Distribution of infectious and parasitic agents among three sentinel bee species across European agricultural landscapes.

Infectious and parasitic agents (IPAs) and their associated diseases are major environmental stressors that jeopardize bee health, both alone and in interaction with other stressors. Their impact on pollinator communities can be assessed by studying multiple sentinel bee species. Here, we analysed the field exposure of three sentinel managed bee species (Apis mellifera, Bombus terrestris and Osmia bicornis) to 11 IPAs (six RNA viruses, two bacteria, three microsporidia). The sentinel bees were deployed at 128 sites in eight European countries adjacent to either oilseed rape fields or apple orchards during crop bloom. Adult bees of each species were sampled before their placement and after crop bloom. The IPAs were detected and quantified using a harmonised, high-throughput and semi-automatized qPCR workflow. We describe differences among bee species in IPA profiles (richness, diversity, detection frequencies, loads and their change upon field exposure, and exposure risk), with no clear patterns related to the country or focal crop. Our results suggest that the most frequent IPAs in adult bees are more appropriate for assessing the bees' IPA exposure risk. We also report positive correlations of IPA loads supporting the potential IPA transmission among sentinels, suggesting careful consideration should be taken when introducing managed pollinators in ecologically sensitive environments.

Functional traits shape plant-plant interactions and recruitment in a hotspot of woody plant diversity.

Understanding and predicting recruitment in species-rich plant communities requires identifying functional determinants of both density-independent performance and interactions. In a common-garden field experiment with 25 species of the woody plant genus Protea, we varied the initial spatial and taxonomic arrangement of seedlings and followed their survival and growth during recruitment. Neighbourhood models quantified how six key functional traits affect density-independent performance, interaction effects and responses. Trait-based neighbourhood models accurately predicted individual survival and growth from the initial spatial and functional composition of species-rich experimental communities. Functional variation among species caused substantial variation in density-independent survival and growth that was not correlated with interaction effects and responses. Interactions were spatially restricted but had important, predominantly competitive, effects on recruitment. Traits increasing the acquisition of limiting resources (water for survival and soil P for growth) mediated trade-offs between interaction effects and responses. Moreover, resprouting species had higher survival but reduced growth, likely reinforcing the survival-growth trade-off in adult plants. Resource acquisition of juvenile plants shapes Protea community dynamics with acquisitive species with strong competitive effects suffering more from competition. Together with functional determinants of density-independent performance, this makes recruitment remarkably predictable, which is critical for efficient restoration and near-term ecological forecasts of species-rich communities.

Alien plant fitness is limited by functional trade-offs rather than a long-term increase in competitive effects of native communities.

Alien plants experience novel abiotic conditions and interactions with native communities in the introduced area. Intra- and interspecific selection on functional traits in the new environment may lead to increased population growth with time since introduction (residence time). However, selection regimes might differ depending on the invaded habitat. Additionally, in high-competition habitats, a build-up of biotic resistance of native species due to accumulation of eco-evolutionary experience to aliens over time may limit invasion success. We tested if the effect of functional traits and the population dynamics of aliens depends on interspecific competition with native plant communities. We conducted a multi-species experiment with 40 annual Asteraceae that differ in residence time in Germany. We followed their population growth in monocultures and in interspecific competition with an experienced native community (varying co-existence times between focals and community). To more robustly test our findings, we used a naïve community that never co-existed with the focals. We found that high seed mass decreased population growth in monocultures but tended to increase population growth under high interspecific competition. We found no evidence for a build-up of competition-mediated biotic resistance by the experienced community over time. Instead, population growth of the focal species was similarly inhibited by the experienced and naïve community. By comparing the effect of experienced and naïve communities on population dynamics over 2 years across a large set of species with a high variation in functional traits and residence time, this study advances the understanding of the long-term dynamics of plant invasions. In our study system, population growth of alien species was not limited by an increase of competitive effects by native communities (one aspect of biotic resistance) over time. Instead, invasion success of alien plants may be limited because initial spread in low-competition habitats requires different traits than establishment in high-competition habitats.

Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR.

Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees (Apis spp.), while Nosema bombi has been described in bumble bees (Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 (RPB1) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee (Apis mellifera) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log10 copies/honey bee. Method accuracy was also verified in bumble bee (Bombus terrestris) and mason bee (Osmia bicornis) homogenates in the range of 5 to 10 log10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite's eco-dynamics in managed and wild bees.

