How reliable is metabarcoding for pollen identification? An evaluation of different taxonomic assignment strategies by cross-validation.

Metabarcoding is a powerful tool, increasingly used in many disciplines of environmental sciences. However, to assign a taxon to a DNA sequence, bioinformaticians need to choose between different strategies or parameter values and these choices sometimes seem rather arbitrary. In this work, we present a case study on ITS2 and rbcL databases used to identify pollen collected by bees in Belgium. We blasted a random sample of sequences from the reference database against the remainder of the database using different strategies and compared the known taxonomy with the predicted one. This in silico cross-validation (CV) approach proved to be an easy yet powerful way to (1) assess the relative accuracy of taxonomic predictions, (2) define rules to discard dubious taxonomic assignments and (3) provide a more objective basis to choose the best strategy. We obtained the best results with the best blast hit (best bit score) rather than by selecting the majority taxon from the top 10 hits. The predictions were further improved by favouring the most frequent taxon among those with tied best bit scores. We obtained better results with databases containing the full sequences available on NCBI rather than restricting the sequences to the region amplified by the primers chosen in our study. Leaked CV showed that when the true sequence is present in the database, blast might still struggle to match the right taxon at the species level, particularly with rbcL. Classical 10-fold CV-where the true sequence is removed from the database-offers a different yet more realistic view of the true error rates. Taxonomic predictions with this approach worked well up to the genus level, particularly for ITS2 (5-7% of errors). Using a database containing only the local flora of Belgium did not improve the predictions up to the genus level for local species and made them worse for foreign species. At the species level, using a database containing exclusively local species improved the predictions for local species by ∼12% but the error rate remained rather high: 25% for ITS2 and 42% for rbcL. Foreign species performed worse even when using a world database (59-79% of errors). We used classification trees and GLMs to model the % of errors vs. identity and consensus scores and determine appropriate thresholds below which the taxonomic assignment should be discarded. This resulted in a significant reduction in prediction errors, but at the cost of a much higher proportion of unassigned sequences. Despite this stringent filtering, at least 1/5 sequences deemed suitable for species-level identification ultimately proved to be misidentified. An examination of the variability in prediction accuracy between plant families showed that rbcL outperformed ITS2 for only two of the 27 families examined, and that the % correct species-level assignments were much better for some families (e.g. 95% for Sapindaceae) than for others (e.g. 35% for Salicaceae).

Plant-pollinator network architecture does not impact intraspecific microbiome variability.

Variation in how individuals interact with food resources can directly impact, and be affected by, their microbial interactions due to the potential for transmission. The degree to which this transmission occurs, however, may depend on the structure of forager networks, which determine the community-scale transmission opportunities. In particular, how the community-scale opportunity for transfer balances individual-scale barriers to transmission is unclear. Examining the bee-flower and bee-microbial interactions of over 1000 individual bees, we tested (1) the degree to which individual floral visits predicted microbiome composition and (2) whether plant-bee networks with increased opportunity for microbial transmission homogenized the microbiomes of bees within that network. The pollen community composition carried by bees was associated with microbiome composition at some sites, suggesting that microbial transmission at flowers occurred. Contrary to our predictions, however, microbiome variability did not differ based on transfer opportunity: bee microbiomes in asymmetric networks with high opportunity for microbial transfer were similarly variable compared to microbiomes in networks with more evenly distributed links. These findings suggest that microbial transmission at flowers is frequent enough to be observed at the community level, but that community network structure did not substantially change the dynamics of this transmission, perhaps due to filtering processes in host guts.

The first ITS2 sequence data set of eDNA from honey of Malaysian giant honeybees (Apis dorsata) and stingless bees (Heterotrigona itama) reveals plant species diversity.

OBJECTIVES: Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species. RESULTS: Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu. DATA DESCRIPTION: Our data offers valuable insights into honey's geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.

Chromosomal-level Genome Assembly of the Coffee Bee Hawk Moth Reveals the Evolution of Chromosomes and the Molecular Basis of Distinct Phenotypes.

