Mite non-reproduction is not a consequence of the brood removal behavior of varroa sensitive hygiene honey bee colonies (Apis mellifera).

A sustainable solution to the global threat of the Varroa destructor mite is the selection of varroa-resistant honey bee (Apis mellifera) colonies. Both "mite non-reproduction" (MNR) and "varroa sensitive hygiene" (VSH) appear to be promising selection traits for achieving the goal of a resistant honey bee. MNR describes colonies that have a high number of non-reproductive mites (no offspring, no males, or delayed development of mite offspring). High numbers of non-reproductive mites have been observed in selected colonies, but the mechanism behind this trait has not yet been identified. The specialized hygienic behavior of selected honey bees, called VSH, is the removal of varroa-infested brood. These traits were thought to be linked by VSH bees preferentially removing reproductive varroa females leaving only non-reproductive mites behind in cells and thus creating colonies with high levels of MNR. To further investigate this link, we used an experimental setup and data sets from a four-year selection project designed to breed for MNR and VSH colonies. In addition, we sought to answer the question of whether non-reproductive mites are a direct consequence of worker removal behavior. To test this, we artificially induced removal behavior, and after providing the mite with enough time to re-enter another cell, we opened all capped cells, relocated the mites, and evaluated their reproduction. As shown in previous studies and in this study, VSH had no effect on MNR levels. Also, the induced removal behavior did not lead to non-reproduction in the subsequent reproductive cycle post interruption. We thus concluded that breeding for non-reproductive mites does not automatically breed for VSH behavior and worker removal behavior does not cause subsequent reproductive failure of the mites forced to flee and find a new cell for reproduction.

Complex European invasion history of Anoplophora glabripennis (Motschulsky): new insights in its population genomic differentiation using genotype-by-sequencing.

Anthropogenic activities like trade facilitate increasing rates of biological invasions. Asian long-horned beetle (ALB), which is naturally distributed in eastern Asia (China, Korean peninsula), was introduced via wood packing materials (WPM) used in trade to North America (1996) and Europe (2001). We used 7810 single nucleotide polymorphisms (SNPs) derived by a genotype-by-sequencing (GBS) approach to decipher the introduction patterns into Europe. This is applied for the first time on European ALB outbreaks from Germany, Switzerland, and Italy, both from still active and already eradicated infestations. The genome-wide SNPs detected signs of small and highly structured populations within Europe, showing clear founder effects. The very high population differentiation is presumably derived from multiple independent introductions to Europe, which are spatially restricted in mating. By admixture and phylogenetic analyses, some cases of secondary dispersal were observed. Furthermore, some populations suggest admixture, which might have been originated by either multiple introductions from different sources into the new sites or recurrent introductions from an admixed source population. Our results confirmed a complex invasion history of the ALB into Europe and the usability of GBS obtained SNPs in invasion science even without source populations.

Parasites, depredators, and limited resources as potential drivers of winter mortality of feral honeybee colonies in German forests.

Wild honeybees (Apis mellifera) are considered extinct in most parts of Europe. The likely causes of their decline include increased parasite burden, lack of high-quality nesting sites and associated depredation pressure, and food scarcity. In Germany, feral honeybees still colonize managed forests, but their survival rate is too low to maintain viable populations. Based on colony observations collected during a monitoring study, data on parasite prevalence, experiments on nest depredation, and analyses of land cover maps, we explored whether parasite pressure, depredation or expected landscape-level food availability explain feral colony winter mortality. Considering the colony-level occurrence of 18 microparasites in the previous summer, colonies that died did not have a higher parasite burden than colonies that survived. Camera traps installed at cavity trees revealed that four woodpecker species, great tits, and pine martens act as nest depredators. In a depredator exclusion experiment, the winter survival rate of colonies in cavities with protected entrances was 50% higher than that of colonies with unmanipulated entrances. Landscapes surrounding surviving colonies contained on average 6.4 percentage points more cropland than landscapes surrounding dying colonies, with cropland being known to disproportionately provide forage for bees in our study system. We conclude that the lack of spacious but well-protected nesting cavities and the shortage of food are currently more important than parasites in limiting populations of wild-living honeybees in German forests. Increasing the density and diversity of large tree cavities and promoting bee forage plants in forests will probably promote wild-living honeybees despite parasite pressure.

Positive selection in cytochrome P450 genes is associated with gonad phenotype and mating strategy in social bees.

The honey bee, Apis mellifera differs from all other social bees in its gonad phenotype and mating strategy. Honey bee queens and drones have tremendously enlarged gonads, and virgin queens mate with several males. In contrast, in all the other bees, the male and female gonads are small, and the females mate with only one or very few males, thus, suggesting an evolutionary and developmental link between gonad phenotype and mating strategy. RNA-seq comparisons of A. mellifera larval gonads revealed 870 genes as differentially expressed in queens versus workers and drones. Based on Gene Ontology enrichment we selected 45 genes for comparing the expression levels of their orthologs in the larval gonads of the bumble bee Bombus terrestris and the stingless bee, Melipona quadrifasciata, which revealed 24 genes as differentially represented. An evolutionary analysis of their orthologs in 13 solitary and social bee genomes revealed four genes with evidence of positive selection. Two of these encode cytochrome P450 proteins, and their gene trees indicated a lineage-specific evolution in the genus Apis, indicating that cytochrome P450 genes may be involved in the evolutionary association of polyandry and the exaggerated gonad phenotype in social bees.

The impact of dietary calcium and phosphorus on mitochondrial-linked gene expression in five tissues of laying hens.

Mitochondria and the energy metabolism are linked to both, the availability of Ca and P to provide the eukaryotic cell with energy. Both minerals are commonly used supplements in the feed of laying hens but little is known about the relationship between the feed content, energy metabolism and genetic background. In this study, we provide a large-scaled gene expression analysis of 31 mitochondrial and nuclear encoded genes in 80 laying hens in the context of dietary P and Ca concentrations. The setup included five tissues and gene expression was analysed under four different diets of recommended and reduced Ca and P concentrations. Our study shows, that mitochondrial gene expression is reacting to a reduction in P and that an imbalance of the nutrients has a higher impact than a combined reduction. The results suggest, that both strains (Lohmann Brown and Lohmann Selected Leghorn) react in a similar way to the changes and that a reduction of both nutrients might be possible without crucial influence on the animals' health or gene expression.

Highly transmissible cytoplasmic incompatibility by the extracellular insect symbiont Spiroplasma.

Cytoplasmic incompatibility (CI) is a form of reproductive manipulation caused by maternally inherited endosymbionts infecting arthropods, like Wolbachia, whereby matings between infected males and uninfected females produce few or no offspring. We report the discovery of a new CI symbiont, a strain of Spiroplasma causing CI in the parasitoid wasp Lariophagus distinguendus. Its extracellular occurrence enabled us to establish CI in uninfected adult insects by transferring Spiroplasma-infected hemolymph. We sequenced the CI-Spiroplasma genome and did not find any homologues of any of the cif genes discovered to cause CI in Wolbachia, suggesting independent evolution of CI. Instead, the genome contains other potential CI-causing candidate genes, such as homologues of high-mobility group (HMG) box proteins that are crucial in eukaryotic development but rare in bacterial genomes. Spiroplasma's extracellular nature and broad host range encompassing medically and agriculturally important arthropods make it a promising tool to study CI and its applications.

Sex- and caste-specific transcriptomes of larval honey bee (Apis mellifera L.) gonads: DMRT A2 and Hsp83 are differentially expressed and regulated by juvenile hormone.

The gonads of honey bee, Apis mellifera, queens and drones are each composed of hundreds of serial units, the ovarioles and testioles, while the ovaries of the adult subfertile workers consist of only few ovarioles. We performed a comparative RNA-seq analysis on early fifth-instar (L5F1) larval gonads, which is a critical stage in gonad development of honey bee larvae. A total of 1834 genes were identified as differentially expressed (Padj < 0.01) among the three sex and caste phenotypes. The Gene Ontology analysis showed significant enrichment for metabolism, protein or ion binding, and oxidoreductase activity, and a KEGG analysis revealed metabolic pathways as enriched. In a principal component analysis for the total transcriptomes and hierarchical clustering of the DEGs, we found higher similarity between the queen and worker ovary transcriptomes compared to the drone testis, despite the onset of programmed cell death in the worker ovaries. Four DEGs were selected for RT-qPCR analyses, including their response to juvenile hormone (JH), which is a critical factor in the caste-specific development of the ovaries. Among these, DMRT A2 and Hsp83 were found upregulated by JH and, thus, emerged as potential molecular markers for sex- and caste-specific gonad development in honey bees.

The dynamics of mitochondrial-linked gene expression among tissues and life stages in two contrasting strains of laying hens.

The cellular energy metabolism is one of the most conserved processes, as it is present in all living organisms. Mitochondria are providing the eukaryotic cell with energy and thus their genome and gene expression has been of broad interest for a long time. Mitochondrial gene expression changes under different conditions and is regulated by genes encoded in the nucleus of the cell. In this context, little is known about non-model organisms and we provide the first large-scaled gene expression analysis of mitochondrial-linked genes in laying hens. We analysed 28 mitochondrial and nuclear genes in 100 individuals in the context of five life-stages and strain differences among five tissues. Our study showed that mitochondrial gene expression increases during the productive life span, and reacts tissue and strain specific. In addition, the strains react different to potential increased oxidative stress, resulting from the increase in mitochondrial gene expression. The results suggest that the cellular energy metabolism as part of a complex regulatory system is strongly affected by the productive life span in laying hens and thus partly comparable to model organisms. This study provides a starting point for further analyses in this field on non-model organisms, especially in laying-hens.

Global population genetic structure and demographic trajectories of the black soldier fly, Hermetia illucens.

BACKGROUND: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents. RESULTS: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions. CONCLUSIONS: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector.

Insights into the Mitochondrial and Nuclear Genome Diversity of Two High Yielding Strains of Laying Hens.

Mitochondria are essential components of eukaryotes as they are involved in several organismic key processes such as energy production, apoptosis and cell growth. Despite their importance for the metabolism and physiology of all eukaryotic organisms, the impact of mitochondrial haplotype variation has only been studied for very few species. In this study we sequenced the mitochondrial genome of 180 individuals from two different strains of laying hens. The resulting haplotypes were combined with performance data such as body weight, feed intake and phosphorus utilization to assess their influence on the hens in five different life stages. After detecting a surprisingly low level of genetic diversity, we investigated the nuclear genetic background to estimate whether the low mitochondrial diversity is representative for the whole genetic background of the strains. Our results highlight the need for more in-depth investigation of the genetic compositions and mito-nuclear interaction in individuals to elucidate the basis of phenotypic performance differences. In addition, we raise the question of how the lack of mitochondrial variation developed, since the mitochondrial genome represents genetic information usually not considered in breeding approaches.

Changes of microorganism composition in fresh and stored bee pollen from Southern Germany.

Analysis of plant pollen can provide valuable insights into the existing spectrum of microorganisms in the environment. When harvesting bee-collected pollen as a dietary supplement for human consumption, timely preservation of the freshly collected pollen is fundamental for product quality. Environmental microorganisms contained in freshly collected pollen can lead to spoilage by degradation of pollen components. In this study, freshly collected bee pollen was sampled at different locations and stored under various storage conditions to examine the hypothesis that storage conditions may have an effect on the composition of microorganisms in pollen samples. The samples were analyzed using 16S and 18S amplicon sequencing and characterized by palynological analysis. Interestingly, the bacterial communities between pollen samples from different locations varied only slightly, whereas for fungal community compositions, this effect was substantially increased. Further, we noticed that fungal communities in pollen are particularly sensitive to storage conditions. The fungal genera proportion Cladosporium and Mycosphaerella decreased, while Zygosaccharomyces and Aspergillus increased during storage. Aspergillus and Zygosaccharomyces fractions increased during storage at 30 °C, which could negatively impact the pollen quality if it is used as a dietary supplement.

Disentangling Ethiopian Honey Bee (Apis mellifera) Populations Based on Standard Morphometric and Genetic Analyses.

The diversity and local differentiation of honey bees are subjects of broad general interest. In particular, the classification of Ethiopian honey bees has been a subject of debate for decades. Here, we conducted an integrated analysis based on classical morphometrics and a putative nuclear marker (denoted r7-frag) for elevational adaptation to classify and characterize these honey bees. Therefore, 660 worker bees were collected out of 66 colonies from highland, midland and lowland agro-ecological zones (AEZs) and were analyzed in reference to populations from neighboring countries. Multivariate morphometric analyses show that our Ethiopian samples are separate from Apis mellifera scutellata, A. m. jemenitica, A. m. litorea and A. m. monticola, but are closely related to A. m. simensis reference. Linear discriminant analysis showed differentiation according to AEZs in the form of highland, midland and lowland ecotypes. Moreover, size was positively correlated with elevation. Similarly, our Ethiopian samples were differentiated from A. m. monticola and A. m. scutellata based on r7-frag. There was a low tendency towards genetic differentiation between the Ethiopian samples, likely impacted by increased gene flow. However, the differentiation slightly increased with increasing elevational differences, demonstrated by the highland bees that showed higher differentiation from the lowland bees (FST = 0.024) compared to the midland bees (FST = 0.015). An allelic length polymorphism was detected (denoted as d) within r7-frag, showing a patterned distribution strongly associated with AEZ (X2 = 11.84, p < 0.01) and found predominantly in highland and midland bees of some pocket areas. In conclusion, the Ethiopian honey bees represented in this study are characterized by high gene flow that suppresses differentiation.

Global allele polymorphism indicates a high rate of allele genesis at a locus under balancing selection.

When selection favours rare alleles over common ones (balancing selection in the form of negative frequency-dependent selection), a locus may maintain a large number of alleles, each at similar frequency. To better understand how allelic richness is generated and maintained at such loci, we assessed 201 sequences of the complementary sex determiner (csd) of the Asian honeybee (Apis cerana), sampled from across its range. Honeybees are haplodiploid; hemizygotes at csd develop as males and heterozygotes as females, while homozygosity is lethal. Thus, csd is under strong negative frequency-dependent selection because rare alleles are less likely to end up in the lethal homozygous form. We find that in A. cerana, as in other Apis, just a few amino acid differences between csd alleles in the hypervariable region are sufficient to trigger female development. We then show that while allelic lineages are spread across geographical regions, allelic differentiation is high between populations, with most csd alleles (86.3%) detected in only one sample location. Furthermore, nucleotide diversity in the hypervariable region indicates an excess of recently arisen alleles, possibly associated with population expansion across Asia since the last glacial maximum. Only the newly invasive populations of the Austral-Pacific share most of their csd alleles. In all, the geographic patterns of csd diversity in A. cerana indicate that high mutation rates and balancing selection act together to produce high rates of allele genesis and turnover at the honeybee sex locus, which in turn leads to its exceptionally high local and global polymorphism.

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.

Phytate degradation, myo-inositol release, and utilization of phosphorus and calcium by two strains of laying hens in five production periods.

The objective of this study was to compare 2 laying hen strains in 5 production periods regarding phytase activity, phytate (InsP6) degradation, and myo-inositol (MI) release in the digestive tract and phosphorus (P) and calcium (Ca) utilization. One offspring of 10 nonrelated roosters per strain (Lohmann Brown-classic (LB) or Lohmann LSL-classic (LSL)) was placed in one of 20 metabolic units in a completely randomized block design in week 8, 14, 22, 28, and 58 of life. All hens were fed the same corn and soybean meal-based diet at one time, but the diet composition was adjusted to the requirements in the respective period. For 4 consecutive days, excreta were collected quantitatively at 24-hour intervals. In week 10, 16, 24, 30, and 60, the blood plasma, digesta of crop, gizzard, jejunum, ileum, and ceca, and mucosa of the jejunum was collected. The concentration of inorganic P in the blood plasma was higher in LB than in LSL hens (P = 0.026). Plasma Ca concentrations increased with each period (P < 0.001) in both strains. In jejunum digesta, the MI concentration did not differ between strains, but InsP6 concentration was higher in LB than in LSL hens (P = 0.002) and the highest in week 30 and 60. Total phosphatase and phytase activities were higher in LB than in LSL hens (P ≤ 0.009). Period effects were also significant for these enzymes. Concentrations of some constituents of the cecal content were different between the strains. The MI concentration in the egg albumen and yolk was higher in LB than in LSL hens. Differences in InsP6- and MI-related metabolism of the 2 hen strains existed. These differences were partly dependent of the period. Especially, week 24 was a period of remarkable change of metabolism. Great differences also existed among individuals, making it worth to have a closer look at the metabolism of individuals in addition to evaluating treatment means. Further studies on metabolic, genetic, and microbiome level may help explain these differences.

Phytate Degradation, Transcellular Mineral Transporters, and Mineral Utilization by Two Strains of Laying Hens as Affected by Dietary Phosphorus and Calcium.

Laying hens require less phosphorus (P) but markedly more calcium (Ca) in their diet than broilers. These differences may cause more distinct interactions with phytate degradation and utilization of minerals in laying hens than those in broilers. The objective of the study was to characterize intestinal phytate degradation, ileal transcript copy numbers of transcellular Ca and P transporters, and mineral utilization by two laying hen strains fed with standard or reduced levels of dietary Ca and P at the laying peak. The strains showed differences regarding several traits driving Ca and P metabolism along the digestive tract. Thus, the two strains may use different mechanisms to meet their respective P demand, i.e., via effective phytate degradation and transcellular transport. Clear effects of the Ca level on myo-inositol concentrations and mineral utilization revealed the significance of this element for the measured traits. The absence of P-mediated effects confirmed the findings of several studies recommending that P concentrations used in laying hen feeds are too high. Differences were noted between individuals within one treatment. The next step would be to evaluate the data in individual birds to identify birds that better cope with a challenging diet.

The trisaccharide melezitose impacts honey bees and their intestinal microbiota.

In general, honey bees (Apis mellifera L.) feed on honey produced from collected nectar. In the absence of nectar, during certain times of the year or in monocultural landscapes, honey bees forage on honeydew. Honeydew is excreted by different herbivores of the order Hemiptera that consume phloem sap of plant species. In comparison to nectar, honeydew is composed of a higher variety of sugars and additional sugars with higher molecular weight, like the trisaccharide melezitose that can be a major constituent of honeydew. However, melezitose-containing honey is known to cause malnutrition in overwintering honey bees. Following the hypothesis that melezitose may be the cause for the so called 'honeydew flow disease', three independent feeding experiments with caged bees were conducted in consecutive years. Bees fed with melezitose showed increased food uptake, higher gut weights and elevated mortality compared to bees fed a control diet. Moreover, severe disease symptoms, such as swollen abdomen, abdomen tipping and impaired movement were observed in melezitose-fed bees. 16S-amplicon sequencing indicated that the melezitose diet changed the species composition of the lactic acid bacteria community within the gut microbiota. Based on these results, we conclude that melezitose cannot be easily digested by the host and may accumulate in the hindgut. Within cages or during winter, when there is no opportunity for excretion, the accumulated melezitose can cause severe intestinal symptoms and death of the bees, probably as result of poor melezitose metabolism capabilities in the intestinal microbiota. These findings confirm the causal relation between the trisaccharide melezitose and the honeydew flow disease and indicate a possible mechanism of pathogenesis.

Positive selection of digestive proteases in Daphnia: A mechanism for local adaptation to cyanobacterial protease inhibitors.

Due to the combined effects of global warming and eutrophication, the frequency of deleterious cyanobacterial blooms in freshwater ecosystems has increased. In line with this, local adaptation of the aquatic keystone herbivore Daphnia to cyanobacteria has received major attention. Besides microcystins, the most frequent cyanobacterial secondary metabolites in such blooms are protease inhibitors (PIs). Recently, it has been shown that a protease gene showed copy number variation between four D. magna populations that differed in tolerance to PIs. From that study, we chose two distinct populations of D. magna which had or had not coexisted with cyanobacteria in the past. By calculating FST values, we found that the two populations were genetically more distant in the protease loci than in neutral loci. Population genetic tests applied to the tolerant population revealed that positive selection was most probably acting on the gene loci of the digestive protease CT448 and CT802. We conclude that the selection of digestive proteases and subsequent reduction in copy number is the molecular basis of evolutionary changes leading to local adaptation to PIs.