Transmission of deformed wing virus between Varroa destructor foundresses, mite offspring and infested honey bees.

BACKGROUND: Varroa destructor is the major ectoparasite of the western honey bee (Apis mellifera). Through both its parasitic life-cycle and its role as a vector of viral pathogens, it can cause major damage to honey bee colonies. The deformed wing virus (DWV) is the most common virus transmitted by this ectoparasite, and the mite is correlated to increased viral prevalence and viral loads in infested colonies. DWV variants A and B (DWV-A and DWV-B, respectively) are the two major DWV variants, and they differ both in their virulence and transmission dynamics. METHODS: We studied the transmission of DWV between bees, parasitic mites and their offspring by quantifying DWV loads in bees and mites collected in in vitro and in situ environments. In vitro, we artificially transmitted DWV-A to mites and quantified both DWV-A and DWV-B in mites and bees. In situ, we measured the natural presence of DWV-B in bees, mites and mites' offspring. RESULTS: Bee and mite viral loads were correlated, and mites carrying both variants were associated with higher mortality of the infected host. Mite infestation increased the DWV-B loads and decreased the DWV-A loads in our laboratory conditions. In situ, viral quantification in the mite offspring showed that, after an initially non-infected egg stage, the DWV-B loads were more closely correlated with the foundress (mother) mites than with the bee hosts. CONCLUSIONS: The association between mites and DWV-B was highlighted in this study. The parasitic history of a mite directly impacts its DWV infection potential during the rest of its life-cycle (in terms of variant and viral loads). Regarding the mite's progeny, we hypothesize that the route of contamination is likely through the feeding site rather than by vertical transmission, although further studies are needed to confirm this hypothesis.

Improving measurements of the falling trajectory and terminal velocity of wind-dispersed seeds.

Seed dispersal by wind is one of the most important dispersal mechanisms in plants. The key seed trait affecting seed dispersal by wind is the effective terminal velocity (hereafter "terminal velocity", V t ), the maximum falling speed of a seed in still air. Accurate estimates of V t are crucial for predicting intra- and interspecific variation in seed dispersal ability. However, existing methods produce biased estimates of V t for slow- or fast-falling seeds, fragile seeds, and seeds with complex falling trajectories. We present a new video-based method that estimates the falling trajectory and V t of wind-dispersed seeds. The design involves a mirror that enables a camera to simultaneously record a falling seed from two perspectives. Automated image analysis then determines three-dimensional seed trajectories at high temporal resolution. To these trajectories, we fit a physical model of free fall with air resistance to estimate V t . We validated this method by comparing the estimated V t of spheres of different diameters and materials to theoretical expectations and by comparing the estimated V t of seeds to measurements in a vertical wind tunnel. V t estimates closely match theoretical expectations for spheres and vertical wind tunnel measurements for seeds. However, our V t estimates for fast-falling seeds are markedly higher than those in an existing trait database. This discrepancy seems to arise because previous estimates inadequately accounted for seed acceleration. The presented method yields accurate, efficient, and affordable estimates of the three-dimensional falling trajectory and terminal velocity for a wide range of seed types. The method should thus advance the understanding and prediction of wind-driven seed dispersal.

Global relationships in tree functional traits.

Due to massive energetic investments in woody support structures, trees are subject to unique physiological, mechanical, and ecological pressures not experienced by herbaceous plants. Despite a wealth of studies exploring trait relationships across the entire plant kingdom, the dominant traits underpinning these unique aspects of tree form and function remain unclear. Here, by considering 18 functional traits, encompassing leaf, seed, bark, wood, crown, and root characteristics, we quantify the multidimensional relationships in tree trait expression. We find that nearly half of trait variation is captured by two axes: one reflecting leaf economics, the other reflecting tree size and competition for light. Yet these orthogonal axes reveal strong environmental convergence, exhibiting correlated responses to temperature, moisture, and elevation. By subsequently exploring multidimensional trait relationships, we show that the full dimensionality of trait space is captured by eight distinct clusters, each reflecting a unique aspect of tree form and function. Collectively, this work identifies a core set of traits needed to quantify global patterns in functional biodiversity, and it contributes to our fundamental understanding of the functioning of forests worldwide.

Seed dispersal by wind decreases when plants are water-stressed, potentially counteracting species coexistence and niche evolution.