Cephonodes hylas, the coffee bee hawk moth is a hawk moth species with unique characteristics, such as larvae feeding on gardenia, overcoming the toxicity of its iridoid glycosides, diurnal adults, and transparent wings. Although C. hylas is a fascinating model for molecular biological research, genome sequence analysis-based genetic approaches to elucidate these peculiarities have not yet been undertaken. We successfully achieved de novo genome assembly at the chromosome level of C. hylas comparable to the Lepidoptera model organism, silkworm. Additionally, 16,854 protein-coding genes were annotated, and the constructed genome sequence and annotated genes were of the highest quality BUSCO completion compared to closely related species. Comparative genome analysis revealed the process of chromosomal evolution from the Bombycoidea ancestral (n = 31) genome and changes in turnover at the chromosome level associated with chromosomal fusion events, such as the rate of repetitive sequence insertion. These analyses were only possible because the genome was constructed at the chromosome level. Additionally, increased the nonsynonymous/synonymous rate (dN/dS) ratios were observed in multiple photoreceptor-related genes that were strongly associated with the acquisition of diurnal activity. Furthermore, tandemly duplicated expanded genes containing many digestive and other enzymes and larval midgut-specific expression were also confirmed. These genes may be involved in the metabolism of genipin, a toxin found in gardenias. Using the genome sequence of C. hylas determined at the chromosome level, we have successfully identified new insights into the chromosomal evolution of Bombycoidea, as well as the relationship between the genome sequence and its characteristic traits.

Survivorship and food consumption of immatures and adults of Apis mellifera and Scaptotrigona bipunctata exposed to genetically modified eucalyptus pollen.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification (GM) of eucalyptus can provide additional characteristics for increasing productivity and protecting wood yield, as well as potentially altering fiber for a diversity of industrial uses. However, prior to releasing a new GM plant, risk assessments studies with non-target organisms must be undertaken. Bees are prominent biological models since they play an important role in varied ecosystems, including for Eucalyptus pollination. The main goal of this study was to evaluate whether a novel event (Eucalyptus 751K032), which carries the cp4-epsps gene that encodes the protein CP4-EPSPS and nptII gene that encodes the protein NPTII, might adversely affect honey bees (Apis mellifera) and stingless bees (Scaptotrigona bipunctata). The experiments were performed in southern Brazil, as follows: (i) larvae and adults were separately investigated, (ii) three or four different pollen diets were offered to bees, depending on larval or adult status, and (iii) two biological attributes, i.e., survivorship of larvae and adults and food intake by adults were evaluated. The diets were prepared with pollen from GM Eucalyptus 751K032; pollen from conventional Eucalyptus clone FGN-K, multifloral pollen or pure larval food. The insecticide dimethoate was used to evaluate the sensitivity of bees to toxic substances. Datasets were analyzed with Chi-square test, survival curves and repeated measures ANOVA. Results indicated no evidence of adverse effects of Eucalyptus pollen 751K032 on either honey bees or stingless bees assessed here. Therefore, the main findings suggest that the novel event may be considered harmless to these organisms since neither survivorship nor food consumption by bees were affected by it.

Differential ability of three bee species to move genes via pollen.

Since the release of genetically engineered (GE) crops, there has been increased concern about the introduction of GE genes into non-GE fields of a crop and their spread to feral or wild cross-compatible relatives. More recently, attention has been given to the differential impact of distinct pollinators on gene flow, with the goal of developing isolation distances associated with specific managed pollinators. To examine the differential impact of bee species on gene movement, we quantified the relationship between the probability of getting a GE seed in a pod, and the order in which a flower was visited, or the cumulative distance traveled by a bee in a foraging bout. We refer to these relationships as 'seed curves' and compare these seeds curves among three bee species. The experiments used Medicago sativa L. plants carrying three copies of the glyphosate resistance (GR) allele as pollen donors (M. sativa is a tetraploid), such that each pollen grain carried the GR allele, and conventional plants as pollen recipients. Different foraging metrics, including the number of GR seeds produced over a foraging bout, were also quantified and contrasted among bee species. The lowest number of GR seeds set per foraging bout, and the GR seeds set at the shortest distances, were produced following leafcutting bee visits. In contrast, GR seeds were found at the longest distances following bumble bee visits. Values for honey bees were intermediate. The ranking of bee species based on seed curves correlated well with field-based gene flow estimates. Thus, differential seed curves of bee species, which describe patterns of seed production within foraging bouts, translated into distinct abilities of bee species to move genes at a landscape level. Bee behavior at a local scale (foraging bout) helps predict gene flow and the spread of GE genes at the landscape scale.

Investigating the use of pollen DNA metabarcoding to quantify bee foraging and effects of threshold selection.

DNA metabarcoding of pollen is a useful tool for studying bee foraging ecology. However, several questions about this method remain unresolved, including the extent to which sequence read data is quantitative, which type of sequence count removal threshold to use and how that choice affects our ability to detect rare flower visits, and how sequence artefacts may confound conclusions about bee foraging behavior. To address these questions, we isolated pollen from five plant species and created treatments comprised of pollen from each species alone and combinations of pollen from multiple species that varied in richness and evenness. We used ITS2 and rbcL metabarcoding to identify plant species in the samples, compared the proportion of pollen by mass to the proportion of sequencing reads for each plant species in each treatment, and analyzed the sequencing data using both liberal and conservative thresholds. We collected pollen from foraging bees, analyzed metabarcoding data from those samples using each threshold, and compared the differences in the pollinator networks constructed from the data. Regardless of the threshold used, the relationship between the proportion of pollen by mass and sequencing reads was inconsistent, suggesting that the number of sequence reads is a poor proxy for pollen abundance in mixed-species samples. Using a liberal threshold resulted in greater detection of original plant species in mixtures but also detected additional species in mixtures and single-species samples. The conservative threshold reduced the number of additional plant species detected, but several species in mixtures were not detected above the threshold, resulting in false negatives. Pollinator networks produced using the two thresholds differed and illustrated tradeoffs between detection of rare species and estimation of network complexity. Threshold selection can have a major effect on conclusions drawn from studies using metabarcoding of bee pollen to study plant-pollinator interactions.

Hawkmoth and bee pollinators impact pollen dispersal at the landscape but not local scales in two species of Oenothera.

PREMISE: Animal pollinators play an important role in pollen dispersal. Here, we assessed differences in pollen and seed dispersal and the role of pollinator functional groups with different foraging behaviors in generating patterns of genetic diversity over similar geographic ranges for two closely related taxa. We focused on two members of Oenothera section Calylophus (Onagraceae) that co-occur on gypsum outcrops throughout the northern Chihuahuan Desert but differ in floral phenotype and primary pollinator: Oenothera gayleana (bee) and O. hartwegii subsp. filifolia (hawkmoth). METHODS: We measured breeding system and floral traits and studied gene flow and population differentiation at the local (<13 km; four populations) and landscape (60-440 km; five populations) scales using 10-11 nuclear (pollen dispersal) and three plastid (seed dispersal) microsatellite markers. RESULTS: Both taxa were self-incompatible and floral traits were consistent with expectations for different pollinators. Seed and pollen dispersal patterns were distinctly different for both species. We found no evidence of genetic structure at the local scale but did at the landscape scale; O. gayleana showed greater differentiation and significant isolation by distance than in O. hartwegii subsp. filifolia. The plastid data were consistent with gravity dispersal of seeds and suggest that pollen dispersal is the principal driver of genetic structure in both species. CONCLUSIONS: We demonstrated that pollinator functional groups can impact genetic differentiation in different and predictable ways. Hawkmoths, with larger foraging distances, can maintain gene flow across greater spatial scales than bees.

Differential bumble bee gene expression associated with pathogen infection and pollen diet.

BACKGROUND: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. RESULTS: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. CONCLUSIONS: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

Elevated temperature affects energy metabolism and behavior of bumblebees.

Bumblebees (Bombus eximius) are one of the most prominent pollinators in the agricultural industry because of their adaptation to temperate climates and pollination behavior (buzz pollination). Several studies have explained the need to increase conservation efforts for bumblebees due to climate change, but studies on the impact of climate change on pollination behavior of bumblebees have been limited. The present study investigated the effect of elevated temperatures on the survival and physiology of bumblebees. The behavioral changes in flight ability and pollen collection were also determined. We found that elevated temperature affects the survival rate and appetite of bumblebees. Gene expression analysis suggested that the energy metabolic pathway tends to involve anaerobic respiration during heat stress. The energy produced is mainly used to maintain essential physiological functions, such as expression of heat shock proteins and conversion of peroxides to harmless molecules. Energy distributed to flight muscles is reduced during heat stress, resulting in lower wing beating frequency. In addition, the flight path of bumblebees is shortened during heat stress, thereby further contributing to reduced pollen collection. These results demonstrate that elevated temperatures cause detrimental effects to bumblebees and can also potentially reduce crop production.

Co-diet supplementation of low density polyethylene and honeybee wax did not influence the core gut bacteria and associated enzymes of Galleria mellonella larvae (Lepidoptera: Pyralidae).

The current plastic pollution throughout the world is a rising concern that demands the optimization of biodegradation processes. One avenue for this is to identify plastic-degrading bacteria and associated enzymes from the gut bacteria of insect models such as Tenebrio molitor, Plodia interpunctella or Galleria mellonella that have the ability to ingest and rapidly degrade polyethylene. Therefore, this study takes part in understanding the role of the gut bacteria by investigating G. mellonella as a biological model feeding with a diet based on honeybee wax mixed or not with low-density polyethylene. Gut microbiome was analyzed by high throughput 16S rRNA sequencing, and Enterococcaceae and Oxalobacteraceae were found to be the major bacterial families. Compared to the control, the supplementation of low-density polyethylene did not cause significant modification of the bacterial microbiota at community and taxa levels, suggesting bacterial microbiome resilience. The bacterial proteome analysis of gut contents was encouraging for the identification of plastic degrading enzymes such as the phenylacetaldehyde dehydrogenase which participate in styrene degradation. This study allowed a better characterization of the gut bacteria of G. mellonella and provided a basis for the further study of biodegradation of polyethylene based on the bacterial microbiota from insect guts.

An integrative bioinformatics pipeline shows that honeybee-associated microbiomes are driven primarily by pollen composition.

The microbiomes associated with bee nests influence colony health through various mechanisms, although it is not yet clear how honeybee congeners differ in microbiome assembly processes, in particular the degrees to which floral visitations and the environment contribute to different aspects of diversity. We used DNA metabarcoding to sequence bacterial 16S rRNA from honey and stored pollen from nests of 4 honeybee species (Apis cerana, A. dorsata, A. florea, and A. laboriosa) sampled throughout Yunnan, China, a global biodiversity hotspot. We developed a computational pipeline integrating multiple databases for quantifying key facets of diversity, including compositional, taxonomic, phylogenetic, and functional ones. Further, we assessed candidate drivers of observed microbiome dissimilarity, particularly differences in floral visitations, habitat disturbance, and other key environmental variables. Analyses revealed that microbiome alpha diversity was broadly equivalent across the study sites and between bee species, apart from functional diversity which was very low in nests of the reclusive A. laboriosa. Turnover in microbiome composition across Yunnan was driven predominantly by pollen composition. Human disturbance negatively impacted both compositional and phylogenetic alpha diversity of nest microbiomes, but did not correlate with microbial turnover. We herein make progress in understanding microbiome diversity associated with key pollinators in a biodiversity hotspot, and provide a model for the use of a comprehensive informatics framework in assessing pattern and drivers of diversity, which enables the inclusion of explanatory variables both subtly and fundamentally different and enables elucidation of emergent or unexpected drivers.

New insights into the criteria of functional heterozygosity of the Apis mellifera complementary sex determining gene-Discovery of a functional allele pair differing by a single amino acid.

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations.

Temporal Patterns of Honeybee Foraging in a Diverse Floral Landscape Revealed Using Pollen DNA Metabarcoding of Honey.

Understanding the plants pollinators use through the year is vital to support pollinator populations and mitigate for declines in floral resources due to habitat loss. DNA metabarcoding allows the temporal picture of nectar and pollen foraging to be examined in detail. Here, we use DNA metabarcoding to examine the forage use of honeybees (Apis mellifera L.) within a florally diverse landscape within the UK, documenting the key forage plants used and seasonal progression over two years. The total number of plant taxa detected in the honey was 120, but only 16 of these were found with a high relative read abundance of DNA, across the main foraging months (April-September). Only a small proportion of the available flowering genera in the landscape were used by the honeybees. The greatest relative read abundance came from native or near-native plants, including Rubus spp., Trifolium repens, the Maleae tribe including Crataegus, Malus, and Cotoneaster, and Hedera helix. Tree species were important forage in the spring months, followed by increased use of herbs and shrubs later in the foraging season. Garden habitat increased the taxon richness of native, near-native and horticultural plants found in the honey. Although horticultural plants were rarely found abundantly within the honey samples, they may be important for increasing nutritional diversity of the pollen forage.

Biomonitoring via DNA metabarcoding and light microscopy of bee pollen in rainforest transformation landscapes of Sumatra.

BACKGROUND: Intense conversion of tropical forests into agricultural systems contributes to habitat loss and the decline of ecosystem functions. Plant-pollinator interactions buffer the process of forest fragmentation, ensuring gene flow across isolated patches of forests by pollen transfer. In this study, we identified the composition of pollen grains stored in pot-pollen of stingless bees, Tetragonula laeviceps, via dual-locus DNA metabarcoding (ITS2 and rbcL) and light microscopy, and compared the taxonomic coverage of pollen sampled in distinct land-use systems categorized in four levels of management intensity (forest, shrub, rubber, and oil palm) for landscape characterization. RESULTS: Plant composition differed significantly between DNA metabarcoding and light microscopy. The overlap in the plant families identified via light microscopy and DNA metabarcoding techniques was low and ranged from 22.6 to 27.8%. Taxonomic assignments showed a dominance of pollen from bee-pollinated plants, including oil-bearing crops such as the introduced species Elaeis guineensis (Arecaceae) as one of the predominant taxa in the pollen samples across all four land-use types. Native plant families Moraceae, Euphorbiaceae, and Cannabaceae appeared in high proportion in the analyzed pollen material. One-way ANOVA (p > 0.05), PERMANOVA (R² values range from 0.14003 to 0.17684, for all tests p-value > 0.5), and NMDS (stress values ranging from 0.1515 to 0.1859) indicated a lack of differentiation between the species composition and diversity of pollen type in the four distinct land-use types, supporting the influx of pollen from adjacent areas. CONCLUSIONS: Stingless bees collected pollen from a variety of agricultural crops, weeds, and wild plants. Plant composition detected at the family level from the pollen samples likely reflects the plant composition at the landscape level rather than the plot level. In our study, the plant diversity in pollen from colonies installed in land-use systems with distinct levels of forest transformation was highly homogeneous, reflecting a large influx of pollen transported by stingless bees through distinct land-use types. Dual-locus approach applied in metabarcoding studies and visual pollen identification showed great differences in the detection of the plant community, therefore a combination of both methods is recommended for performing biodiversity assessments via pollen identification.

Zinc supplementation modifies brain tissue transcriptome of Apis mellifera honeybees.

BACKGROUND: Bees are the most important group of pollinators worldwide and their populations are declining. In natural conditions, Apis mellifera depends exclusively on food from the field to meet its physiological demands. In the period of scarcity, available resources are insufficient and artificial supplementation becomes essential for maintaining the levels of vitamins, proteins, carbohydrates, and minerals of colonies. Among these minerals, zinc is essential in all living systems, particularly for the regulation of cell division and protein synthesis, and is a component of more than 200 metalloenzymes. RESULTS: The total RNA extracted from the brain tissue of nurse bees exposed to different sources and concentrations of zinc was sequenced. A total of 1,172 genes in the treatment that received an inorganic source of zinc and 502 genes that received an organic source of zinc were found to be differentially expressed among the control group. Gene ontology enrichment showed that zinc can modulate important biological processes such as nutrient metabolism and the molting process. CONCLUSIONS: Our results indicate that zinc supplementation modulates the expression of many differentially expressed genes and plays an important role in the development of Apis mellifera bees. All the information obtained in this study can contribute to future research in the field of bee nutrigenomics.

Antibiotics Alter the Expression of Genes Related to Behavioral Development in Honey Bees (Hymenoptera: Apidae).

Honey bees, as many species of social insects, display a division of labor among colony members based on behavioral specializations related to age. Adult worker honey bees perform a series of tasks in the hive when they are young (such as brood care or nursing) and at ca. 2-3 wk of age, shift to foraging for nectar and pollen outside the hive. The transition to foraging involves changes in metabolism and neuroendocrine activities. These changes are associated with a suite of developmental genes. It was recently demonstrated that antibiotics influence behavioral development by accelerating or delaying the onset of foraging depending on timing of antibiotic exposure. To understand the mechanisms of these changes, we conducted a study on the effects of antibiotics on expression of candidate genes known to regulate behavioral development. We demonstrate a delay in the typical changes in gene expression over the lifetime of the individuals that were exposed to antibiotics during immature stage and adulthood. Additionally, we show an acceleration in the typical changes in gene expression on individuals that were expose to antibiotics only during immature stage. These results show that timing of antibiotic exposure alter the typical regulation of behavioral development by metabolic and neuroendocrine processes.

Morphological characteristics of pollen from triploid watermelon and its fate on stigmas in a hybrid crop production system.

Hybrid crop production is more reliant on pollinators compared to open-pollinated crops because they require cross-pollination between a male-fertile and a male-sterile line. Little is known about how stigma receipt of pollen from male-sterile genotypes affects reproduction in hybrids. Non-viable and non-compatible pollen cannot fertilise plant ovules, but may still interfere with pollination success. Here we used seedless watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) as a model hybrid plant, to evaluate the morphology, physiology, and movement of pollen from inter-planted genotypes (diploids and triploids). We found that pollen from triploids ('Exclamation' and 'Royal Armada') and diploids ('SP-6', 'Summer Flavor 800', and 'Tiger') was visually distinguishable. Pollen in triploids had more deformities (42.4-46%), tetrads (43-44%), and abnormal growth of callose plugs in pollen tubes. The amount of pollen in triploids to germinate on stigmas was low (8 ± 3%), and few pollen grains produced pollen tubes (6.5 ± 2%). Still, contrary to previous reports our results suggest that some viable pollen grains are produced by triploid watermelons. However, whilst honey bees can collect and deposit pollen from triploids onto stigmas, its effect on hybrid watermelon reproduction is likely to be minimal due to its low germination rate.

Maine's Bumble Bees (Hymenoptera: Apidae)-Part 2: Comparisons of a Common (Bombus ternarius) and a Rare (Bombus terricola) Species.

As part of a quantitative survey of Maine's bumble bee fauna (Butler et al. 2021), we compared and contrasted genetic diversity, parasite and pathogen burdens, and pesticide exposure of the relatively common Bombus ternarius Say, 1937 and the spatially rare Bombus terricola Kirby, 1837. We recorded 11 Bombus species at 40 survey sites across three Maine ecoregions, and B. ternarius was the most common species, while B. terricola was spatially rare. Nonmetric multidimensional scaling indicated that B. terricola was associated with higher elevation sites in Maine, while B. ternarius was more broadly distributed in the state. Pollinator networks constructed for each bee indicated B. ternarius foraged on more plant species than B. terricola, but that there was considerable overlap (73%) in plant species visited. Genetic diversity was greater in the spatially restricted B. terricola, whereas the widely distributed B. ternarius was characterized by greater genetic differentiation among regions. Bombus terricola had higher molecular marker levels of the microsporidian fungi Nosema spp. and the trypanosome Crithidia spp., and both species had high levels of Trypanosoma spp. exposure. No Western Honey Bee (Apis mellifera, Linnaeus, 1758) viruses were detected in either species. Pesticides were not detected in pollen samples collected from workers of either species, and B. ternarius worker tissue samples exhibited only trace levels of diflubenzuron.

Herbaceous perennial ornamental plants can support complex pollinator communities.

Human-designed landscapes can host diverse pollinator communities, and the availability of floral resources is central to supporting insect biodiversity in highly modified environments. However, some urban landscapes have relatively few pollinator-attractive plant species and management in urban environments rarely considers the function of these plants in generating and supporting a stable ecological community. Evaluations of 25 cultivars within five commercially popular herbaceous perennial ornamental plant genera (Agastache, Echinacea, Nepeta, Rudbeckia, and Salvia) revealed variation in the total and proportional abundance of visitors attracted. These varieties supported multiple pollinator functional groups, however bees were the primary visitors to in this system. Cultivars were assessed according to their function within a plant-pollinator network. Comparisons of artificial networks created with the six most attractive and six least attractive cultivars demonstrated that a planting scheme using the most attractive cultivars would attract nearly four times as many bee species, including several specialists and rare species. Plant diversity in the landscape was correlated with abundance and diversity of pollinator visitors, demonstrating that community context shapes a plant's relative attractiveness to pollinators. We conclude that herbaceous perennial cultivars can support an abundance and diversity of pollinator visitors, however, planting schemes should take into consideration the effects of cultivar, landscape plant diversity, floral phenology, floral area, and contribution to a stable ecological community.