In contrast, the floral odors that attract moth pollinators have been more extensively researched. In this study we determined that CMV infection induced changes in olfactory cues emitted by Arabidopsis thaliana and tomato plants in ways that could be perceived by the bumblebee Bombus terrestris, and confirmed in tomato that this was associated with quantitative and qualitative changes in the blend of plant-emitted volatile organic compounds . We also elucidated a role for the host microRNA pathway in regulating the emission of bee-perceivable olfactory cues. Our data indicated that bumblebees possess an innate preference for olfactory signals emitted by CMV-infected tomato plants and we mathematically modeled what the possible wider implications of this might be if a similar preference occurred in wild host plants under natural conditions.In ‘free-choice’ assays, bumblebees encountered flight arenas containing ten tomato plants concealed within towers designed to allow odors to diffuse out but prevent the bees from seeing or touching the plants . Cups that were placed on top of towers hiding plants of both treatment groups offered bumblebees the identical ‘incentive’ of a 30% sucrose solution. Nonetheless, plastic planter pot when presented with mock-inoculated and CMV-infected tomato plants, bumblebees preferred to visit the towers that were hiding infected plants .
Bumblebees showed similar preferences for flowering and non-flowering CMV-infected plants, indicating that leaves were the main source of attractive volatiles . Bumblebees also displayed a preference for CMV-infected tomato plants over plants infected with CMVΔ2b, a viral mutant lacking the gene for the 2b VSR , a factor that also influences CMV-plant-aphid interactions.The results obtained in free-choice assays with tomato plants infected with CMVΔ2b suggested that the 2b protein, which is a VSR, may be exerting effects on the metabolism of plant volatiles by interfering with host small RNA pathways. The model plant Arabidopsis is the best higher plant system to use to investigate the effects of small RNA pathways. However, whilst Arabidopsis plants emit potentially pollinator-influencing volatiles, this species is not bee-pollinated. Consistent with this, bumblebees showed no significant difference in preference for volatiles emitted by CMV-infected versus mock-inoculated Arabidopsis plants in free-choice assays . An alternative approach to investigate the ability of bees to recognise differences in olfactory or other stimuli is to set up a differential conditioning or ‘learning curve’ assay. A differential conditioning assay can reveal whether bees can perceive cues that would not normally induce any behavioural responses and that could not be studied in free-choice assays.
In our differential conditioning assays, cups on towers offered bumblebees either a 30% sucrose solution ‘reward’ for choosing one treatment group or a ‘punishment’ for choosing the other group. Bumblebees cannot distinguish quinine from sucrose except by taste. Thus, increasing frequency of visits to sucrose-offering towers over the course of an experiment indicated that bees have learned to use plant odor as a cue to identify and avoid drinking from cups placed on towers offering quinine solutions. In these assays, a steep learning curve shows that bumblebees can easily distinguish between two treatment groups, and indicates that the volatile blends are likely to be qualitatively and/or quantitatively very distinct, whereas less steep curves indicate that differences between blends are less marked, and that bees find it more difficult to learn to distinguish between them based on odor. An illustration of the power of this approach is shown in Fig 2 . Although bumblebees displayed an innate preference for volatiles emitted by CMV-infected tomato plants in free choice assays , they could be trained by differential conditioning to overcome their innate preference and instead preferentially visit mock-inoculated tomato plants and avoid CMV-infected plants . Although we had observed that bumblebees had no innate preference for, or aversion to, volatiles emitted by Arabidopsis plants , differential conditioning assays revealed that the insects could recognize differences between volatiles emitted by Arabidopsis plants that had been mock-inoculated and by plants that were infected with CMV . Bumblebees could also distinguish between CMV-infected and CMVΔ2b-infected Arabidopsis plants .
Hence, although they exhibit no innate behavioural response to the volatile blends emitted by Arabidopsis plants, differential conditioning assays showed that bumblebees could perceive differences in volatiles emitted by these plants. This meant that differential conditioning assays could permit further dissection of the mechanisms underlying CMV-induced changes in volatile emission using Arabidopsis as a model system. Bumblebees could learn to differentiate transgenic plants constitutively expressing the 2b VSR from non-transgenic plants and from control-transgenic plants that were expressing an untranslatable 2b transcript . However, the insects displayed less ability to learn to distinguish mock-inoculated from CMVΔ2b-infected plants . Comparison of the learning curves in Fig 3A versus Fig 3E by logistic regression indicated that bumblebees were better at distinguishing mock-inoculated plants from CMV-infected plants than from CMVΔ2b-infected plants = 40.17, p < 0.0001. Bees could not be trained to differentiate non-transgenic plants from control-transgenic plants expressing a non-translatable 2b transcript . The results with CMVΔ2b suggested that the 2b VSR plays an important role in altering the emission of bee-perceivable olfactory cues emitted by tomato and Arabidopsis plants . However, CMVΔ2b accumulates to lower levels in plants than wild-type CMV and in previous work it was found that viral titer, as well as the presence of the 2b protein, plays a role in modification of the interactions of Arabidopsis with aphids. Hence, it was conceivable that differences in virus titer might affect the emission of bee-perceivable volatiles by plants infected by CMV or CMVΔ2b and explain why the bees found it difficult to distinguish CMVΔ2b-infected plants from mock-inoculated plants. However, it is known that CMVΔ2b accumulates to levels comparable to those of wild type CMV in Arabidopsis plants carrying mutations in the genes encoding the Dicer-like endoribonucleases DCL2 and DCL4, which are important factors in antiviral silencing. Therefore, we examined the ability of bumblebees to learn to distinguish between volatile blends emitted by CMVΔ2b-infected and mock-inoculated dcl2/4 double mutant plants . The resulting learning curve was not significantly different from that obtained using wild-type plants that had been mock inoculated or infected with CMVΔ2b = 0.66, p = 0.42, indicating that an increase in CMVΔ2b titer did not enhance bee learning. Although we cannot rule out a role for other CMV gene products, the results indicate that the 2b VSR is the most significant viral factor conditioning changes in the emission of bee-perceivable volatiles.One of the host molecules that interact with the 2b VSR is the Argonaute 1 ‘slicer’ protein. AGO1 is required for silencing directed both by short-interfering RNAs and by miRNAs, which are generated by a specific host endoribonuclease from miRNA precursor transcripts encoded by nuclear genes. In differential conditioning assays, bumblebees were able to learn to distinguish between volatiles emitted by wild-type plants versus those emitted by ago1 mutant plants and those emitted by dcl1 mutant Arabidopsis plants . However, bumblebees showed little or no ability to learn to distinguish between volatile blends emitted by ago1 and dcl1 mutant plants, 30 litre plant pots indicating that the volatile blends emitted by plants of these two mutant lines were very similar . Thus, the miRNA-directed silencing pathway regulates the emission of bee-perceivable volatile compounds. Double mutant dcl2/4 plants are unable to generate CMV-derived short-interfering RNAs but are not affected in miRNA biogenesis. In CMV-infected dcl2/4 plants a higher proportion of the 2b protein is available to bind AGO1 and inhibit its miRNA-directed activity, which is likely to enhance virus-induced changes in emission of bee-perceivable volatiles. In line with this, bumblebees were able to learn to distinguish between volatiles emitted by CMV-infected wild-type and dcl2/4 double mutant Arabidopsis plants . As an additional control we showed that bumblebees could not learn to distinguish between volatiles emitted by mock-inoculated plants covered by towers offering sucrose rewards or quinine punishments .
The responses of bumblebees to CMV-infected tomato plants that were hidden from the insects indicated that changes in the emission of volatiles were affecting bee behavior and were responsible for the innate preference of these insects for CMV-infected plants . To confirm that CMV infection caused changes in the emission of VOCs, tomato plant headspace volatiles were collected and analysed by gas chromatography coupled to mass spectrometry . VOCs were collected from non-flowering mock-inoculated plants, plants infected with CMV-Fny and plants infected with the 2b gene deletion mutant of CMV-Fny, CMVΔ2b. The emitted VOCs were distinct from each other when compared by principal component analysis on the relative intensity of ions within the samples . PC1 explained 80.3% of the variation and discriminated between samples from mock-inoculated and CMV-infected plants, whereas PC2 discriminated between samples from mockinoculated and CMVΔ2b-infected plants . Thus, the VOC blend emitted by CMVinfected tomato plants was more distinct from that released by mock-inoculated plants than it was from the volatiles emitted by CMVΔ2b-infected plants. Nevertheless, VOC emission byCMVΔ2b-infected tomato plants was distinct from either mock-inoculated plants or CMV infected plant VOC emission , despite this mutant virus accumulating to markedly lower levels than CMV . Although CMV-infected plants were smaller than either mock-inoculated or CMVΔ2binfected plants, the emission of the combined volatiles on a whole plant basis was similar between mock-inoculated and CMV-infected plants . Indeed, expressing the emission of the combined VOCs by mass of tissue revealed that CMV-infected plants released greater quantities of volatiles compared to mock-inoculated and CMVΔ2b-infected plants . Thus, despite being stunted, CMV-infected plants generated a greater total quantity of VOC than either mock-inoculated or CMVΔ2b-infected tomato plants. Identification by GC-MS of the most abundant VOC by g dry weight of tomato plant tissue showed that terpenoids dominated the profile, with α-pinene, 2-carene, p-cymene, β-phellandrene and the sesquiterpene -caryophyllene being apparent . CMV infection caused quantitative changes in the profile of these VOCs; α-pinene and p-cymene emission increased markedly, whereas 2-carene and β-phellandrene did not, and -caryophyllene almost disappeared from the profile . Isomeric composition was not further determined than that stated here. When VOC emission was compared on a whole plant basis, α- pinene and p-cymene emission rates from CMV-infected plants appeared similar to mockinoculated or CMVΔ2b-infected plants, while 2-carene and β-phellandrene levels appeared to be lower . Bumblebees of a closely related species are known to be repelled by β-phellandrene and 2-carene. Thus, lower emission values of these VOCs from CMV-infected plants may explain why bumblebees displayed an innate preference for CMV-infected tomato plants over mock-inoculated plants in free choice assays . The VOC profiles of mock-inoculated and CMVΔ2b-infected plants were similar, although not identical , and this could explain the bees’ lack of preference in free choice assays . However, optimal self-fertilization requires sonication of the flower to release pollen from the anthers onto the stigma, which is provided either by buzz-pollination from a bee or simulated buzzpollination using mechanical vibration. This is illustrated in Fig 5A, which shows how mechanical buzz-pollination of flowers increased seed production by around a third. Seed production by tomato was very dramatically decreased in plants infected with CMV-Fny to less than 10% of the yield in mock-inoculated plants . Remarkably, artificial buzz-pollination of flowers of CMV-infected plants rescued seed production to a significant degree with seed numbers reaching approximately half the level seen for non-buzzed flowers of mock inoculated plants and about 6- to 7-fold greater than the number of seeds produced in nonbuzzed, CMV-infected plants. The difference in seed yield between mock-inoculated and CMV-infected plants that had been vibrated was less marked than between non-buzzed, mock inoculated and CMV-infected plants . Although CMV-infected plants produced fewer seeds, the mass of individual seeds was unaffected by infection and was not affected whether or not flowers were vibrated . Additionally, the number of flowers produced by CMV-infected plants was similar to the number produced by mock-inoculated plants, and tomato flower morphology was also not markedly altered by infection . Overall plant growth was stunted by CMV infection but, interestingly, virus infection appeared to accelerate the appearance of flowers by a small but statistically significant degree . A recent report indicated that flowers of squash plants infected with the potyvirus zucchini yellow mosaic virus yielded decreased quantities of pollen. However, we found no significant differences in the quantity or viability of pollen released from mock-inoculated and CMV-infected tomato flowers .We investigated the effects of CMV infection on bumblebee-mediated pollination under glasshouse conditions in which the insects could see and interact with flowers .