isms, since it is based on metabolites used by melanogaster, Anopheles gambiae), the three plants against pests and diseases (e.g., Simone basic immune pathways – the Toll-, IMD- and et al., 2009; Simone and Spivak, 2010). Hon- JAK/STAT-pathway have been identified in the eybees also have innate molecular defenses honeybee genome (Evans et al., 2006). One ar- against pathogens, such as antimicrobial pep- gument for the deficit of immune genes was tides and nucleic acids, whose activity can be that colony level pathogen resistance mecha- stimulated by appropriate molecular therapies. nisms would be more important in social in- Propolis constituents, recombinant honeybee sects compared to solitary insects. However, peptides, and molecular therapies will be as- individual resistance mechanisms are equally sayed at individual and colony level, singly essential in a social context as in the solitary ffi and in combination, for e cacy against hon- one. It is therefore likely that the gene cas- eybee diseases. cades in these pathways are differently regu- lated compared to the other insect model sys- To control diseases efficiently with novel, tems, yielding similar results yet in a colony sustainable strategies, we must understand the context. In this context, it may be highly re- infection processes at all relevant levels: from warding to compare genes identified in the the apiary, via the colony and individual bee, honeybee with homologues in the well studied down to the molecular immune mechanisms fruit fly Drosophila. There are even reports of at the genome level. The interactions among a specific memory in insect immune systems pathogens driving virulence and transmission (Kurtz and Franz, 2003), albeit with a lower of diseases need to be comprehensively under- adaptability compared to vertebrate immune stood if we want to design more efficient treat- systems. Hence, we expect to find very specific ments that are also effective at the population modifications of the already known immune level (Evans and Spivak, 2010). We will need gene cascades for evolutionary stable insect to develop strategies that not only increase bee host parasite systems, and also highly species tolerance to specific diseases, but those that specific, yet unknown, mechanisms for con- can reduce pathogen virulence by blocking trolling the pathogen resistance of bees. Since critical infection pathways or harmful interac- oligonucleotide microarrays with all annotated tions among pathogens. This will enable the genes of the honeybee (∼13 400 genes) are development of complementary strategies for available, the transcriptome can be screened disease control to prevent colony losses and se- to reveal differential genome responses to spe- cure the quality and safety of honey and other cific infections. Although such studies can- bee products. not identify the function of novel honeybee 236 R.F.A. Moritz et al. specific genes, they can reveal the function of to be most informative (Kocher et al., 2008; gene cascades in response to various environ- Alaux et al., 2009). With this tool the genomic mental conditions (Grozinger et al., 2003)in- responses towards pathogens and pesticides cluding bee health stressors (Navajas et al., at the transcriptome level can be used to ex- 2008), which are essential to understand gene plore host-pathogen interactions at the molec- control of the phenotype. ular level.

3.3.1. Candidate genes known from 3.3.2. Novel genes specific to honeybees sequence homology In addition, it will be important to identify Genes already known from model or- novel genes that control host – pathogen inter- ganisms can be identified by standard bio- actions. Many pathogens are highly specific to informatics: those specific for honeybees still the honeybee system and hence very special require mapping and gene expression studies. solutions for pathogen resistance may have This is greatly facilitated with the complete evolved. A key element will be to use drones genome sequence for the honeybee at hand. for mapping of quantitative trait loci (QTL) For example the gene “thelytoky”(th)was that are relevant for disease resistance (Moritz mapped down to 20 cM within a few months and Evans, 2007). Candidate loci for all brood and was recently identified as a transcription diseases including AFB susceptibility, Varroa factor homologous to gemini in Drosophila resistance, DWV resistance and Nosema resis- (Lattorff et al., 2007). Two genes, th and csd tance will need to be identified. This will com- (the sex locus), are currently the only two plete the set of resistance genes to the main known honeybee specific genes. This may honeybee diseases, which will greatly facili- seem to be a low number of genes, but only in tate breeding programs. a few cases will a single gene determine a specific phenotype. Most phenotypes will be controlled by several genes, so called quantitative 3.4. Diagnostics trait loci (QTL). If there are only few major QTLs for a trait these can be mapped by test- The different operational levels of BEE ing linkage with segregation of a large number DOC (experimental, surveillance, applied) re- of variable markers (e.g. microsatellite mark- quire different tools for identifying the vari- ers) which saturate the genome. Because the ous parasites, pathogens and chemicals. This recombination rate of the honeybee genome is internal requirement will allow BEE DOC to 19 cM/Mb, an order of magnitude higher than provide diagnostic tools to different groups of in Drosophila, honeybee mapping studies re- stakeholders in the bee industry, from the require a large number of marker loci to saturate searcher to the beekeeper. the genome (Weinstock et al., 2006). For gene identification, the high recombination rate is an advantage, because linked