Pollen Diet and Sterol Provisioning Differentially Affected Larval Development in a Generalist Solitary Bee

Pollen Diet and Sterol Provisioning Differentially Affected Larval Development in a Generalist Solitary Bee | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Pollen Diet and Sterol Provisioning Differentially Affected Larval Development in a Generalist Solitary Bee Carlos Martel , Josef Kreidt , Samuel Furse , Jennifer Scott , Janine Griffiths-Lee , Geraldine A. Wright , Philip C. Stevenson doi: https://doi.org/10.1101/2025.04.21.649687 Carlos Martel 1 Royal Botanic Gardens , Kew, Richmond, Surrey, TW9 3AE, United Kingdom 2 Instituto de Ciencias Ómicas y Biotecnología Aplicada, Pontificia Universidad Católica del Perú , San Miguel 15088, Lima, Peru Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: c.martel{at}kew.org Josef Kreidt 1 Royal Botanic Gardens , Kew, Richmond, Surrey, TW9 3AE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Samuel Furse 1 Royal Botanic Gardens , Kew, Richmond, Surrey, TW9 3AE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Scott 3 Department of Biology, University of Oxford, Oxford , OX1 3SZ, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Janine Griffiths-Lee 1 Royal Botanic Gardens , Kew, Richmond, Surrey, TW9 3AE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Geraldine A. Wright 3 Department of Biology, University of Oxford, Oxford , OX1 3SZ, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Philip C. Stevenson 1 Royal Botanic Gardens , Kew, Richmond, Surrey, TW9 3AE, United Kingdom 4 Natural Resources Institute, University of Greenwich , Chatham, Kent, ME4 4TB, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Pollen sterols are essential micronutrients for bees as membrane components, hormone precursors and gene regulation. Sterols vary among plant species; therefore, bee development may be influenced by or adapted to specific pollen diets. To better understand the roles of pollen diet and sterol provisioning, we investigated the effect of different pollen types with sterol supplementation (i.e., pollen provided by the bee mother, Castanea sativa pollen, C. sativa pollen supplemented with sterols, polyfloral pollen, and a combination of polyfloral and C. sativa pollen) on the development, and sterolome of the generalist, solitary bee Osmia bicornis . Pollen diet significantly affected bee development (i.e., final weight, feeding period, growth rate) and larvae fed on pollen supplemented with sterols had an increased growth rate compared to those from the other treatments. Despite sterolomes being distinctive among pollen diets, sterolomes were more similar within some bees than between bees and their pollen diets. Moreover, larvae fed on polyfloral pollen had much higher relative concentrations of campesterol (∼10%) than the pollen itself (∼0.2%), indicating Osmia bees are highly efficient in nutrient accumulation or can metabolically produce campesterol. Our results contrast with previous work which suggests bees cannot modify sterols and highlight the complexity of bee nutrition. 1. Introduction Around 90% of angiosperms rely on animal pollination for reproduction [ 1 ] with bees recognised as the most important pollinators [ 2 ]. While social bees, including honey bees ( Apis mellifera ) and bumble bees ( Bombus spp.), are well-established species globally for providing pollination services [ 3 , 4 ], they only represent a small proportion (5%) of bee diversity which is comprised mainly of solitary species. Solitary bees are somewhat overlooked pollinators but are highly efficient [ 2 ]. Both social and solitary bees feed exclusively on nectar and pollen throughout their development [ 5 ], with pollen being the primary food source of protein and lipids along with vitamins and minerals, [ 6 - 9 ]. Whereas social bees are polylectic (pollen generalists), solitary bee species are oligolectic (pollen specialists) or polylactic (pollen generalists). Sterols are a group of amphipathic lipids characterised by a triterpenoid skeleton with a hydroxy substitution and a hydrocarbon tail [ 10 , 11 ]. Sterols exhibit significant diversity, with over 250 documented structures. While sterols are present across all eukaryotic organisms, the sterol profile differs between taxa [ 12 ]. Sterols are cell membrane components and hormone precursors and also regulate gene expression and modulate responses to environmental conditions [ 13 , 14 ]. In pollen, β-sitosterol, isofucosterol, cycloartenol, 24-methylenecholesterol and campesterol are among the most abundant sterols [ 15 , 16 ]. Sterols are essential nutrients for insects since they are required for development but insects lack the biochemical scaffolding to synthesise sterols de novo and therefore rely on dietary sources to meet their metabolic needs [ 17 , 18 ]. The reliance of bees on sterols is crucial not only to critical physiological processes like pupation, moulting or the development of ovaries but also to the expression of reproductive behaviours such as caste determination [ 19 , 20 ]. For example, honey bees rely on 24-methylenecholesterol to survive and develop during the larval phase [ 18 ]; therefore, honey bee workers must gather pollen from plant species that produce 24-methylenecholesterol to nurture a healthy brood [ 21 , 22 ]. In B. terrestris , high concentrations of either 24-methylenecholesterol, β-sitosterol or isofucosterol in the diet resulted in bigger larvae [ 19 ], while 24-methylenecholesterol, campesterol and cholesterol have been suggested to be essential for both A. mellifera and B. terrestris [ 22 ]. However, some sterols may not be usable and therefore might be unsuitable for the development of some bees [ 18 , 20 ]. While social bee nutrition is fairly well established [ 21 - 23 ], the effects of sterol availability for developmental stages of solitary bees have been overlooked. Thus, the consequences for bee development and challenges to metabolic health resulting from a limited range of sterols in pollen remain unknown [ 22 ]. A floral landscape lacking the sterols required by a particular bee species may result in nutritional deficiencies [ 15 ], which might in turn contribute to the decline in bee diversity, especially of those species with specific dietary requirements. Understanding how sterols impact the development and health of solitary bees is therefore crucial. Although previous studies have suggested that bees are unable to produce or modify sterols, none of those studies manipulated the sterol provision only. Consequently, in this study we aimed to shed light on the effect of pollen diet variation and sterol supplementation on the development of solitary bees using the red mason bee, Osmia bicornis , as a model system. Bee larvae were experimentally fed pollen with distinctive sterol profiles and sterol supplementation to determine their effect on bee feeding period, weight, and growth rate. Furthermore, we elucidated the effects of a species-rich pollen diet on the bees’ development compared to pollen from a single species. The sterol profiles of the bees were also chemically analysed to elucidate how O. bicornis bees are using the sterol provisioned in the pollen. The primary result of this study is that O. bicornis larvae were affected by sterol availability, but they can shape their sterol profile by selective accumulation and transformation of sterols. 2. Materials and Methods (a) Study organism Osmia bicornis is a solitary, polylectic, cavity-nesting bee. The species ranges throughout the Western Palearctic region, extending from Iberia in the west, through Europe and northern Africa, to Iran in the east, and is the commonest Osmia species in the United Kingdom [ 24 ]. Osmia bicornis is renowned for its exceptional pollination abilities, and it has been estimated that 500 O. bicornis females have a comparable pollination efficiency to 2–4 robust honey bee colonies [ 25 ]. Although the species is considered polylectic, O. bicornis prefers pollen from Quercus and Ranunculus [ 26 ]. Nevertheless, knowledge about how different pollen diets affect larval development in O. bicornis has yet to be established. The sterolome of O. bicornis bee is comprised of several sterols, and contrary to honey bees and bumble bees the sterol profile is dominated by campesterol instead of 24-methylenecholesterol [Baker et al. unpublished data]. (b) Recruitment of Osmia bicornis larvae Osmia bicornis is an abundant species at the Royal Botanic Gardens Kew (RBG Kew) where we provided wooden nest boxes located in different areas of