Hydrology is a major environmental factor determining plant fitness, and hydrological niche segregation (HNS) has been widely used to explain species coexistence. Nevertheless, the distribution of plant species along hydrological gradients does not only depend on their hydrological niches but also depend on their seed dispersal, with dispersal either weakening or reinforcing the effects of HNS on coexistence. However, it is poorly understood how seed dispersal responds to hydrological conditions. To close this gap, we conducted a common-garden experiment exposing five wind-dispersed plant species (Bellis perennis, Chenopodium album, Crepis sancta, Hypochaeris glabra, and Hypochaeris radicata) to different hydrological conditions. We quantified the effects of hydrological conditions on seed production and dispersal traits, and simulated seed dispersal distances with a mechanistic dispersal model. We found species-specific responses of seed production, seed dispersal traits, and predicted dispersal distances to hydrological conditions. Despite these species-specific responses, there was a general positive relationship between seed production and dispersal distance: Plants growing in favorable hydrological conditions not only produce more seeds but also disperse them over longer distances. This arises mostly because plants growing in favorable environments grow taller and thus disperse their seeds over longer distances. We postulate that the positive relationship between seed production and dispersal may reduce the concentration of each species to the environments favorable for it, thus counteracting species coexistence. Moreover, the resulting asymmetrical gene flow from favorable to stressful habitats may slow down the microevolution of hydrological niches, causing evolutionary niche conservatism. Accounting for context-dependent seed dispersal should thus improve ecological and evolutionary models for the spatial dynamics of plant populations and communities.

The importance of individual movement and feeding behaviour for long-distance seed dispersal by red deer: a data-driven model.

BACKGROUND: Long-distance seed dispersal (LDD) has strong impacts on the spatiotemporal dynamics of plants. Large animals are important LDD vectors because they regularly transport seeds of many plant species over long distances. While there is now ample evidence that behaviour varies considerably between individual animals, it is not clear to what extent inter-individual variation in behaviour alters seed dispersal by animals. METHODS: We study how inter-individual variation in the movement and feeding behaviour of one of Europe's largest herbivores (the red deer, Cervus elaphus) affects internal seed dispersal (endozoochory) of multiple plant species. We combine movement data of 21 individual deer with measurements of seed loads in the dung of the same individuals and with data on gut passage time. These data serve to parameterize a model of passive dispersal that predicts LDD in three orientations (horizontal as well as upward and downward in elevation).With this model we investigate to what extent per-seed probabilities of LDD and seed load vary between individuals and throughout the vegetation period (May-December). Subsequently, we test whether per-seed LDD probability and seed load are positively (or negatively) correlated so that more mobile animals disperse more (or less) seeds. Finally, we examine whether non-random associations between per-seed LDD probability and seed load affect the LDD of individual plant species. RESULTS: The studied deer dispersed viable seeds of at least 62 plant species. Deer individuals varied significantly in per-seed LDD probability and seed loads. However, more mobile animals did not disperse more or less seeds than less mobile ones. Plant species also did not differ significantly in the relationship between per-seed LDD probability and seed load. Yet plant species differed in how their seed load was distributed across deer individuals and in time, and this caused their LDD potential to differ more than twofold. For several plant species, we detected non-random associations between per-seed LDD probability and seed load that generally increased LDD potential. CONCLUSIONS: Inter-individual variation in movement and feeding behaviour means that certain deer are substantially more effective LDD vectors than others. This inter-individual variation reduces the reliability of LDD and increases the sensitivity of LDD to the decline of deer populations. Variation in the dispersal services of individual animals should thus be taken into account in models in order to improve LDD projections.

Shifts in plant functional community composition under hydrological stress strongly decelerate litter decomposition.

Litter decomposition is a key process of nutrient and carbon cycling in terrestrial ecosystems. The decomposition process will likely be altered under ongoing climate change, both through direct effects on decomposer activity and through indirect effects caused by changes in litter quality. We studied how hydrological change indirectly affects decomposition via plant functional community restructuring caused by changes in plant species' relative abundances (community-weighted mean (CWM) traits and functional diversity). We further assessed how those indirect litter quality effects compare to direct effects. We set up a mesocosm experiment, in which sown grassland communities and natural turf pieces were subjected to different hydrological conditions (dryness and waterlogging) for two growing seasons. Species-level mean traits were obtained from trait databases and combined with species' relative abundances to assess functional community restructuring. We studied decomposition of mixed litter from these communities in a common "litterbed." These indirect effects were compared to effects of different hydrological conditions on soil respiration and on decomposition of standard litter (direct effects). Dryness reduced biomass production in sown communities and natural turf pieces, while waterlogging only reduced biomass in sown communities. Hydrological stress caused profound shifts in species' abundances and consequently in plant functional community composition. Hydrologically stressed communities had higher CMW leaf dry matter content, lower CMW leaf nitrogen content, and lower functional diversity. Lower CWM leaf N content and functional diversity were strongly related to slower decomposition. These indirect effects paralleled direct effects, but were larger and longer-lasting. Species mean traits from trait databases had therefore considerable predictive power for decomposition. Our results show that stressful soil moisture conditions, that are likely to occur more frequently in the future, quickly shift species' abundances. The resulting functional community restructuring will decelerate decomposition under hydrological stress.