markers can be 3.4.1. Experimental physically very close to a target gene. Once a genomic region with a QTL has been iden- Real time RT-qPCR is the standard tech- tified, the sequence allows for saturating this nique for quantitative diagnosis of the bee target region with a large number of novel mi- pathogens. Microarrays (Navajas et al., 2008; crosatellite markers for fine mapping. Com- Johnson et al., 2009), metagenomic analyses parisons across microarray experiments show (Cox-Foster et al., 2007) and next genera- differential transcriptome responses towards tion DNA sequencing are more comprehen- infestations with Varroa (Navajas et al., 2008). sive (and expensive) screening technologies, to The novel oligonucleotide microarray com- be used only in limited experimental setting. prising all annotated 13 440 genes of the hon- DNA chip technology (Whitfield et al., 2006) eybee has already been validated and found allows a rapid yet cost efficient diagnosis of all Research strategies to improve honeybee health in Europe 237 known pathogens in experimental samples, as to identify the underlying factors and mech- well as a number of honeybee genes of interest anisms (e.g. ring tests). Indeed, efforts by (Evans, 2007). Pesticides and bio-chemicals individual countries to reveal the drivers of will be quantified by a number of chromato- colony losses have small chance of success graphic techniques. due to the high number of interacting factors. Therefore, the development of emergency measures and sustainable management strate- 3.4.2. Surveillance gies will require an international network. The COLOSS network does not directly support The real-time quantitative PCR-based diag- science but aims at coordinating national re- nostic tools for pathogen detection, as well as search activities across Europe and the world. the pesticide and biochemical analyses, will be COLOSS comprises all three groups of stake- adapted for routine use in large-scale surveys holders, scientists, beekeepers and industry by extension labs. with the aims of complementing and not du- plicating research approaches, and of creating trans-national synergies. The tight networking 3.4.3. Applied among science and the industry is facilitated through conferences, and more importantly, Robust, immunochromatography-based sti- through a large series of hands-on workshops ck assays, common in medical and veterinary for extension specialists and apiculturists. The disease diagnosis (Abhyankar et al., 2006;Pu- European and global strategy for the preven- gia et al., 2004), will need to be developed to tion of colony losses is therefore clearly based facilitate accurate, sensitive and instant diag- on a broad transnational platform with a strong nosis of honeybee diseases in the field. focus on the transfer of science into practice. Only if we succeed to bridge the gap between bee scientists and apiculture will we 4. IMPLEMENTION OF SCIENTIFIC achieve sustainable progress in the prevention PROGRESS INTO APICULTURAL of colony losses at a continental scale. PRACTICE AND COORDINATION OF RESEARCH ACKNOWLEDGEMENTS The transfer from science into application Funding for the BEE DOC research network is typically a major problem. In Europe this has been provided by the EU Commission (RTD transfer is greatly facilitated through one of REG/E.4(2009)D/561221) and for the COLOSS the largest programs in history, COST (Eu- network by the COST action FA0803. ropean COoperation in Science and Tech- nology). COLOSS (Prevention of Honeybee // / Stratégies de recherche pour améliorer la santé Colony Losses, http: www.coloss.org )isa des abeilles en Europe. global network with currently over 150 part- ners in 39 countries (most of Europe, Aus- Apis mellifera / pathologie / diagnostic / résis- tralia, Canada, Israel, Jordan, PR China, South tance aux maladies Korea, Republic of South Africa, USA). The aim of COLOS is to coordinate national and ff Zusammenfassung – Forschungsstrategien international e orts to explain and prevent zur Verbesserung der Bienengesundheit in large scale losses of honeybee colonies. For Europa. Die letzten Jahrzehnte waren durch that purpose, international standards will be einen konstanten Rückgang von Bienenvölkern in developed for both monitoring and research den Mitgliedstaaten der EU gekennzeichnet (cf. activities in form of a BEE BOOK (analo- Abb. 1). Insbesondere dramatische und unkontrol- lierbare Völkerverluste entwickelten sich zu einer gous to the RED BOOK of the Drosophila akuten Insolvenz - Bedrohung für Imkereibetriebe. community). This effort will enable combined Nach wie vor sind die Ursachen dieser großen, and large-scale international research efforts flächendeckenden Völkerverluste auf nationaler 238 R.F.A. Moritz et al.

Ebene unklar und daher sind zielgerichtete kausale projekte zur Bienengesundheit koordinieren und Therapien nicht möglich. Oft wurden daher unnöti- aufeinander abstimmen. Nur wenn es gelingt, die ge, prophylaktisch medikamentöse Behandlungen Forschungsergebnisse in der Imkerei umzusetzen, durchgeführt, um regionale Völkerbestände zu werden wir Fortschritte bei der nachhaltigen sichern. Dies hat bislang jedoch noch nicht zu einer Prävention von Völkerverlusten auf einer europa- nachhaltigen Bekämpfung von Bienenkrankheiten und weltweiten Ebene erzielen können. geführt, allerdings regelmäßig zur Kontamination des Honigs. Apis mellifera / Pathologie / Diagnose / Krank- Die Forschungspolitik der EU zielt daher darauf ab, heitsresistenz die Honigbelastung zu reduzieren, die Rassevielfalt europäischer Honigbienen zu erhalten, Völker- verluste zu vermeiden und die Bedeutung der Interaktionen zwischen Parasiten, Pathogenen und Pestiziden für die Koloniegesundheit zu verstehen. 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