the gardens for O. bicornis females to nest (The Red Beehive Company, Bishops Waltham, UK). In preparation for the feeding experiment, 100 O. bicornis cocoons harvested in August 2023 were placed in the wooden nest boxes in April 2024 to allow them to emerge under natural conditions and near to the nest boxes. Emerged, mated females use the cavities of the boxes to build bee cells and lay eggs, which were provisioned with pollen by the mother bees. The nest boxes were checked daily to identify and remove newly deposited eggs and to collect the pollen from the bee cells. Eggs were carefully removed and placed in glass vials and transported to the lab for the feeding experiment. The pollen from each individual bee cell was collected for the experiment using a mini portable handheld vacuum cleaner, mixed and stored in glass vials. To assess the mass of pollen provisioned by the Osmia mother, the pollen from five bee cells was weighted using an analytical balance, resulting in a mass of ∼450 mg of pollen per bee cell. (c) Feeding experiments with different types of pollen diet We used polypropylene well microtube racks (for 1.5–2 ml microtubes, well i.d. 12 mm, 22.5 × 6.7 × 2.8 cm) to rear the bees. In each well, an O. bicornis egg was placed alongside one of the five dietary treatments containing 450 mg of pollen. Pollen treatments included: (i) Osmia female provisioned pollen [ Osmia treatment] (n = 15), (ii) Castanea sativa pollen (Apicoltura Metalori Aldo, Italy) [chestnut treatment] (n = 15), (iii) C. sativa pollen supplemented with sterols (PureBulk, Roseburg, Oregon) [chesnut+sterol treatment] (n = 14), (iv) polyfloral pollen (Agralan Ltd, Wiltshire, GB; an Asteraceae dominated-pollen mixture and containing only minute amounts of campesterol) [polyfloral treatment] (n = 14), and (v) a 50:50 mixture of C. sativa and polyfloral pollen [polyfloral+chestnut treatment] (n = 15). Pollen selection was based on its sterolome and that of the O. bicornis bees (see results for details of the sterols profiles of pollen and bees). Furthermore, to test the effects of changes in the sterol profile and abundance only, we added commercial soy-extracted sterols (a combination of β-sitosterol, campesterol, stigmasterol, etc.; total sterols ≥95%) to the pollen in a mass proportion of 0.01%, since this is the average sterol concentration in pollen; thus, this treatment contains around twice the amount of total natural sterol in pollen. Since the Osmia treatment contains water and nectar added by the mother bees, we added sugar syrup (i.e., 1 M solution of sucrose/glucose/fructose) to each pollen treatment to get a consistency similar to that of the control (sugar syrup supplement represented 23.6%, 23.6%, 18.6%, and 15.09% of the pollen mass of the chestnut, chesnut+sterol, polyfloral and polyfloral+chestnut treatment, respectively). Rearing racks containing the bees and treatment were placed in a dark room and reared at an average temperature of 22°C. The hatching date, the weight on the first day and the date on which cocoon spinning began were recorded each morning. After hatching, the bees were weighed every fifth day until larvae started spinning their cocoon; the final measurement was taken when pupation was completed. Pupae continued their development within the cocoon for six weeks and then were frozen at -80°C for later sex identification and chemical analysis. Sex determination could only be performed on fully developed bees; however, larvae that had not reached this stage were sexed based on their weight. The feeding period was calculated as the number of days between hatching and the onset of cocoon spinning. (d) Chemical analyses of sterols in pollen and bees Pollen samples (10 mg per treatment) were transferred to Eppendorf Safe-Lock micro test tubes and then frozen at -20°C. Bees were transferred to glass vials filled with deionised water and frozen at - 20°C in glass vials. Pollen and bee samples were then transferred to a freeze-dryer (MechaTech Systems, LyoDry Midi) for 72 hours. 1.0 ml of GCTU (Guanidine 6 M guanidinium chloride and thiourea 1·5 M) was added to both pollen and bees before homogenisation was done for bee samples using a hand-held Tissue Tearor (BioSpec Products, Inc.). Samples were then transferred to the freezer (-20°C). Solutions of homogenised samples (50 μL) were combined with internal standards (150 μL, d7-cholesterol [Avanti Polar Lipids, Alabama, USA], in methanol at 0.1 mg/mL), DMT (500 μL, dichloromethane: methanol: triethylammonium chloride 3:1:0.002 v/v/w), and water (500 μL, ddH 2 O) using a pipette (Eppendorf Research Plus pipette 1000 μL fixed volume) before they were agitated and centrifuged (500 g, 2 min) using an EZ2 evaporator (Genevac EZ-2, GeneVac Ltd, Ipswich, UK). 50 μL of the solution were transferred to micro test tubes and left to dry. The dried c Mass spectra were obtained using a Thermo Scientific Orbitrap Fusion mass spectrometer, which was equipped with an Atmospheric Pressure Chemical Ionization (APCI) probe and integrated with a Thermo Scientific Vanquish liquid chromatography system (Thermo Scientific, UK). Chromatographic separations were performed on a Thermo Scientific Hypersil GOLD LCMS C18 column, measuring 50 × 2.1 mm and featuring a particle size of 1.9 μm (Thermo Scientific, UK), maintained at a temperature of 55 °C for all analyses. A 1 μL injection volume was used, with a mobile phase flow rate set at 0.60 mL/min. The eluents consisted of A (methanol, LCMS grade) and B (double-distilled, deionised water). The sterol chromatography protocol involved a gradient of 16% B from 0 to 11 minutes, followed by a shift to 0% B from 11.1 to 16.4 minutes, and then returned to 16% B from 16.5 to 19 minutes. The overall duration of each sample run, including transitions and washing steps, was 20 minutes. Mass spectrometric data were acquired in positive ionisation mode via APCI, operating at a resolution of 120,000. The spray voltage was set to 2.86 kV, with sheath nitrogen gas flows of 45 (sheath), 5 (auxiliary), and 1 (sweep) arbitrary units. The ion transfer tube and vaporizer temperatures were maintained at 300 °C and 350 °C, respectively. The mass acquisition range was calibrated to m/z 197– 500, with the lower limit adjusted to detect the fluoranthene cation ( m/z 202.077), which served as the internal mass calibration reference. Under these conditions, the internal standard used for the samples, d7-cholesterol, eluted at 5.6 minutes, with the primary ion species identified as [M - H 2 O + H] + at m/z 376.395. This measurement was accomplished within a mass variance of less than 1 ppm. Both samples and external standards were analysed in a single sequence. The bee and pollen samples were randomised among themselves, while the external standards were run sequentially, beginning with the least concentrated standard. The data file (*.raw), was processed using Analyzer PRO XD (SpectralWorks Ltd, Cheshire, UK). Signals were extracted over a retention time of 3 to 11 minutes within a mass range of 360–442 m/z . An area threshold of 500,000 was applied, along with smoothing set to 15 arbitrary units, a width of 0.01 minutes, and mass accuracy of 3 decimal places. Mass spectrum signals with an average size that was three times greater than that of the signal’s average in the blanks, and with retention times between 3 and 11 minutes, were identified as sterols. The signal intensity of sterols in all samples was adjusted by dividing the signal area of each metabolite by the total area of all sterol signals for that sample, expressed in percentage (%). The signals’ intensity of the internal standard was used to calculate the absolute concentration of specific sterol in pollen and bee samples. (e) Statistical analyses Prior to analysing data on the larvae development (total number of days, total weight, and weight increase), they were normalised using a fourth root transformation. The feeding period (days), final total weight (mg), and growth rate (mg/5-day) were fitted to a generalised linear mixed model (GLMM) with Gaussian distribution. Pollen diet treatment was assigned as the fixed effect, and the bee sex was the random effect. Models were then subsequently subject to post-hoc tests. All these analyses were performed using the “car” [ 27 ], “emmeans” [ 28 ], and “lme4” [ 29 ] packages of the software R version 4.4.1 [ 30 ]. To identify differences in sterol profiles among pollen and bee treatments, we evaluated the relative proportions of all detected compounds for each sample to determine semi-quantitative (dis)similarities by calculating the Bray-Curtis similarity index. A non-metric multidimensional scaling (NMDS) was performed to display the sterol (dis)similarities among samples graphically. To test for statistical differences in the sterol profiles among pollen and bees from different treatments, a permutational multivariate analysis of variance (PERMANOVA) with 10,000 permutations was performed, followed by pair-wise comparisons among bee and pollen treatments. A similarity percentage (SIMPER) was also used to identify which sterols explained most of the differences between treatments. These calculations were performed using the “vegan” [ 31 ] and “pairwiseAdonis” [ 32 ] packages of R, and “ggplot2” [ 33 ] was used to produce the figures. 3. Results (a) Pollen diet on bee development Diet types affect bee feeding period (GLMM, χ 2 = 35.95, df = 4, p < 0.0001; Figure 1 ). The polyfloral treatment resulted in the longest feeding period and was significantly extended compared to all the other treatments (i.e., Osmia [ p < 0.0001], chestnut+sterol [ p 0.05). Download figure Open in new tab Figure 1. Duration of feeding periods (in days) by pollen diet treatment. Red points indicate the observations, blue circles represent the estimated mean, and blue bars represent the standard error. Different letters indicate statistical differences. Pollen diets affect the final weight of the O. bicornis larvae (GLMM, χ 2 = 31.75, df = 4, p < 0.0001; Figure 2 ). While the weight increase of bees from the Osmia treatment was significantly lower compared to the chestnut ( p = 0.025) and chestnut+sterol ( p = 0.018) treatments, it was not significantly different when compared with the other treatments (all p > 0.05). The weight increase was lowest in the polyfloral treatment, although only significantly lesser than the chestnut ( p = 0.001) and chestnut+sterol ( p = 0.001) treatments. Bees of the polyfloral+chestnut treatment also had a lower final weight than those of the chestnut+sterol ( p = 0.007) and chestnut ( p = 0.011) treatments. Download figure Open in new tab Figure 2. Final total weight of the bees (in mg) by pollen diet treatment. Red points indicate the observations, blue dots are the estimated mean, and blue bars represent the standard error. Different letters indicate statistical differences. Download figure Open in new tab Figure 3. Weight (in mg) in relation to the age of development (in days) by pollen diet treatment. Colours indicate the pollen diet treatments, circles represent the mean, and bars represent the standard error. The growth rate of bees from the chestnut+sterol treatment was significantly higher than the weight increase of the Osmia ( p < 0.01), polyfloral ( p < 0.0001), and polyfloral+chestnut ( p < 0.001) treatments ( Figure 2 ). However, no significant differences were detected between the chestnut+sterol treatment compared to the chestnut treatment ( p = 0.161). Similarly, no differences were detected when comparing the Osmia treatment with the polyfloral ( p = 0.830) and polyfloral+chestnut ( p = 0.998) treatments. (b) Sterol profile patterns of pollen and bees Seventeen different sterols of which fifteen were unequivocally identified were detected in the five treatment groups of bees and pollen samples ( Figure 4 , Table 1). Bees from the Osmia , chestnut and chestnut+sterol treatments had a similar sterol profile ( Figure 4 ), which was dominated by campesterol; but was different to the sterol profile of the pollen diet which was dominated by other sterols (e.g., isofucosterol in the Osmia treatment, β-sitosterol in the chestnut and chestnut+sterol) treatments. Interestingly, the sterol profiles of bees from the polyfloral and polyfloral+chestnut treatments were dominated by β-sitosterol and isofucosterol, despite the pollen diets used in these treatments were largely dominated by 24-methylenecholesterol. Bees from the polyfloral treatment contain 10% campesterol in their sterol profile, highly different to the 0.2% in their pollen diet. Download figure Open in new tab Figure 4. Sterol profiles of bees and pollen diets. Colours indicate a specific sterol and its height the relative proportion in the sterol profile. Bee symbol represents the bee samples and the pollen grain represents the pollen diet. High variability in the sterol profiles among all treatments was shown after a multivariate analysis and was also observable in the NMDS plot ( Figure 5 ). When testing for the effects of the pollen diets on the sterol composition, we detected significant effects (PERMANOVA: pseudo-F = 3.1908, R 2 = 0.39, p < 0.001), indicating that the observed sterol profiles were partially shaped by the pollen diet. In pairwise comparisons, we detected that sterol profiles of bees from the Osmia , chestnut and chestnut+sterol treatments were indistinct from each other ( p > 0.05), but they were significantly different from those of bees from the polyfloral and polyfloral+chestnut treatments ( p < 0.05). According to the SIMPER, the greatest dissimilarities in sterol profile were detected between bees from the chestnut+sterol and polyfloral treatments; campesterol accounted for 18.7% of the dissimilarity, followed by stigmasterol (6.6%), both being relatively more abundant in bees from the chestnut+sterol treatment. Sterol profile dissimilarities of bees from the polyfloral treatment and from chestnut and chestnut+sterol treatments were accounted for primarily by campesterol. This is also the case for bees from chestnut+sterol and polyfloral+chestnut treatments. Download figure Open in new tab Figure 5. Non-metric multidimensional scaling (NMDS) plot depicting the sterol composition of samples based on Bray-Curtis distances (stress = 0.17). Ellipses represent 95% confidence intervals around treatments. Circles represent the bee samples and triangles the pollen samples. 4. Discussion In this study, we elucidated the effects of different pollen diets on the development of O. bicornis and the resulting differences in the bees’ sterol profiles. We demonstrated that the type of pollen and its sterol profiles affect the feeding period, total weight, and growth rate of O. bicornis larvae. Our results also confirm that O. bicornis can feed on a variety of pollen types and still successfully develop and pupate across all tested diets. The feeding period was longest for bees fed with polyfloral pollen, a result that was mirrored in the slower growth rate and reduced total weight increase in this treatment compared to the four other treatments. Although one might expect that a more diverse pollen diet leads to a faster rate of development, we found that bees fed with polyfloral pollen experienced a significantly longer feeding period and with lower growth rates compared to bees of other treatments. Given that most of the pollen grains in the polyfloral treatment were identified as Asteraceae [Kreidt unpublished data], physical and chemical defences associated with Asteraceae pollen could have contributed to the delayed development observed in O. bicornis larvae fed with polyfloral pollen as previously shown on Bombus impatiens [ 34 ]. Asteraceae pollen is characterised by their spinous exine, which can reduce feeding capabilities in generalist bees [ 35 ]. Asteraceae pollen is also known for its high diversity of secondary metabolites which may delay bee development as reported for toxic compounds affecting Osmia cornifrons larvae [ 36 ]. Thus, our results indicate that pollen diversity is not a prerequisite for optimised development in a generalist bee but is more dependent on the physical and lipid composition of the pollen. This aligns with a recent study showing that honeybees fed with polyfloral pollen consumed more pollen and had a higher survival rate but weighed less than those fed with low-diversity pollen [ 37 ], suggesting that developmental performance may not necessarily reflect overall bee health. The sterol profiles of bees varied significantly, with the polyfloral treatment displaying the greatest dissimilarities to other treatments, particularly to the Osmia , chestnut and chestnut+sterol treatments. Campesterol, a sterol suggested to be essential for bee development and involved in steroid hormone synthesis [ 22 ], accounted for a notable portion of the dissimilarity among treatments, particularly between the chestnut and polyfloral treatments. Its low concentration in the polyfloral treatment suggests campesterol significantly impacts Osmia larvae performance but also Osmia bees may be able to modify sterols. This also contrasts with other reports on social bees in which variations in campesterol concentrations did not affect colony performance [ 6 ]. Interestingly, 24-methylenecholesterol, a major and essential sterol in honey bees [ 23 , 38 ], did not dominate in our pollen and bee samples, indicating that O. bicornis may have different sterol requirements to honey bees and development is not significantly influenced by or dependent on this sterol. Although campesterol appears to play a role, other sterols also contribute to development. While sterols are essential for bee development [ 22 ], macronutrients, including proteins, other lipids, and carbohydrates, also significantly impact growth and overall health [ 39 , 40 ]. In our experiment, the variation in pollen types may have also provided differences in these other nutrients, and also secondary metabolites, that could explain some of the observed differences in bee development as previously reported for other solitary bees [ 41 ]. The balance of nutrients, particularly the protein-to-lipid ratio, is known to influence bee larval development [ 42 ]. Therefore, bees selectively forage for pollen containing the ideal protein-to-lipid ratio, which differs from one species to another. All this indicates the nutritional requirements differ between bees and between life stages. Future research should aim to isolate the effects of different types of sterols and nutrient types to better understand their roles in bee development. Overall, our study provides valuable insights into the complex interactions between pollen diet, sterol composition, and development in a solitary bee, highlighting the importance of considering multiple nutritional and environmental factors in understanding bee health and performance. Our results suggest that sterol diversity and concentration play crucial roles in larval development, but also the high sterol selectivity and potential sterol metabolism in bees, which has previously been ruled out. In summary, pollen diversity may provide some nutrient diversity, but the specific physical and metabolic nature of pollen must be considered to fully understand what is required for an optimal diet and bee nutrition is more species specific than previous research suggests. Data accessibility The processed data will be available as Supplementary material and available also online. Declaration of AI use We have not used AI-assisted technologies in creating this article. Authors’ contributions C.M.: conceptualization, data curation, formal analysis, investigation, methodology, software, validation, visualization, writing—original draft, and writing—review and editing; J.K.: formal analysis, methodology, writing—original draft, and writing—review and editing; S.F.: data curation, investigation, methodology and writing—review and editing; J.S.: methodology, and writing—review and editing; J.G-L.: investigation, methodology, and writing—review and editing; G.A.W.: conceptualization, funding acquisition, project administration, methodology and writing—review and editing; P.C.S.: conceptualization, funding acquisition, project administration, supervision, resources and writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Conflict of interest declaration We declare we have no competing interests. Funding This study was supported by the NERC (NE/V012282/1 to P.C.S. and G.A.W.) and the MSc program at RBG Kew. Footnotes Electronic supplementary material will be available online when the paper is accepted for publication. Acknowledgements We thank the NERC (NE/V012282/1) for their financial support of the project. We also thank Melina Domingues-Olivares and Katie Perry for their help with the lab work. References ↵ Tong Z.-Y. , Wu L.-Y. , Feng H.-H. , Zhang M. , Armbruster W.S. , Renner S.S. , Huang S.-Q. 2023 New calculations indicate that 90% of flowering plant species are animal-pollinated . National Science Review 10 ( 10 ), nwad219. ( doi: 10.1093/nsr/nwad219 ). OpenUrl CrossRef ↵ Khalifa S.A.M. , Elshafiey E.H. , Shetaia A.A. , El-Wahed A.A.A. , Algethami A.F. , Musharraf S.G. , AlAjmi M.F. , Zhao C. , Masry S.H.D. , Abdel-Daim M.M. , et al. 2021 Overview of bee pollination and its economic value for crop production . Insects 12 ( 8 ), 688 . ( doi: 10.3390/insects12080688 ). 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