DOI: 10.5061/dryad.wh70rxwnd
title: The impact of dietary breadth on bumblebee colony fitness

description: The current decline of pollinators may disrupt ecosystems and ecosystem
 services with potentially harmful effects on nature and human society.
 While the importance of habitat loss and fragmentation, pollution and
 increased disease risk in driving pollinator decline has been clearly
 demonstrated, the impact of resource diversity is less well understood. In
 this study, we investigated the effect of pollen diversity and composition
 on reproductive success and fitness of Bombus terrestris colonies. We
 asked the question whether a higher plant diversity results in a more
 diverse diet, lower pathogen incidence and a higher colony fitness. To
 answer these questions, colonies of lab-reared bumblebees were placed in
 species-poor heathland and species-rich semi-natural grasslands that
 strongly differed in plant community composition and diversity. We
 examined pollen loads on the bodies of foragers and identified the plant
 taxa present in the realized diet via DNA metabarcoding of the ITS2
 marker. Liquid chromatography-mass spectrometry (LC-MS) was used to
 compare peptide composition of pollen samples from both habitats. Colony
 fitness was assessed by counting the number of sexuals produced by the
 colony at the end of its cycle. At the same time, colonies were examined
 for parasite incidence. Pollen composition and diversity on pollinators’
 bodies differed significantly between bees foraging in grasslands and
 heathlands. Concomitantly, peptide composition differed significantly
 between pollen samples from grasslands and heathlands. Colonies developed
 significantly better in heathland sites than in grasslands. In addition,
 colony fitness was only weakly related to pollen diversity and effects in
 some cases depended on the habitat where the bees were foraging. Pathogen
 incidence was very low and not affected by habitat. Overall, our results
 indicate that plant diversity is not necessarily a good predictor of
 colony fitness, and that vegetation composition and associated differences
 in both the quantity and quality of pollen are more important than pollen
 diversity per se.
description: <em>Colony
 performance</em> After collection of the hives at
 the end of the experiment, all bumblebees were killed and stored at -20°C
 to conserve the hives’ structure. All hives were dissected, and individual
 caste members were counted. The number of queens, workers, males, regular
 pupae and queen pupae were counted to assess colony development. The
 production of sexuals is mainly indicative for colony performance and
 directly related to fitness. Therefore, the total number of queens
 (emerged and pupae), the number of males, and the ratio of gynes:males was
 used as proxy for colony performance. A higher ratio in the latter
 indicates better colony performance because of the higher overall
 investment in queens than males (Duchateau et al, 2004). In addition, we
 recorded other information such as colony net weight gain, presence of
 involucrum, and the weight of the pollen mass stored in the pollen pots
 inside the colony. <em>DNA metabarcoding of pollen
 samples</em> Pollen DNA was extracted using the
 Norgen Biotek Corp Plant/ Fungi DNA Isolation Kit following the
 manufacturer’s instructions. Given the powdery nature of pollen, no
 grinding in liquid nitrogen was performed. DNA quantity and quality were
 assessed using the Thermo Scientific NanoDrop Spectrophotometer
 (Desjardins & Conklin, 2010). Species identification was done via
 DNA metabarcoding of the internal transcribed spaced 2 nuclear ribosomal
 fragment (ITS2) (Suchan et al., 2019), using the primer pair ITS-S2F and
 ITS-S3R (Kozich et al., 2013; Sickel et al., 2015). The PCR consisted of 1
 minute at 95 °C for initial denaturation, followed by 30 cycles of 15
 seconds denaturation at 95°C, 15 seconds at 55 °C for hybridization and 15
 seconds of elongation at 72 °C.  The ALLin HiFi DNA Polymerase (HLE0205
 van highQu) master mix was used. The PCR product was purified
 following the Agencourt® AMPure ® XP kit, with a single 70% ethanol wash.
 DNA yield quantification was done on the Invitrogen Qubit Fluorometer
 (Thermofisher). The aliquot DNA library was sent to Genomics Core Leuven
 for Illumina MiSeq sequencing. Data processing consisted of matching
 forward and reverse Illumina reads followed by omitting low quality
 samples. Reads with a similarity of 97% or higher were clustered in
 Operational Taxonomic Units (OTU). From each OTU, a reference sequence was
 computed and Blasted against the NCBI Gen database for species
 identification. If no species match was found, the higher taxonomic level
 was assigned to each OTU (genus or family). Only OTUs with a frequency
 larger than 0.0005% were kept. This way, the majority of rare OTUs from
 potential mismatches and high intraspecific variation for the marker were
 omitted from the data. The relative number of Illumina
 sequence reads of a plant species or OTU per sample was considered to be a
 good approximation of the relative abundance of that taxonomic entity in
 the diet of <em>Bombus terrestris</em> (Deagle et al, 2018).
 All OTU abundances were transformed from number of sequence reads to
 relative abundances per sample. <em>Protein
 analyses</em> Complementary to DNA metabarcoding,
 liquid chromatography-mass spectrometry (LC-MS) was used to unravel
 possible compositional differences in protein content and to assess the
 quality of the pollen diet. Twenty milligrams of fresh pollen were
 grounded with pestle and mortar in liquid nitrogen and transferred to a
 clean Eppendorf tube. Total pollen protein content was extracted using a
 phenol extraction for recalcitrant plant tissue (Carpentier et al., 2005;
 Faurobert et al., 2007), because plants protect the pollen genetic
 material with the sporopollenin protein, one of the most inert biological
 polymers (Mackenzie et al., 2015) and the method also efficiently deals
 with polysaccharides, lipids and other phenolic compounds (Faurobert et
 al., 2007). Whilst working on ice to inhibit protease activity, 500µl of
 extraction buffer was mixed with the ground pollen. Next, 500µl buffered
 phenol was added to each sample and vortexed thoroughly on ice. The
 mixture was then centrifuged for 10 min at 12 000 rpm at 4 °C. The
 phenolic phase containing the proteins was collected and an equal volume
 of extraction buffer was added to the phenolic phase. The mixture was
 centrifuged for 5 min at 12,000 rpm at 4 °C. Proteins were precipitated
 overnight at -20 °C from the residual phenol phase using 100 mM ammonium
 acetate in cold methanol (5*Volume of phenolic phase) followed by
 centrifuging 60 min at 13,000 rpm at 4 °C. The supernatant was carefully
 removed to not resuspend the protein pellet. The protein pellet was rinsed
 twice in 2 ml rinsing solution without resuspension. Each rinse with an
 incubation for 1 hour at -20 °C, followed by centrifuging for 30 min on
 13,000 rpm at 4 °C. After rinsing, the pellet was dried under the fume
 hood before resuspension in 90 µl in lysis buffer, without overheating to
 avoid carbamylation. Samples were centrifuged for 30 min at 13 000 rpm at
 30 °C and stored at -80 °C until further purification and mass
 spectrometry preparation. After the last extraction step, peptide
 quantification was done using the procedure for the 2-D Quant Kit (GE
 Healthcare Life Sciences). Proteins were selectively digested
 between lysine and arginine with the endoprotease Pierce™ Trypsin Protease
 (MS Grade) from ThermoFisher Scientific. Cleavage to polypeptides reduced
 the polypeptides' length to an average of 700–1500 dalton, which is a
 suitable range for MS. Cleaved samples were resuspended and
 1,4-Dithiothreitol (DTT, Sigma, final working concentration 20 mM) was
 added followed by 15min incubation. Next, iodoacetamide (IAA, final
 working concentration 50 mM) was added to each sample as an irreversible
 cysteine peptidase and incubated in the dark for 30min. Ammonium
 bicarbonate (150mM, 3* sample volume) was added for protein reduction. 0.2
 µg trypsin was added to each sample followed by overnight incubation at 37
 °C. After overnight digestion, 1/10th of sample volume of 1%
 trifluoroacetic Acid (TFA) was added to stop trypsin activity (SyBioMa,
 2019). Samples were purified using Pierce® C18 Spin Columns (Thermo
 Scientific). Cleaned samples were dried in a SpeedVac vacuum drier (Thermo
 Scientific) for approximately 40 min. Peptide separation was performed on
 the Ultimate 3000 HPLC Liquid Chromatography (Thermo Fisher Scientific).
 Peptide charging was done with Electrospray Ionization (ESI) followed by
 mass analysis with Thermo Fisher Scientific Orbitrap Q. In total,
 32 pollen
 samples from the first and second sampling (10/08 & 13/08 and
 18/08 & 19/08/2019) were analyzed. 27 samples went successfully
 through the analysis pipeline and yielded usable
 results. <em>Incidence of
 parasites</em> Parasite incidence can strongly
 influence colony fitness. At the end of the five-week field experiment,
 bumblebees were therefore checked for the presence of endo- and
 ectoparasites. Infection of queens by <em>Parasitellus</em>
 mites was noted for each hive and was assigned a rank. Four categories
 were distinguished: ‘no mites’ meaning that not a single queen was found
 to be carrying mites, ‘few mites’ for low infection rate (&lt;25%) or
 low number of mites on the queens, ‘medium infection’ for up to 50% of
 queens affected or medium densities per queen and ‘high infection’ for
 everything above the previous. From each hive, three workers were
 randomly selected for the detection of endoparasites, yielding to 216
 dissected workers. Because workers are the most motile cast, the chance of
 encountering parasites is higher in workers than in the other castes. The
 workers were screened for incidence of <em>Nosema</em>
 <em>bombi</em> and <em>N. ceranae</em>,
 <em>Crithidia</em> sp. and <em>Apicystis</em> sp.
 using quantitative PCR (qPCR). The hindgut, possibly with some remains of
 trachea or the poison gland, of three workers from the same hive were
 pooled in a clean Eppendorf tube and stored at -20°C before qPCR. qPCR was
 performed using the primers described by Huang et al. (2016) and the
 protocol specified in Bosmans et al. (2018). Each reaction was performed
 in duplicate. In each analysis, a positive (pathogen DNA) and negative
 control (template DNA replaced by sterile water) were included.
 Additionally, six gut samples from wild bumblebees that were known to be
 either contaminated or free of pathogens (screened by microscopy and
 regular PCR) were included as additional controls. To verify amplification
 specificity, a melting curve analysis was performed. CT-values were used
 to calculate DNA concentration and to assess parasite load. Quantification
 was based on standard curves from amplification of cloned target sequences
 in a TOPO-TA vector (Invitrogen). All samples exceeding the DNA
 concentration of the negative control (water) were considered positive.
 From three of the 72 hives, the qPCR was not successful (2 heathland and 1
 grassland hive). The presence of
 <em>Physocephala</em> sp. was not expected prior to dissection
 of the workers but was frequently encountered by visual encounter of the
 larvae or pupae (Schmid-Hempel and Schmid-Hempel, 1996). Thorough colony
 infection rates could not be extrapolated from the incidence data of the
 five randomly selected workers nor can the parasitoids be transferred
 horizontally. Incidence per colony was therefore only categorically
 (‘present’ vs ‘absent’) recorded.
description: See README file. There are separate files for each section above.
 R code refers to any of these sections and datasets.