Quantitative microbiome profiling of honey bee (Apis mellifera) guts is predictive of winter colony loss in northern Virginia (USA).

For the past 15 years, the proportion of honey bee hives that fail to survive winter has averaged ~ 30% in the United States. Winter hive loss has significant negative impacts on agriculture, the economy, and ecosystems. Compared to other factors, the role of honey bee gut microbial communities in driving winter hive loss has received little attention. We investigate the relationship between winter survival and honey bee gut microbiome composition of 168 honey bees from 23 hives, nine of which failed to survive through winter 2022. We found that there was a substantial difference in the abundance and community composition of honey bee gut microbiomes based on hive condition, i.e., winter survival or failure. The overall microbial abundance, as assessed using Quantitative Microbiome Profiling (QMP), was significantly greater in hives that survived winter 2022 than in those that failed, and the average overall abundance of each of ten bacterial genera was also greater in surviving hives. There were no significant differences in alpha diversity based on hive condition, but there was a highly significant difference in beta diversity. The bacterial genera Commensalibacter and Snodgrassella were positively associated with winter hive survival. Logistic regression and random forest machine learning models on pooled ASV counts for the genus data were highly predictive of winter outcome, although model performance decreased when samples from the location with no hive failures were excluded from analysis. As a whole, our results show that the abundance and community composition of honey bee gut microbiota is associated with winter hive loss, and can potentially be used as a diagnostic tool in evaluating hive health prior to the onset of winter. Future work on the functional characterization of the honey bee gut microbiome's role in winter survival is warranted.

The fitness consequences of synonymous mutations in Escherichia coli: Experimental evidence for a pleiotropic effect of translational selection.

Codon usage bias (CUB) is a universal feature of genomes, and in most species CUB of protein coding genes is positively correlated with expression level and degree of evolutionary conservation. There is mounting experimental evidence that CUB is due in part to selection for translational efficiency and/or accuracy, i.e., translational selection. However, there is a paucity of experimental data on whether and how CUB acts in trans - does the usage of preferred codons in a highly expressed gene affect the translation of other genes by freeing up more ribosomes, thereby increasing their availability to translate all mRNA transcripts in the cell? We investigated this question by creating two extreme versions of the highly expressed Escherichia coli ß-lactamase (bla) gene, one comprised almost entirely of unpreferred codons, and a second comprised almost entirely of preferred codons. We monitored the fitness effects of these synonymous mutations over hundreds of generations in two selective environments that allowed us to disentangle translational effects acting in cis from those acting in trans. In a selective environment for maximizing translational efficiency in trans of a gene (tetA) encoding a tetracycline resistance protein, unpreferred synonymous mutations had a negative impact on long-term fitness, whereas preferred mutations had a positive impact on long-term fitness, providing strong experimental evidence for a pleiotropic effect of translational selection.

Morphology and life history divergence in cave and surface populations of Gammarus lacustris (L.).

Cave animals provide a unique opportunity to study contrasts in phenotype and life history in strikingly different environments when compared to surface populations, potentially related to natural selection. As such, we compared a permanent cave-living Gammarus lacustris (L.) population with two lake-resident surface populations analyzing morphology (eye- and antennal characters) and life-history (size at maturity, fecundity and egg-size). A part of the cytochrome c oxidase subunit I gene in the mitochondrion (COI) was analyzed to contrast genetic relationship of populations and was compared to sequences in GenBank to assess phylogeography and colonization scenarios. In the cave, a longer life cycle was implied, while surface populations seemed to have a shorter life cycle. Egg size, and size at maturity for both sexes, were larger in the cave than in surface populations, while fecundity was lower in the cave than in surface populations. The cave population had longer first- and second antennae with more articles, longer first- and second peduncles, and fewer ommatidia than surface populations. The cold low-productive cave environment may facilitate different phenotypic and life-history traits than in the warmer and more productive surface lake environments. The trait divergences among cave and surface populations resembles other cave-surface organism comparisons and may support a hypothesis of selection on sensory traits. The cave and Lake Ulvenvann populations grouped together with a sequence from Slovenia (comprising one genetic cluster), while Lake Lille Lauarvann grouped with a sequence from Ukraine (comprising another cluster), which are already recognized phylogenetic clusters. One evolutionary scenario is that the cave and surface populations were colonized postglacially around 9 000-10 000 years ago. We evaluate that an alternative scenario is that the cave was colonized during an interstadial during the last glaciation or earlier during the warm period before onset of the last glaciation.

The transcriptomes of cave and surface populations of Gammarus minus (Crustacea: Amphipoda) provide evidence for positive selection on cave downregulated transcripts.

Gammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is an excellent model for investigating evolutionary adaptation to the subterranean environment. RNA-Seq was conducted on one pair of morphologically distinct sister populations inhabiting surface and cave habitats to identify genes that were differentially expressed in the two populations, as well as to compare levels and patterns of genetic variation within and between populations. Of the 104,630 transcripts identified in the transcriptome assembly, 57% had higher average levels of expression in the cave population. After Benjamini-Hochberg correction for multiple tests, 1517 and 551 transcripts were significantly upregulated or downregulated, respectively, in the cave population, indicating an almost three-fold enrichment of cave-upregulated genes. The average level of nucleotide diversity across all transcripts was significantly lower in the cave population. Within the cave population, where the average nucleotide diversity of cave-downregulated transcripts was 75% that of the cave-upregulated transcripts, a highly significant difference, whereas within the spring population the nucleotide diversities of cave-downregulated and cave-upregulated transcripts was virtually identical. Three lines of evidence suggest that the reduced variation in cave downregulated transcripts is due to positive selection in the cave population: 1) the average neutrality index of cave-downregulated genes was < 1, consistent with positive selection, and significantly less than that of cave-upregulated genes; 2) Tajima's D was positively correlated with the cave:surface expression ratio, and 3) cave-downregulated transcripts were significantly more likely to be highly diverged from their surface homologs than cave-upregulated transcripts. Five transcripts had fixed premature termination codons in the cave population. The expression patterns and sequence variation in one such transcript, encoding the DNA repair protein photolyase, were examined in more detail and provide the first evidence for the relaxation of functional constraint in this light-dependent protein in a subterranean population.

Codon bias and gene ontology in holometabolous and hemimetabolous insects.

The relationship between preferred codon use (PCU), developmental mode, and gene ontology (GO) was investigated in a sample of nine insect species with sequenced genomes. These species were selected to represent two distinct modes of insect development, holometabolism and hemimetabolism, with an aim toward determining whether the differences in developmental timing concomitant with developmental mode would be mirrored by differences in PCU in their developmental genes. We hypothesized that the developmental genes of holometabolous insects should be under greater selective pressure for efficient translation, manifest as increased PCU, than those of hemimetabolous insects because holometabolism requires abundant protein expression over shorter time intervals than hemimetabolism, where proteins are required more uniformly in time. Preferred codon sets were defined for each species, from which the frequency of PCU for each gene was obtained. Although there were substantial differences in the genomic base composition of holometabolous and hemimetabolous insects, both groups exhibited a general preference for GC-ending codons, with the former group having higher PCU averaged across all genes. For each species, the biological process GO term for each gene was assigned that of its Drosophila homolog(s), and PCU was calculated for each GO term category. The top two GO term categories for PCU enrichment in the holometabolous insects were anatomical structure development and cell differentiation. The increased PCU in the developmental genes of holometabolous insects may reflect a general strategy to maximize the protein production of genes expressed in bursts over short time periods, e.g., heat shock proteins. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 686-698, 2015. © 2015 Wiley Periodicals, Inc.

Parallel reduction in expression, but no loss of functional constraint, in two opsin paralogs within cave populations of Gammarus minus (Crustacea: Amphipoda).

BACKGROUND: Gammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is a premier candidate for studying the evolution of troglomorphic traits such as pigmentation loss, elongated appendages, and reduced eyes. In G. minus, multiple pairs of genetically related, physically proximate cave and surface populations exist which exhibit a high degree of intraspecific morphological divergence. The morphology, ecology, and genetic structure of these sister populations are well characterized, yet the genetic basis of their morphological divergence remains unknown. RESULTS: We used degenerate PCR primers designed to amplify opsin genes within the subphylum Crustacea and discovered two distinct opsin paralogs (average inter-paralog protein divergence ≈ 20%) in the genome of three independently derived pairs of G. minus cave and surface populations. Both opsin paralogs were found to be related to other crustacean middle wavelength sensitive opsins. Low levels of nucleotide sequence variation (< 1% within populations) were detected in both opsin genes, regardless of habitat, and dN/dS ratios did not indicate a relaxation of functional constraint in the cave populations with reduced or absent eyes. Maximum likelihood analyses using codon-based models also did not detect a relaxation of functional constraint in the cave lineages. We quantified expression level of both opsin genes and found that the expression of both paralogs was significantly reduced in all three cave populations relative to their sister surface populations. CONCLUSIONS: The concordantly lowered expression level of both opsin genes in cave populations of G. minus compared to sister surface populations, combined with evidence for persistent purifying selection in the cave populations, is consistent with an unspecified pleiotropic function of opsin proteins. Our results indicate that phototransduction proteins such as opsins may have retained their function in cave-adapted organisms because they may play a pleiotropic role in other important processes that are unrelated to vision.

Experimentally increased codon bias in the Drosophila Adh gene leads to an increase in larval, but not adult, alcohol dehydrogenase activity.

Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

Molecular genetic variation and population structure in morphologically differentiated cave and surface populations of the freshwater amphipod Gammarus minus.

Gammarus minus is an important component of surface spring and cave ecosystems throughout Appalachia, and is a useful indicator of the hydrology and gene flow in freshwater communities. Gammarus minus populations occupying large cave passages (> 2 km) are usually troglomorphic, having reduced eyes, fewer ommatidia, larger body size, longer antennae, and reduced pigmentation relative to surface populations. We surveyed five cave stream and 10 surface spring populations for DNA sequence variation in the cytochromec oxidase I (COI) and internal transcribed spacer 1 (ITS-1) genes with an aim towards characterizing phylogeographical structure and comparing the nature of genetic variation in cave vs. surface populations. Although standing variation at both loci was rather low within populations, a significant degree of divergence and spatial structuring of populations was observed. Levels of genetic variation within cave and spring populations differed substantially, with caves harbouring significantly less variation at the COI locus than surface springs. Codon usage bias was significantly lower in caves, indicating that cave streams harbour smaller and/or more recently colonized populations. Overall these data indicate limited gene flow among populations and suggest that the cave populations sampled in this study are prone to bottlenecks, possibly due to larger temperature fluctuations and more frequent incidence of drought conditions associated with these particular cave habitats.

Nitrifier and denitrifier molecular operational taxonomic unit compositions from sites of a freshwater estuary of Chesapeake Bay.

Temporal and spatial changes in the molecular operational taxonomic unit (OTU) compositions of bacteria harboring genes for nitrification and denitrification were assessed using denaturing gradient gel electrophoresis (DGGE), clone-based DNA sequencing of selected PCR products, and analyses of ammonium and organic matter concentrations. Sediment, overlying water, and pore-water samples were taken from different vegetated sites of Jug Bay National Estuarine Research Reserve, Maryland, during spring, summer, and fall 2006. OTU richness and the diversities of nitrifiers and denitrifiers were assessed by the presence of bands on DGGE gels, both ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were seasonally dependent. AOB OTU richness was highest in the summer when NOB richness was decreased, whereas NOB richness was highest in the spring when AOB richness was decreased. The OTU diversities of nitrifiers did not correlate with ammonium concentrations, organic matter concentrations, or the presence of vegetation. The OTU diversities of denitrifiers possessing either the nirK or nosZ genes were not seasonally dependent but were positively correlated with organic matter content (p = 0.0015, r2 = 0.27; p < 0.0001, r2 = 0.39, respectively). Additionally, the presence of vegetation significantly enhanced nosZ species richness (Wilcoxon/Kruskal-Wallis test, p < 0.008), but this trend was not seen for nirK OTU richness. Banding patterns for nirK OTUs were more similar within sites for each season compared with any of the other genes. Over all seasons, nirK OTU richness was highest and AOB and nosZ OTU richness were lowest (Wilcoxon/Kruskal-Wallis test, p < 0.0001). High levels of sequence divergence among cloned nirK PCR products indicate a broad diversity of nirK homologs in this freshwater estuary.