Mismatches between demographic niches and geographic distributions are strongest in poorly dispersed and highly persistent plant species.

The ecological niche of a species describes the variation in population growth rates along environmental gradients that drives geographic range dynamics. Niches are thus central for understanding and forecasting species' geographic distributions. However, theory predicts that migration limitation, source-sink dynamics, and time-lagged local extinction can cause mismatches between niches and geographic distributions. It is still unclear how relevant these niche-distribution mismatches are for biodiversity dynamics and how they depend on species life-history traits. This is mainly due to a lack of the comprehensive, range-wide demographic data needed to directly infer ecological niches for multiple species. Here we quantify niches from extensive demographic measurements along environmental gradients across the geographic ranges of 26 plant species (Proteaceae; South Africa). We then test whether life history explains variation in species' niches and niche-distribution mismatches. Niches are generally wider for species with high seed dispersal or persistence abilities. Life-history traits also explain the considerable interspecific variation in niche-distribution mismatches: poorer dispersers are absent from larger parts of their potential geographic ranges, whereas species with higher persistence ability more frequently occupy environments outside their ecological niche. Our study thus identifies major demographic and functional determinants of species' niches and geographic distributions. It highlights that the inference of ecological niches from geographical distributions is most problematic for poorly dispersed and highly persistent species. We conclude that the direct quantification of ecological niches from demographic responses to environmental variation is a crucial step toward a better predictive understanding of biodiversity dynamics under environmental change.

Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.

Co-exposure to pesticides and viruses is likely to occur in honey bee colonies. Pesticides can be present in pollen, nectar, and persist in stored food (honey and bee bread), and viruses can be highly prevalent in honey bee colonies. Therefore, the present study describes the influence of chronic co-exposure to thiamethoxam and Chronic bee paralysis virus (CBPV) on bee survival, virus loads, expression level of immune and detoxication genes, and pesticide metabolism Experiments were performed on honey bees collected from a winter apiary with reduced viral contaminations. No synergistic effect of co-exposure was observed on bee survival, nor on the ability of bees to metabolise the pesticide into clothianidin. However, we found that co-exposure caused an increase in CBPV loads that reached the viral levels usually found in overt infections. The effect of co-exposure on CBPV replication was associated with down-regulation of vitellogenin and dorsal-1a gene transcription. Nevertheless, the observed effects might be different to those occurring in spring or summer bees, which are more likelyco-exposed to thiamethoxam and CBPV and exhibit a different physiology.

Validation of quantitative real-time RT-PCR assays for the detection of six honeybee viruses.

Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus 1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were taken to determine the viral load ranges for which quantification uncertainty was below ± 1 log10 equivalent of genome copies per bee (hereafter reported as genome copies/bee). The accuracy range of RT-qPCR was found to be between 6.4 and 10.4 log10 genome copies/bee for ABPV, between 3.0 and 10.0 log10 genome copies/bee for BQCV, between 2.4 and 10.4 log10 genome copies/bee for DWV and between 3.4 and 10.4 log10 genome copies/bee for SBV. Outside these ranges, the results' uncertainty is higher. VDV1 RT-qPCR accuracy was outside accuracy limits for all viral loads. Using these RT-qPCR methods, we quantified viral loads in naturally-infected honeybees. The viral load distribution and clinical signs reported with the honeybee samples allowed us to define a threshold that could be used to differentiate between covert and overt infections. These methods will be useful in diagnosing the main viral infections impairing honeybee health.

The geometry of habitat fragmentation: Effects of species distribution patterns on extinction risk due to habitat conversion.

Land-use changes, which cause loss, degradation, and fragmentation of natural habitats, are important anthropogenic drivers of biodiversity change. However, there is an ongoing debate about how fragmentation per se affects biodiversity in a given amount of habitat. Here, we illustrate why it is important to distinguish two different aspects of fragmentation to resolve this debate: (a) geometric fragmentation effects, which exclusively arise from the spatial distributions of species and habitat fragments, and (b) demographic fragmentation effects due to reduced fragment sizes, and/or changes in fragment isolation, edge effects, or species interactions. While most empirical studies are primarily interested in quantifying demographic fragmentation effects, geometric effects are typically invoked as post hoc explanations of biodiversity responses to fragmentation per se. Here, we present an approach to quantify geometric fragmentation effects on species survival and extinction probabilities. We illustrate this approach using spatial simulations where we systematically varied the initial abundances and distribution patterns (i.e., random, aggregated, or regular) of species as well as habitat amount and fragmentation per se. As expected, we found no geometric fragmentation effects when species were randomly distributed. However, when species were aggregated, we found positive effects of fragmentation per se on survival probability for a large range of scenarios. For regular species distributions, we found weakly negative geometric effects. These findings are independent of the ecological mechanisms which generate nonrandom species distributions. Our study helps to reconcile seemingly contradictory results of previous fragmentation studies. Since intraspecific aggregation is a ubiquitous pattern in nature, our findings imply widespread positive geometric fragmentation effects. This expectation is supported by many studies that find positive effects of fragmentation per se on species occurrences and diversity after controlling for habitat amount. We outline how to disentangle geometric and demographic fragmentation effects, which is critical for predicting the response of biodiversity to landscape change.

The dimensionality of stability depends on disturbance type.

Ecosystems respond in various ways to disturbances. Quantifying ecological stability therefore requires inspecting multiple stability properties, such as resistance, recovery, persistence and invariability. Correlations among these properties can reduce the dimensionality of stability, simplifying the study of environmental effects on ecosystems. A key question is how the kind of disturbance affects these correlations. We here investigated the effect of three disturbance types (random, species-specific, local) applied at four intensity levels, on the dimensionality of stability at the population and community level. We used previously parameterized models that represent five natural communities, varying in species richness and the number of trophic levels. We found that disturbance type but not intensity affected the dimensionality of stability and only at the population level. The dimensionality of stability also varied greatly among species and communities. Therefore, studying stability cannot be simplified to using a single metric and multi-dimensional assessments are still to be recommended.

Life-History Traits Evolved Jointly with Climatic Niche and Disturbance Regime in the Genus Leucadendron (Proteaceae).

Organisms have evolved a diversity of life-history strategies to cope with variation in their environment. Persistence as adults and/or seeds across recruitment events allows species to dampen the effects of environmental fluctuations. The evolution of life cycles with overlapping generations should thus permit the colonization of environments with uncertain recruitment. We tested this hypothesis in Leucadendron (Proteaceae), a genus with high functional diversity native to fire-prone habitats in the South African fynbos. We analyzed the joint evolution of life-history traits (adult survival and seed-bank strategies) and ecological niches (climate and fire regime), using comparative methods and accounting for various sources of uncertainty. In the fynbos, species with canopy seed banks that are unable to survive fire as adults display nonoverlapping generations. In contrast, resprouters with an underground seed bank may be less threatened by extreme climatic events and fire intervals, given their iteroparity and long-lasting seed bank. Life cycles with nonoverlapping generations indeed jointly evolved with niches with less exposure to frost but not with those with less exposure to drought. Canopy seed banks jointly evolved with niches with more predictable fire return, compared to underground seed banks. The evolution of extraordinary functional diversity among fynbos plants thus reflects, at least in part, the diversity of both climates and fire regimes in this region.

Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study.

The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log10 CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log10 CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n=270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (SR) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log10 CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/- 1.96SR) at the diagnostic threshold (8 log10 CBPV/bee) was+/- 2.08 log10 CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log10 CBPV/bee may be considered to indicate probable cases of chronic paralysis.

RNA 1 and RNA 2 Genomic Segments of Chronic Bee Paralysis Virus Are Infectious and Induce Chronic Bee Paralysis Disease.

Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1 (3,674 nt) and RNA 2 (2,305 nt). Three minor RNAs (about 1,000 nt each) have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of in vivo inoculation of the two purified CBPV major RNAs are presented and demonstrate that RNA 1 and RNA 2 are infectious. Honeybees inoculated with 10(9) RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV.