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BEExact classifies more ASVs and at higher confidence compared to the widely implemented SILVA database. (A) Overall classification rates at each taxonomic level for all data sets evaluated. Data depict means 6 standard deviations at each level for n = 32 data sets with statistics shown for two-way ANOVA with Tukey's multiple comparisons. (B) IDTAXA bootstrap confidence scores on the total set of 4,957 unique ASVs from all data sets combined. The dotted line showing the cutoff (20%) used for all other comparisons shown. (C and D) Classification rates broken down by bee species (grouped by eusocial or solitary type membership) (C) and by sample type irrespective of background bee type (D). Data depict means 6 standard deviations per sample classified in each of the categories shown (two-way ANOVA with Tukey's multiple comparisons). (E and F) Scatterplots demonstrate that BEExact outcompetes SILVA more often in assigning taxonomy to ASVs found at either high prevalence or abundance across all data sets evaluated. Nested visualization plots above show how classification rates change based on differences in ASV prevalence. (G) Heat trees display the weighted classification (Continued on next page)
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The failure of current universal taxonomic databases to support the rapidly expanding field of bee microbiota research has led to many investigators relying on “in-house” reference sets or manual classification of sequence reads (usually based on BLAST searches), often with vague identity thresholds and subjective taxonomy choices. This time expens...
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... different studies, we utilized an established next-generation sequence simulator (ART [62]) to emulate several MiSeq runs at various read depths using a subset of in silico-extracted V4 sequences from BEEx-FL-refs (total of 718 unique sequences used as inputs). Based on mimicked error rates calculated from sequencing data evaluated in this study (Fig. S5), less than half of the 718 unique input sequences were detectable by the denoising algorithm implemented in DADA2 at a per sequence read depth of 32,000. As read depth doubled to 64,000, approximately 70% of ASVs were detectable, and at a read depth of 256,000, .80% of ASVs were detectable (Fig. S4C). These trends in the simulated data ...
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... by the denoising algorithm implemented in DADA2 at a per sequence read depth of 32,000. As read depth doubled to 64,000, approximately 70% of ASVs were detectable, and at a read depth of 256,000, .80% of ASVs were detectable (Fig. S4C). These trends in the simulated data sets strongly recapitulated empirical observations (Data Set S1G to J; Fig. 5B) and suggest that a majority (approximately 50% or more) of rare or low-abundance bee host-associated sequence variants are likely missed in studies sampling at a read depth of ,50,000 reads per sample. Corroborating the reported importance of sequencing depth on characterization of microbial communities (63), the number of ASVs shared ...
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... sets when considering mean 6 SE classification rates at the phylum (97.8% 6 0.5% versus 99.01% 6 0.1%; P = 0.9666), class (97.02% 6 0.6% versus 99.8% 6 0.1%; P = 0.8105), order (94.13% 6 1.2% versus 99.0% 6 0.2%; P = 0.0728), family (92.5% 6 1.5% versus 97.4% 6 0.4%; P = 0.0740), and genus (89.2% 6 1.8% versus 87.5% 6 1.0%; P = 0.9949) levels (Fig. 5A). However, at the species level, BEExact enabled strikingly higher classification rates compared to SILVA (81.0% 6 1.8% versus 28.4% 6 1.6%; P , ...
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... the true taxonomy of ASVs is unknown, classifier confidence thresholds were used as a proxy to gauge the certainty at which taxonomic predictions were made. BEExact produced significantly higher overall mean 6 SE confidence scores for species-level classifications compared to SILVA (40.59% 6 0.32% versus 25.5% 6 0.18%; P , 0.0001; Fig. 5B, Data Set S1I and ...
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... of classification rates after accounting for background differences in bee host and sample type also demonstrated that BEExact outperformed SILVA in all instances ( Fig. 5C and D). Notably, SILVA demonstrated a general trend toward higher classification on samples from eusocial corbiculate bee hosts rather than those from solitary bee origin-an effect potentially due to a higher sequence representation associated with the former as a result of the extensive characterization of social bee gut microbial ...
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... cutoff (0.1%), which can be expected simply based on the sheer reduction in classifiable sequences (Fig. S6B). Prevalence thresholds demonstrated a similar trend (Fig. S6C and D), and importantly appear to be better suited for data set noise reduction based on visualization of these relationships shown in the prevalence-abundance scatterplots in Fig. 5E and ...
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... when considering only ASVs found at a prevalence of .1.0% in any given data set, there is never an instance when applying additional abundance cutoffs would yield better classification rates without concurrently eliminating a large majority of ASVs found with a relative abundance between 0.0001 and 0.01% (Fig. 5F). In contrast, applying an abundance cutoff of 0.00001% favorably avoids the large undercut of ASVs (mostly classified by BEExact) found at low abundance and high prevalence, while reducing low-abundance ASVs which BEExact was unable to classify, and thus likely represent environmental contaminants or transient taxa. From these ...
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... classify, and thus likely represent environmental contaminants or transient taxa. From these observations and assuming an adequate sample size, a combined prevalence cutoff of #0.05% (frequency # 5 Â 10 24 ) and abundance cutoff of #0.00001% (frequency # 10 27 ) appear justified for general purposes. Taxonomic heat trees for BEExact and SILVA in Fig. 5G display the phylogenetic relatedness of ASVs remaining unclassified after applying the aforementioned cutoffs. Visual inspection demonstrated that despite classifying far more ASVs at the species level, BEExact left twice as many taxon groups (12 versus 6) completely unclassified at the family level or higher (i.e., no lower common ...
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... unclassified after applying the aforementioned cutoffs. Visual inspection demonstrated that despite classifying far more ASVs at the species level, BEExact left twice as many taxon groups (12 versus 6) completely unclassified at the family level or higher (i.e., no lower common rank members in any of the lineage were classified) compared to SILVA (Fig. ...
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... previously published 16S rRNA gene sequencing data from 50 different bee hosts across 32 independent studies. Specifically, we report that despite SILVAbased training sets offering nearly identical performance compared to that of BEExact down to the genus level (;90% or higher), classification rates dropped sharply to ;28% at the species level (Fig. 5A), which is nearly identical to the in silico estimates of ;30% using the same confidence thresholds (Fig. 4B). In contrast, BEExact enabled persistently higher classification of ;80% at the species level across most data sets (Fig. 5), which is expectedly lower than in silico estimates, but nonetheless demonstrates the habitat-specify ...
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... down to the genus level (;90% or higher), classification rates dropped sharply to ;28% at the species level (Fig. 5A), which is nearly identical to the in silico estimates of ;30% using the same confidence thresholds (Fig. 4B). In contrast, BEExact enabled persistently higher classification of ;80% at the species level across most data sets (Fig. 5), which is expectedly lower than in silico estimates, but nonetheless demonstrates the habitat-specify and comprehensiveness of the containing database reference sequences from bee host-associated microbial communities. Moreover, we identified several additional advantages, including increased classifier confidence scores when using ...
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... BEExact (indicator of accuracy), marked improvement in classification of ASVs derived from bee sample origins besides that of gut tissue (e.g., surface, food, larvae), and the classification of 845 ASVs representing novel species which were identifiable by the phylogenetically consistent placeholder names developed in this study (Table 1 and Fig. ...
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... using the DADA2 (v1.8) pipeline to infer exact amplicon sequence variants (ASVs) from amplicon data (7). Briefly, sequence reads were filtered (reads truncated after a quality score of #2 and forward/reverse reads truncated after 170/160 bases, respectively) using optimized parameter settings as recommended for the quality profiles (shown in Fig. S5). Next, sequence reads were dereplicated, denoised, and merged using DADA2 default parameters with pooled sample inference implemented for each study data set. A total of 234,567,560 raw reads were processed across the 32 data sets. Following quality assurance measures described in the DADA2 pipeline (27), ASVs were dereplicated using ...
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... how sequence depth impacts the overall quality and comprehensiveness of surveying bee-associated microbial communities. We determined that the total number of detectable ASVs per study was strongly and positively correlated (R 2 = 0.9337) with per sample read counts (i.e., read depth; Fig. S4A and B). For outgroup human gut comparisons in Fig. 5C, preprocessed ASV tables from the American Gut Project were downloaded from the ftp site (ftp://ftp.microbio.me/ ...
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The purpose of this study was to investigate the purification effect of a new adsorption material containing bioreactor and the critical role of viable but non-culturable (VBNC) bacteria in a eutrophication ecosystem. Major water quality parameters of the prepared eutrophic water were determined, and the microbial community was analyzed during 2 ye...
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... Targeted amplification of the V3-V4 region of the 16S rRNA gene was achieved using the established Bakt_341F (5'-CCTACGGGNGGCWGCAG-3') and Bakt_805R (5'-GACTACHVGGGTATCTAATCC-3') primer set, shown to be optimal for characterization of honey bee-associated bacterial communities [29]. For fungal community profiling, the ITS1f (5'-CTTGGTCATTTAGAG-GAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') modified primer set was used as specified in the Earth Microbiome Project (EMP) ITS amplicon protocol (https://earthmicrobiome.org/protocols-and-standards/its). ...
... package in R with the BEExact (v2021.0.2; https://github.com/ bdaisley/BEExact) pre-trained V3V4 database [29], which enabled specieslevel classification for 97.9% of sequences (including "bxid" annotations of taxa lacking formal nomenclature; global pairwise identity scores in Supplementary Data 1D). Fungal ASVs were assigned taxonomy in a similar manner using the UNITE database (v8.3-RefS; https://doi.org/ ...
... 10.15156/BIO/1280049), which enabled species-level classification for 35.17% of sequences (Supplementary Data 1E). To enable further predictive analysis with unclassified ASVs (belonging to bacterial and fungal dark matter) we applied phylogenetically-consistent placeholder names based on closest identities with known species representatives as previously described [29]. Briefly, all unclassified ASVs were crossreferenced at their lowest common ancestor (LCA) rank using NCBI's Bacterial 16S rRNA and Fungal ITS RefSeq Targeted Loci project (https:// ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci) ...
Managed honey bee ( Apis mellifera ) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems—standard pollen patty infusion and a novel spray-based formulation—affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus , Bifidobacterium , Lactobacillus , and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.
... Only OTUs represented by more than 0.5 % of the reads within each sample were retained. Taxonomic identifications were assigned to OTUs using the BEExact database (Daisley & Reid, 2021) with the IDTAXA algorithm (Murali, Bhargava, & Wright, 2018) using the DECIPHER R package (Wright, 2016). ...
Gut microbial communities confer protection against natural pathogens in important pollinators from the genera Bombus and Apis . In commercial species B. terrestris and B. impatiens , the microbiota increases their resistance to the common and virulent trypanosomatid parasite Crithidia bombi . However, the mechanisms by which gut microorganisms protect the host are still unknown. Here, we test two hypotheses: microbiota protect the host 1) through stimulation of its immune response or protection of the gut epithelium and 2) by competing for resources with the parasite inside the gut. To test them, we reduced the microbiota of workers and fed part of them with microbiota supplements. We exposed them to an infectious dose of C. bombi and characterised gene expression and gut microbiota composition. We examined the expression of three antimicrobial peptide (AMP) genes and Mucin-5AC , a gene with a putative role in gut epithelium protection, using qPCR. Although a protective effect against C. bombi was observed in bumblebees with supplemented microbiota, we did not observe an effect of the microbiota on gene expression that could explain alone the protective effect observed. On the other hand, we found an increased relative abundance of Lactobacillus bacteria within the gut of infected workers and a negative correlation of this genus with Gilliamella and Snodgrassella genera. Therefore, our results point to a displacement of bumblebee endosymbionts by C. bombi that might be caused by competition for space and nutrients between the parasite and the microbiota within the gut.
... Across the total sequence read data set (n = 345), many OTUs were highly inversely correlated with microbiome size (r-square values), and with one another (File S2). Much of the taxonomy returned for these suspect OTUs was associated with various environmental sources, not order Hymenoptera or the pollination environment 44 . Based on results from the combined criteria we designated the following OTUs as the top eight contaminants: Ralstonia, Caulobacter, Bradyrhizobium, Pelomonas, Cyanobacteria, Lysinibacillus, Shigella, and Nevskia, and more generally, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Burkholderiaceae, and Bradyrhizobiaceae. ...
... Microbial community analysis. The BEExact classifier placed many of the top OTUs to species level44 .From the diseased apiaries, eight OTUs contributed the majority of variation to changes in relative and absolute abundance accounting for 90% of the sequence total in the curated data set, from most to least abundant; M. plutonius, Bo. apis, A. kunkeei, E. faecalis, F. perrara, Lachnospiraceae, F. fructosus and L. apis (File S2). The top 17 ...
As essential pollinators of ecosystems and agriculture, honey bees (Apis mellifera) are host to a variety of pathogens that result in colony loss. Two highly prevalent larval diseases are European foulbrood (EFB) attributed to the bacterium Melissococcus plutonius, and Varroosis wherein larvae can be afflicted by one or more paralytic viruses. Here we used high-throughput sequencing and qPCR to detail microbial succession of larval development from six diseased, and one disease-free apiary. The disease-free larval microbiome revealed a variety of disease-associated bacteria in early larval instars, but later developmental stages were dominated by beneficial symbionts. Microbial succession associated with EFB pathology differed by apiary, characterized by associations with various gram-positive bacteria. At one apiary, diseased larvae were uniquely described as “melting and deflated”, symptoms associated with Varroosis. We found that Acute Bee Paralysis Virus (ABPV) levels were significantly associated with these symptoms, and various gram-negative bacteria became opportunistic in the guts of ABPV afflicted larvae. Perhaps contributing to disease progression, the ABPV associated microbiome was significantly depleted of gram-positive bacteria, a likely result of recent antibiotic application. Our results contribute to the understanding of brood disease diagnosis and treatment, a growing problem for beekeeping and agriculture worldwide.
... Then, paired reads were merged. Taxonomy was assigned to amplicon sequence variants (ASVs) using the BEExact database (Daisley and Reid, 2021) by assignTaxonomy. Reads belonging to mitochondria, chloroplast, and eukaryotes were excluded from further analyses ("phyloseq" package version 1.28.0 (McMurdie and Holmes, 2013), "subset_taxa" function). ...
Insect pollinators are threatened worldwide, being the exposure to multiple pesticides one of the most important stressor. The herbicide Glyphosate and the insecticide Imidacloprid are among the most used pesticides worldwide, although different studies evidenced their detrimental effects on non-target organisms. The emergence of glyphosate-resistant weeds and the recent ban of imidacloprid in Europe due to safety concerns, has prompted their replacement by new molecules, such as glufosinate-ammonium (GA) and sulfoxaflor (S). GA is a broad-spectrum and non-selective herbicide that inhibits a key enzyme in the metabolism of nitrogen, causing accumulation of lethal levels of ammonia; while sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and generates excitatory responses including tremors, paralysis and mortality. Although those molecules are being increasingly used for crop protection, little is known about their effects on non-target organisms. In this study we assessed the impact of chronic and acute exposure to sublethal doses of GA and S on honey bee gut microbiota, immunity and survival. We found GA significantly reduced the number of gut bacteria, and decreased the expression of glucose oxidase, a marker of social immunity. On the other hand, S significantly increased the number of gut bacteria altering the microbiota composition, decreased the expression of lysozyme and increased the expression of hymenoptaecin. These alterations in gut microbiota and immunocompetence may lead to an increased susceptibility to pathogens. Finally, both pesticides shortened honey bee survival and increased the risk of death. Those results evidence the negative impact of GA and S on honey bees, even at single exposition to a low dose, and provide useful information to the understanding of pollinators decline.
... Two other species clusters are related to Alphaproteobacteria: Alpha 1, which is similar to the Bartonella species, and Alpha 2 (Alpha 2-1, which is a gut specialist, and Alpha 2-2 Parasaccharibacter apium that commonly grows outside in the environment and is present in the larval gut, adult crop, nectar, honey, and hive materials, but is absent from the adult hindgut). Lactobacillus kunkei has been found in the hive, larval gut, adult crop, honey, and nectar, but not in the adult hindgut [11,23,24,60,62,63,67,[69][70][71][72]76,77,79,82,83,87,[129][130][131]. Some other clusters of Bacteroidetes may be present in low abundance, or Enterobacteriaceae may be present with common insect pathogens [59,60]. ...
... They favor the production of AMP and regulate the immune system. They play a crucial role in the detoxification of ingested toxins and in resistance against pathogens via effect on the immune system, AMP, and biomass effect [52,56,58,[65][66][67]. Due to its close contact with gut epithelium, the microbiota conveys information to the host. ...
Climate change, loss of plant biodiversity, burdens caused by new pathogens, predators, and toxins due to human disturbance and activity are significant causes of the loss of bee colonies and wild bees. The aim of this review is to highlight some possible strategies that could help develop bee resilience in facing their changing environments. Scientists underline the importance of the links between nutrition, microbiota, and immune and neuroendocrine stress resistance of bees. Nutrition with special care for plant-derived molecules may play a major role in bee colony health. Studies have highlighted the importance of pollen, essential oils, plant resins, and leaves or fungi as sources of fundamental nutrients for the development and longevity of a honeybee colony. The microbiota is also considered as a key factor in bee physiology and a cornerstone between nutrition, metabolism, growth, health, and pathogen resistance. Another stressor is the varroa mite parasite. This parasite is a major concern for beekeepers and needs specific strategies to reduce its severe impact on honeybees. Here we discuss how helping bees to thrive, especially through changing environments, is of great concern for beekeepers and scientists.
... Hence, we used the V3-V4 hypervariable region as described in Klindworth et al. (2013) who selected a primer pair targeting the V3-V4 region as the one providing the best representation of the bacterial diversity. This was more recently confirmed for a dataset of 16S rRNA gene sequences from different bee hosts (including solitary bees) during an in silico evaluation of different primer sets and demonstrated that the V1-V3 region performed most poorly whereas the V3-V4 region gave the most accurate representation of the community (Daisley and Reid, 2021). ...
... Specifically for honey bees, there is the HBDB (Newton and Roeselers, 2012), HoloBee or the BGM-Db (Zhang et al., 2019a). Recently, Daisley and Reid (2021) constructed the BEExact database based on available 16S rRNA gene sequences from bee-associated microbiota studies, including both social and solitary bee associated sequences. The database outperformed existing universal and honey bee reference databases for species level classification of ASVs retrieved from social and solitary bee associated 16S rRNA amplicon sequencing datasets. ...
The current Anthropocene epoch is characterized by major changes in the environment and biogeochemical systems at the global scale. Within this changing landscape, pollinators, including bees and wasps, are threatened by a variety of stressors, such as changes in land use, introduction of pathogens and alien species, use of pesticides, and climate change. While most species are negatively affected by the effects of the Anthropocene, some species seem to thrive well in the changing environmental conditions by expanding their geographical range. A better understanding of the effects of climate change on pollinators and their adaptability was the objective of the interuniversity ‘Climate change and effect on Pollination Services’ (CliPS) project. In order to fully understand the effect of climate change and the adaptability of pollinators, it is necessary to also study the gut microbiota. Therefore, different gut microbiota studies were performed during the present PhD, as part of the CliPS project, and aimed to characterize and identify the microbial communities associated with native solitary bees and invasive aculeate species. Solitary bees account for the majority of approximately 20,000 known bee species. However, their gut microbial communities have been rarely studied compared to those of social bees. In a first study, we described the bacterial and fungal gut microbiota of the crop, midgut, hindgut, and ovaries of four solitary bee species commonly occurring in Belgium, i.e. Andrena vaga, Anthophora plumipes, Colletes cunicularius and Osmia cornuta, using a combination of amplicon sequencing and culturomics. The microbial communities were dominated by endosymbionts of the genera Wolbachia and Spiroplasma, and environmental bacteria and yeasts with high metabolic versatility. The bacterial communities varied between gut fractions and appeared to be species-specific. Additionally, we obtained a total of 1,510 isolates from the different fractions of each bee species during a large-scale isolation campaign. The obtained reference cultures were identified at the species-level and can be used for functional analyses in future studies. In a second study, we characterized the gut microbiota of eight solitary bee species sampled in apple orchards along a latitudinal axis in Europe, thus representing a climate gradient. The aim of this study was to determine the factors involved in shaping the solitary bee bacterial and fungal communities. Host species and location were the main factors that influenced the microbiota composition of these bees, and infections with parasites led to changes in the microbial community. The bacterial community was more host specific and most strongly impacted by bee community and landscape variables, compared to the fungal community which was more strongly influenced by the local environment and climate variables. Parasite infection appeared to be host specific and resulted in a state of dysbiosis, which was characterized by increased richness and diversity and which changed the microbial community composition. The two invasive aculeate species currently occurring in Europe, the solitary bee Megachile sculpturalis and the Asian hornet Vespa velutina, belong to the few species that are thriving during the current environmental changes. They were the subjects of two additional studies of the present PhD. Both species were accidentally introduced to Europe from Asia as a result of globalization and trading of goods. They have successfully expanded their range in Europe since their arrival. Yet, the mechanisms underlying the invasion success of these species are not fully understood. We wanted to evaluate to what extent the microbial communities may contribute to the adaptability and fitness of these invasive species in the novel environments, as the gut microbiota can exhibit important functionalities and given its superior plasticity to changing conditions. We characterized the bacterial, fungal and parasite communities of M. sculpturalis sampled from native (Japan) and invaded regions (New York, USA and Marseille, France). Native, co-foraging bee species from Marseille (France) were additionally analyzed to assess the transmission of microbiota and pathogens between native and invasive bees. The gut microbiota of M. sculpturalis bees from the two invaded regions was highly similar and differed strongly from those obtained from the native region. In Marseille (France), one of the invaded regions, the microbiota of M. sculpturalis was significantly different from that of native co-foraging bees, yet native and invasive bees shared core amplicon sequence variants suggesting a potential for horizontal transmission of microbes and common environmental sources. M. sculpturalis bees examined in the present study did not harbor known bee pathogens. We proposed two hypotheses that might explain the similarity of the microbial community in bees from the two invaded regions and the absence of parasites: a common shift in gut microbiota in the invaded regions as a response to changed environmental conditions, or a founder effect in gut microbial composition caused by the introduction events. In a final study, we provided a comprehensive characterization of the bacterial communities of the gut fractions and ovaries of V. velutina through a combination of 16S rRNA amplicon sequencing and culturomics. The bacterial community was dominated by highly specialized core lactic acid bacteria (Convivina and Fructobacillus), generalist core lactic acid bacteria (Lactococcus and Lactiplantibacillus), and Sphingomonas and Spiroplasma. The four sample types revealed distinct bacterial communities with similar richness and Shannon diversity. We isolated a total of 861 isolates including two species of the core symbiont Convivina: C. intestini, which was previously isolated from a bumble bee, and the novel species C. praedatoris. We analyzed both species through comparative functional genomics and biochemical assays to describe their metabolic capabilities within the hornet gut. Our results revealed that C. intestini was adapted towards amino acid metabolism and the novel species C. praedatoris was adapted towards carbohydrate metabolism.
... For example, the honey bee AMP apidaecin exerts a minimum inhibitory concentration (MIC) of 1.56 μgml 1 on E. coli, whereas the MIC against 'core' bacterial symbionts (such as Bifidobacterium, Lactobacillus, Snodgrassella, and certain Gilliamella spp. found in social bees [56,57]) exceeds >50 μg ml 1 in most cases [58]. This preferential immune response is thought to play a key role in helping to shape their gut microbiota [59], which in effect provides an added layer of protection against infectious disease. ...
Paenibacillus larvae is a spore-forming bacterial entomopathogen and causal agent of the important honey bee larval disease, American foulbrood (AFB). Active infections by vegetative P. larvae are often deadly, highly transmissible, and incurable for colonies but, when dormant, the spore form of this pathogen can persist asymptomatically for years. Despite intensive investigation over the past century, this process has remained enigmatic. Here, we provide an up-to-date synthesis on the often overlooked microbiota factors involved in the spore-to-vegetative growth transition (corresponding with the onset of AFB disease symptoms) and offer a novel outlook on AFB pathogenesis by focusing on the 'collaborative' and 'competitive' interactions between P. larvae and other honey bee-adapted microorganisms. Furthermore, we discuss the health trade-offs associated with chronic antibiotic exposure and propose new avenues for the sustainable control of AFB via probiotic and microbiota management strategies.
... command. Sequences were aligned to BEExact (Daisley and Reid, 2021) database using the align.seqs command. ...
Honey bees exhibit an elaborate social structure based in part on an age-related division of labor. Young workers perform tasks inside the hive, while older workers forage outside the hive, tasks associated with distinct diets and metabolism. Critical to colony fitness, the work force can respond rapidly to changes in the environment or colony demography and assume emergency tasks, resulting in young foragers or old nurses. We hypothesized that both task and age affect the gut microbiota consistent with changes to host diet and physiology. We performed two experiments inducing precocious foragers and reverted nurses, then quantified tissue-specific gut microbiota and host metabolic state associated with nutrition, immunity and oxidative stress. In the precocious forager experiment, both age and ontogeny explained differences in midgut and ileum microbiota, but host gene expression was best explained by an interaction of these factors. Precocious foragers were nutritionally deficient, and incurred higher levels of oxidative damage relative to age-matched nurses. In the oldest workers, reverted nurses, the oxidative damage associated with age and past foraging was compensated by high Vitellogenin expression, which exceeded that of young nurses. Host-microbial interactions were evident throughout the dataset, highlighted by an age-based increase of Gilliamella abundance and diversity concurrent with increased carbonyl accumulation and CuZnSOD expression. The results in general contribute to an understanding of ecological succession of the worker gut microbiota, defining the species-level transition from nurse to forager.
... Bacterial sequences were collapsed at specie level using qiime-taxa-collapse, and SILVA data base ver. 132-NR-97 (Quast et al. 2013) and BEExact v2020.0.2 (Daisley and Reid 2021) were used as reference to classify them. UNITE database with all eukaryotes sequences ver. ...
Bacterial and fungal communities in the honey of sympatric populations of the bee species Apis mellifera and Melipona beecheii were profiled by amplicon sequencing of the 16S gene and the ITS of the ribosomal DNA. Results showed that the structure of the honey microbiota of these two bee species was very different from each other. Both the bacterial and fungal species in A. mellifera honey were more similar to those of A. mellifera honey reported for other parts of the world than to those in M. beecheii honey. Nevertheless, in both, the most abundant bacterial species belonged to the family Lactobacillaeae.
... Furthermore, the accurate taxonomic classification of species in complex samples remains a challenging task, which depends on many factors, such as the selected primers for the variable 16S rRNA gene region, the taxonomy assignment method, and the database used [4][5][6][7]. Ecosystem-based databases for taxonomy assignment can achieve higher resolution at the species level [8][9][10][11] as shown by an improvement in species level classification obtained with a specific and manually curated database for milk and cheese analysis as compared to more general databases [9]. ...
Background
Next-generation sequencing (NGS) methods and especially 16S rRNA gene amplicon sequencing have become indispensable tools in microbial ecology. While they have opened up new possibilities for studying microbial communities, they also have one drawback, namely providing only relative abundances and thus compositional data. Quantitative PCR (qPCR) has been used for years for the quantification of bacteria. However, this method requires the development of specific primers and has a low throughput. The constraint of low throughput has recently been overcome by the development of high-throughput qPCR (HT-qPCR), which allows for the simultaneous detection of the most prevalent bacteria in moderately complex systems, such as cheese and other fermented dairy foods. In the present study, the performance of the two approaches, NGS and HT-qPCR, was compared by analyzing the same DNA samples from 21 Raclette du Valais protected designation of origin (PDO) cheeses. Based on the results obtained, the differences, accuracy, and usefulness of the two approaches were studied in detail.
Results
The results obtained using NGS (non-targeted) and HT-qPCR (targeted) show considerable agreement in determining the microbial composition of the cheese DNA samples studied, albeit the fundamentally different nature of these two approaches. A few inconsistencies in species detection were observed, particularly for less abundant ones. The detailed comparison of the results for 15 bacterial species/groups measured by both methods revealed a considerable bias for certain bacterial species in the measurements of the amplicon sequencing approach. We identified as probable origin to this PCR bias due to primer mismatches, variations in the number of copies for the 16S rRNA gene, and bias introduced in the bioinformatics analysis.
Conclusion
As the normalized microbial composition results of NGS and HT-qPCR agreed for most of the 21 cheese samples analyzed, both methods can be considered as complementary and reliable for studying the microbial composition of cheese. Their combined application proved to be very helpful in identifying potential biases and overcoming methodological limitations in the quantitative analysis of the cheese microbiota.
