David B. Carlini

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.

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 cytochrome c 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.

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.

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave‐adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog‐related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic ‘hotspots’ of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.

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.

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.