To avoid confusion with the fruit fly NO zones, which are named by anatomical position Wolff and Rubin, , we have called these the small unit NOs , main unit NOm , and the cap NOc. To reveal the detailed cellular architecture of the bumblebee NO, we traced all input neurons as well as all columnar neurons that supply the NO in our high-resolution data 24 nm resolution.
Based on these analyses, the projections from tangential neurons delineated three domains in the bumblebee NO: the small unit NOs , main unit NOm , and the cap NOc; Figure 4f—g. These regions were supplied by distinct types of tangential cells. Finally, a set of 15—20 smaller input cells projected to the NOc region.
These cells did not have neurites projecting from the direction of the ipsilateral lateral accessory lobes LAL , but emerged from a fiber bundle that vertically passed the EB and originated in the contralateral anterior protocerebrum Figure 4d. The same bundle contained numerous tangential cells of the FB, suggesting that the tangential neurons supplying the NOc region also belong to this group of neurons. This proposed morphology was confirmed by an intracellular dye fill from Megalopta Figure 4—figure supplement 1d and we named these neurons FB-NOc cells.
Additional cells with smaller fiber diameters originated alongside the larger PFN neurons, and mostly projected exclusively to the NOc region. They thus most likely constitute the only identifiable subtype of PFN cells. In our low resolution data, we identified PFN cells total, while in the nm data we identified a minimum of PFN neurons in a single hemisphere, with many still left to be traced. Based on these numbers, we estimate the total numbers of PFN cells to be at least for the entire CX, double the number found in Drosophila Hulse et al.
At least two thirds of PFN neurons had very small fiber diameters, making them impossible to trace using the nm resolution data set. Additionally, as branches within the FB could only be traced over short distances and no layering was present in the NO, we were unable to subtype this large group of neurons further, leaving PFNc and PFN main as the only obvious subtypes.
However, clear and consistent differences in the diameter of the main PFN neurites in each columnar bundle demonstrated that the population of PFN cells is not homogeneous and many subtypes likely exist. With regard to their columnar projection pattern, PFN cells are laterally shifted by one column in the FB and are entirely absent from the innermost PB columns Figure 4c.
While in other species, the former can be further classified into two subtypes based on their opposite polarity, this distinction could not be made in our data, owing to the limited resolution of the data set. Blue neurons project from right hemisphere and red from left. Figure 6 with 1 supplement see all Download asset Open asset A fly-like head direction circuit in the bumblebee EB.
PEN cells are shifted contralaterally by one column in the EB. Akin to a biological compass, EPGs track the flies rotational movement as a single bump of activity in the EB and two bumps of activity in the PB, one in each hemisphere Seelig and Jayaraman, Tangential EB neurons feed predominantly visual information from the anterior visual pathway to EPGs in the EB, tethering the bump to features of the environment Fisher et al. While activity in the EB input cells directly generates an activity bump in the EPG network that is based on allothetic information, idiothetic information laterally shifts the bump position via the PEN activity.
This offset has the effect whereby PEN activation results in shifting the activity bump either to the left or to the right, thereby translating clockwise or counterclockwise body rotations into counterclockwise or clockwise movements of the neural activity in the EPG population Turner-Evans et al.
In the nm data set, we identified 34 PEN cells at two per PB column, with the exception of the outermost columns that contained three PEN cells and the innermost columns that were entirely devoid of PEN neurons Figure 6a—b,d ; Figure 3—figure supplement 1. Interestingly however, we found 20 PEN cells projecting from a single hemisphere in the 24 nm data set, which suggest a total of 40 PEN cells are present in the entire bee CX, nearly identical to numbers to those found in Drosophila Hulse et al.
The fan-shaped body The FB is the largest and arguably the most functionally mysterious of the CX neuropils. In our data set, the majority of FB columnar cell types were found to connect the PB with the FB and onward to tertiary regions. Hanesch et al. As with the EB, we focused our reconstructions on the columnar cells with large neurite diameters. One group of cells, the already mentioned PFN cells, innervated the posterior portion of the FB and represented the most numerous columnar neurons of the FB.
Even considering only the largest PFN cells obtained in the low resolution data individuals their number exceeded the total count of all remaining columnar FB cells combined neurons. This ratio is likely even more biased toward PFN cells when considering that the PFN cells likely comprise only one fourth of the total number of these cells as identified in our high-resolution data set.
As the extent of the fine processes within the FB was not traceable, the exact layer these cells project to could not be identified. Although fiber diameters of different sizes present in each columnar bundle of PFN cells demonstrates the existence of subtypes, these could not be reliably established. The limits imposed by the unresolved neuronal terminals apply to all neuron types of the FB and were amplified by the fact that neural processes outside the imaged tissue block were truncated.
Two types of neurons had main neurites located near the dorsal edge of the bundle connecting the PB with the FB and possessed characteristically large fiber diameters when leaving the PB Figure 7 ; Figure 8. This main neurite entered the FB dorsally giving rise to numerous thin branches.
While passing through the FB in a ventral direction this neurite dramatically, but consistently, thinned before turning toward the contralateral lateral complex Figure 7f ; Figure 8e. Due to this thinning, many cells were lost at that point. All these cells followed a projection pattern that exhibited shifted innervations of the PB. Based on these features as well as a single dye filled example from the halictid sweat bee Megalopta genalis these cells were identified as PFL neurons, the main output cells of the CX.
More detailed comparisons of the projection patterns with data from other insects Heinze et al. While the latter has a bifurcating neurite in all other species, the bifurcation was not identified in our data, very likely because of the extremely thin neurite diameter at the point where the bifurcation should be located. PFL1,3 neurons are characterized by large diameter fibers leaving the PB white arrowheads which thin substantially as they exit the FB black arrowheads.
They are ipsilaterally offset by a single column in the FB. Therefore, this schematic is simplified. PFL2 neurons were unique in other aspects as well. Although not shown in the idealized schematics Figure 8d , these cells did not typically stick within well defined columns in the PB.
The first set of cells was a second type of columnar cell intrinsic to the CX, as no fibers were found that exited the CX after branching in the FB Figure 9a—d. These cells were termed PF1 cells and existed in two copies per PB column.
Whereas fibers within the PB were not resolvable in each individual, in some columns these fibers were sufficiently clear to suggest their existence across all individuals of this set. These neurons were the only cells innervating predominantly the anterior and ventral layers of the FB with numerous processes Figure 9f. While this needs to be confirmed by higher resolution data, PF1 cells might be uniquely innervating both the FB and the EB Figure 9d,f—g.
Figure 9 Download asset Open asset PF1 cells. Note that some cells have ventral branches that extend into the EB asterisk. Finally, a set of four related cell types was found in more ventral regions of the W, X, Y, Z bundles and traversed the FB in a shared fiber tract Figure Their main neurite was comparably thin when entering the FB dorsally, but substantially thickened when giving rise to projections inside the FB, making these cells clearly distinct from PFL neurons.
As their main neurite was very thin when entering the PB, it could rarely be traced inside the PB, yielding only a few examples with verifiable branches within the PB Figure This leaves some doubt as to whether all these cell types indeed posses significant PB arborizations.
For two of the four cell types, PB branches were confirmed by intracellular dye fills Figure 10 ; Figure 10—figure supplement 1. Assuming that PB branches are a feature of all these cells, the four types were named PFx Figure Figure 10 with 1 supplement see all Download asset Open asset PFx cells. Arrowheads point to fibers entering the PB. Reconstruction is partially incomplete due to weak signal in overview confocal scan asterisk , see Figure 10—figure supplement 1d for additional high resolution image data.
Lateral schematics underneath show approximate innervation regions of PFx1,2,3,4 cells within the FB. Similarly, but without bifurcation, PFx1 neurons leave the FB on the anterior side, but turn toward the contralateral anterior brain, wrapping around the anterior surface of the medial lobe of the mushroom body Figure The target of these cells was unclear and might either be the lateral complex, or the anterior regions of the inferior protocerebrum.
An additional, unusual type of cell included neurons with large diameter fibers that occurred in two individuals in one hemisphere and four in the other. These cells resembled PFL neurons in that they possessed very large neurites near the PB, which then dramatically thinned after passing the FB and turns toward the contralateral lateral complex.
These neurons did not give rise to resolvable branches within the FB, but the existence of fibers below our resolution limit cannot be ruled out. Interestingly, this cell type shows a clear asymmetry in that one individual in column L1 projecting to the ipsilateral lateral complex does not have a counterpart on the contralateral side.
These neurons contain input fibers in one column and output fibers in a column in the opposite hemisphere Heinze and Homberg, ; Hulse et al. Historically, these cells have been termed pontine cells Hanesch et al. Here, we adhere to the new nomenclature proposed by Hulse et al.
More cell types may exist but would require full cell morphologies from high-resolution tracing data. Frontal views left and horizontal views right. This high number makes them the second most numerous cell type of the CX after the columnar PFN cells. Their numbers were surprisingly unevenly distributed across the bundles of origin. This suggested an overall branching system consisting of twelve lateral sectors across the width of the FB.
Indeed, the cells originating in the X and Y bundles could be segregated into two adjacent arborization domains each Figure The collective arborizations of the majority of these posterior cells followed the described twelve vertical column system across the FB Figure These cells shared projections within the anteriormost layer of the FB. The branching fibers which innervated the FB furthest away from the location of their cell bodies, that is, their likely output fibers, were positioned anteriorly relative to the domains of their putative input fibers.
Further, these cells had wider branches and collectively formed eight columns as opposed to the twelve formed by Types 1, 2, and 3. Interestingly, the putative input fibers of these neurons entered the FB posteriorly, but their putative outputs enter dorsally and are reminiscent in morphology of a claw crane Figure Due to the limited resolution of our data set, we were unable to trace finer branches and could not determine the FB layer in which these cells send their terminal branches.
Discussion In this first study of a hymenopteran CX based on 3D EM data, we asked what neuroarchitectural information would be attainable by tracing a relatively low resolution SBEM data set of the entire CX of a Bombus terrestris worker. To this aim we manually traced more than columnar and pontine neurons of the bumblebee CX and established neural projection patterns underlying overall information flow within this brain region. Based on these patterns, we extrapolated whether anatomically constrained computational algorithms identified in particular in the fruit fly could also exist in the bumblebee.
The present study is therefore a first step toward evaluating how conserved the neural circuits uncovered by the Drosophila connectome Scheffer et al. What information is missing from our low-resolution projectome? Additionally to the low-resolution data, we collected higher resolution data sets of the NO from a different individual.
This not only allowed us to trace the fine neurites of PFN cells, which have been proposed to integrate heading with distance information in bees and likely play a major role in path integration Stone et al. The comparison between the three data sets revealed that the lowest resolution data clearly misses all neurons with neurite diameters smaller than a certain cutoff, in our case 0.
This cutoff was not identical across all regions of the data, as especially toward the boundaries of the main image stack, resolution was much poorer compared to the center. Additionally, the inability to resolve fine details also led to the loss of all but the main branches of arborizations within neuropils.
Synapses were not clearly visible in any of the three data sets. This not only has implications for detailed maps of overlapping arborizations, but limits cell type identification. The information needed to resolve these cell types clearly lies in higher resolution data. Without higher resolution and the associated ability to reconstruct full branching trees, we are unable to discern the subtype identity of any of these cells in the bumblebee. Adding a different perspective, we also compared the skeletons of our low-resolution EM-based data to detailed neuron morphologies obtained by intracellular dye injections and confocal microscopy.
This analysis not only demonstrated that regions of cells that lie outside of the imaged volume are obviously missing from our data, but that our low-resolution EM data can be supplemented by full arborization trees obtained by light microscopical data, as long as neurons are selectively labeled. The complete morphologies obtained with this method confirmed the result of the comparison between low- and high-resolution EM data and highlighted that neither polarity indicators fine versus beaded endings , nor the full size of branches were captured in the EM-based reconstructions based on low-resolution images.
However, mapping of EM-based skeletons and neuron skeletons based on confocal data yielded surprisingly good matches, even in the occasional cases when data from M. He thought he could see Deke's Camaro, but he wasn't sure. I guess. He thought he heard a noise for a moment-a rough noise, like a roll of canvas being pulled through a narrow window-but that might have only been nerves.
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Strong competition selects for staggered flowering phenologies of co-occurring plant species [ 17 — 19 ]. As deceptive species are typically more pollinator limited than rewarding species [ 5 ], their flowering time should be most responsive to such selection. In temperate areas, flowering during early spring may allow GFD orchids to avoid the flowering peaks of most co-occurring rewarding species and thus to experience lower competition for pollinators [ 13 ].
Additionally, the proportion of inexperienced pollinators should generally be higher early during the flowering season than later. All deceitful orchids are pollinated by insects with annual life cycles, so that in each flowering season all potential pollinators have a naive period before they learn which plant species are rewarding [ 1 , 20 , 21 ].
Flowering early when most pollinators are naive should expose GFD orchids to more frequent visits, even though more individuals of pollinating species are active later. This advantage would be accentuated when late-emerging insects learn faster than early-emerging ones, because the former learn from the latter [ 22 ]. We assessed several general hypotheses concerning floral longevity and flowering time in GFD orchids, using Calypso bulbosa L. Oakes var. This GFD orchid flowers in spring and is pollinated primarily by queen Bombus and Psithyrus bumble-bees [ 23 , 24 ], before the emergence of worker bees.
Each reproductive C. To test whether flowering early for a protracted period enhances pollinator visitation and reproductive success, we manipulated flowering time early versus late and duration brief versus long in a natural population. To determine how flowering time affects pollination of deceptive orchids, we additionally assessed the relative influences of associative learning through positive experience with rewarding Arctostaphylos uva-ursi L.
Ericaceae , and avoidance learning during previous experience with C. Material and methods a Study site We used populations of C. In this area, both species are among the first to flower in spring and are pollinated by the same bee species. Arctostaphylos uva-ursi occurs along the edges of mossy, coniferous forests occupied by C.
During , the C. Flowers opened continuously between mid and late May, and average flower lifespan was Nearby, A. Bumble-bee queens were first observed in mid-May, roughly coincident with the beginning of C. We selected 19 patches containing at least four reproductive ramets. Before anthesis, we enclosed each flower in a mesh bag to prevent pollinator visits.
Within each patch, we randomly selected four flowering individuals i. This design ensured that two flowering ramets in each patch were exposed simultaneously to pollinators during the 9 days, hence avoiding potential confounding effects of flower abundance, while allowing availability of fresh flowers within each patch during the experimental period.
We measured plant height and labellum length and width for each plant during the day prior to its exposure to pollinators and recorded flower age days since anthesis on the exposure day. In particular, flowers exposed early averaged 2. Starting on 20 May day 0 , plants were unbagged between Because tree cover can affect pollen removal and the risk of frost damage in C.
We estimated bumble-bee abundance by counting the bees that foraged at the study site during 3 h each day. Average daily bumble-bee abundance did not vary among the four treatment combinations permutation tests on mean squares; treatment: m.
At the end of each exposure period, we bagged each flower between On 6 June, a snow storm damaged most flowering stems in our study population, precluding measurement of fruit and seed set. Nevertheless, the aggregation of pollen into solid pollinia in many orchids, including C.
In particular, a previous study of the population we examined found that We used a generalized linear mixed model to assess the effects on pollinarium removal of flowering time, flowering duration crossed, fixed factors , plant height, labellum length, labellum width, percentage of tree cover and flower age fixed covariates , and patch random factor.
This analysis employed the glmmPQL function in R v. Each bumble-bee was exposed individually to one or two of the following treatments: — Naive. Naive bees exposed to 10 C. To follow the trends of the market we provide a set of free forex trend indicators. The free forex trend indicators can be installed on any broker charting system, including Meta trader.
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