Synonymous SNPs provide evidence for selective constraint on human exonic splicing enhancers.

The human SNP database was used to detect selection on 238 hexamers previously identified as exonic splicing enhancers (ESEs). We compared the distribution of the 238 putative ESEs in biallelic and triallelic SNPs within five different functional categories of the SNP database: synonymous, nonsynonymous, introns, UTRs, and nongenic SNPs. Since true ESEs do not function outside of exons, SNPs that disrupt ESE motifs were expected to be more common in nonexonic portions of the genome. Our results supported this expectation: ESEs were least prevalent within synonymous SNPs and most common in nongenic SNPs. There were approximately 11% fewer ESEs within synonymous biallelic SNPs than expected under no selective constraint. We also compared the frequency of neutral SNPs, those where neither allele was an ESE, with deleterious SNPs, those where one or more alleles was an ESE, across the five different functional classes of SNPs. In comparison with the other functional classes of SNPs, synonymous SNPs contained an excess of neutral variants (+1.64% and +6.04% for biallelic and triallelic SNPs, respectively) and a dearth of deleterious variants (-13.11% and -52.39% for biallelic and triallelic SNPs, respectively). The observed patterns were consistent with purifying selection on the 238 hexamers to maintain their function as ESEs. However, in contrast to previous work, we did not find evidence for selection to maintain ESE function at nonsynonymous SNPs because selection at the protein level probably obscured any difference at the level of ESE function.

Context-dependent codon bias and messenger RNA longevity in the yeast transcriptome.

Context-dependent codon bias and its relationship with messenger RNA (mRNA) longevity was examined in 4,648 mRNA transcripts of the Saccharomyces cerevisiae transcriptome for which mRNA half-lives have been empirically determined. Surprisingly, rare codon usage (codons used <13 times per 1,000 codons in the genome) increased with mRNA half-life. However, it is shown that this pattern was not due to preference for rare codon use within codon families containing both rare and nonrare codons. Rather, the pattern was due to an increase in the frequency of amino acids encoded solely by rare codons, and a decrease in the frequency of amino acids never encoded by rare codons, with mRNA half-life. When standardized by open reading frame length, the use of consecutive rare codons was also positively correlated with mRNA half-life. There was negative correlation between the usage of synonymous A|T dinucleotides spanning codon boundaries and mRNA half-life, despite the fact that the frequency of AT dinucleotide usage overall, and AT dinucleotide usage at other codon position contexts (e.g., 1-2, 2-3, or 3|1 total), was not correlated with mRNA half-life. The use of A|T dinucleotides at synonymous dicodon boundaries could potentially allow for more efficient 3'-5' degradation by endonucleolytic cleavage.

Experimental reduction of codon bias in the Drosophila alcohol dehydrogenase gene results in decreased ethanol tolerance of adult flies.

The ethanol tolerance of adult transgenic flies of Drosophila containing between zero and ten unpreferred synonymous mutations that reduced codon bias in the alcohol dehydrogenase (Adh) gene was assayed. As the amino acid sequences of the ADH protein were identical in the four genotypes assayed, differences in ethanol tolerance were due to differences in the abundance of ADH protein, presumably driven by the effects of codon bias on translational efficiency. The ethanol tolerance of genotypes decreased with the number of unpreferred synonymous mutations, and a positive correlation between ADH protein abundance and ethanol tolerance was observed. This work confirms that the fitness effects of unpreferred synonymous mutations that reduce codon bias in a highly expressed gene are experimentally measurable in Drosophila melanogaster.

In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein.

The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s >or= 10(-5)) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster.