Low Diversity in theMitogenome of Sperm Whales Revealed by Next-Generation Sequencing

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20 mitogenomes from 17 sperm whales representative of worldwide diversity using Next Generation Sequencing (NGS) technologies (Illumina GAIIx, Roche 454 GS Junior). Resequencing of three individuals with both NGS platforms and partial Sanger sequencing showed low discrepancy rates (454-Illumina: 0.0071%; Sanger-Illumina: 0.0034%; and Sanger-454: 0.0023%) confirming suitability of both NGS platforms for investigating low mitogenomic diversity. Using the 17 sperm whale mitogenomes in a phylogenetic reconstruction with 41 other species, including 11 new dolphin mitogenomes, we tested two hypotheses for the low CR diversity. First, the hypothesis that CR-specific constraints have reduced diversity solely in the CR was rejected as diversity was low throughout the mitogenome, not just in the CR [overall diversity pi]=0.096%; protein-coding 3rd codon =0.22%; CR =0.35%), and CR phylogenetic signal was congruent with protein-coding regions. Second, the hypothesis that slow substitution rates reduced diversity throughout the sperm whale mitogenome was rejected as sperm whales had significantly higher rates of CR evolution and no evidence of slow coding region evolution relative to other cetaceans. The estimated time to most recent common ancestor for sperm whale mitogenomes was 72,800 to 137,400 years ago (95% highest probability density interval), consistent with previous hypotheses of a bottleneck or selective sweep as likely causes of low mitogenome diversity.Keywords: Nucleotide diversity, Mitochondrial genome, Population genetics, Cetacean, Substitution rates, Physeter macrocephalus, mtDNA, Bayesian phylogenetic

Sex pheromone evolution is associated with differential regulation of the same desaturase gene in two genera of leafroller moths.

peer reviewedChemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation

Sequence Comparisons of Odorant Receptors among Tortricid Moths Reveal Different Rates of Molecular Evolution among Family Members

In insects, odorant receptors detect volatile cues involved in behaviours such as mate recognition, food location and oviposition. We have investigated the evolution of three odorant receptors from five species within the moth genera Ctenopseustis and Planotrotrix, family Tortricidae, which fall into distinct clades within the odorant receptor multigene family. One receptor is the orthologue of the co-receptor Or83b, now known as Orco (OR2), and encodes the obligate ion channel subunit of the receptor complex. In comparison, the other two receptors, OR1 and OR3, are ligand-binding receptor subunits, activated by volatile compounds produced by plants - methyl salicylate and citral, respectively. Rates of sequence evolution at non-synonymous sites were significantly higher in OR1 compared with OR2 and OR3. Within the dataset OR1 contains 109 variable amino acid positions that are distributed evenly across the entire protein including transmembrane helices, loop regions and termini, while OR2 and OR3 contain 18 and 16 variable sites, respectively. OR2 shows a high level of amino acid conservation as expected due to its essential role in odour detection; however we found unexpected differences in the rate of evolution between two ligand-binding odorant receptors, OR1 and OR3. OR3 shows high sequence conservation suggestive of a conserved role in odour reception, whereas the higher rate of evolution observed in OR1, particularly at non-synonymous sites, may be suggestive of relaxed constraint, perhaps associated with the loss of an ancestral role in sex pheromone reception

Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation

Sequence variation determining stereochemistry of a delta-11 desaturase active in moth sex pheromone biosynthesis

A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution

Crystal structure of Epiphyas postvittana pheromone binding protein 3

The insect olfactory system operates as a well-choreographed ensemble of molecules which functions to selectively translate volatile chemical messages present in the environment into neuronal impulses that guide insect behaviour. Of these molecules, binding proteins are believed to transport hydrophobic odorant molecules across the aqueous lymph present in antennal sensilla to receptors present in olfactory sensory neurons. Though the exact mechanism through which these proteins operate is still under investigation, these carriers clearly play a critical role in determining what an insect can smell. Binding proteins that transport important sex pheromones are colloquially named pheromone binding proteins (PBPs). Here, we have produced a functional recombinant PBP from the horticultural pest, Epiphyas postvittana (EposPBP3), and experimentally solved its apo-structure through X-ray crystallography to a resolution of 2.60 Å. Structural comparisons with related lepidopteran PBPs further allowed us to propose models for the binding of pheromone components to EposPBP3. The data presented here represent the first structure of an olfactory-related protein from the tortricid family of moths, whose members cause billions of dollars in losses to agricultural producers each year. Knowledge of the structure of these important proteins will allow for subsequent studies in which novel, olfactory molecule-specific insecticides can be developed

Structure of an antennally-expressed carboxylesterase suggests lepidopteran odorant degrading enzymes are broadly tuned

Insects rely on the detection of chemical cues present in the environment to guide their foraging and reproductive behaviour. As such, insects have evolved a sophisticated chemical processing system in their antennae comprised of several types of olfactory proteins. Of these proteins, odorant degrading enzymes are responsible for metabolising the chemical cues within the antennae, thereby maintaining olfactory system function. Members of the carboxyl/cholinesterase gene family are known to degrade odorant molecules with acetate-ester moieties that function as host recognition cues or sex pheromones, however, their specificity for these compounds remains unclear. Here, we evaluate expression levels of this gene family in the light-brown apple moth, Epiphyas postvittana, via RNAseq and identify putative odorant degrading enzymes. We then solve the apo-structure for EposCCE24 by X-ray crystallography to a resolution of 2.43 Å and infer substrate specificity based on structural characteristics of the enzyme's binding pocket. The specificity of EposCCE24 was validated by testing its ability to degrade biologically relevant and non-relevant sex pheromone components and plant volatiles using GC–MS. We found that EposCCE24 is neither capable of discriminating between linear acetate-ester odorant molecules of varying chain length, nor between molecules with varying double bond positions. EposCCE24 efficiently degraded both plant volatiles and sex pheromone components containing acetate-ester functional groups, confirming its role as a broadly-tuned odorant degrading enzyme in the moth olfactory organ

Predicted transmembrane topologies of OR1 (A), OR2 (B) and OR3 (C), with variable sites highlighted in red.

<p>The double line indicates the membrane region, with extracellular and cytoplasmic sides labelled.</p

Estimating the number of whales entering trade using DNA profiling and capture-recapture analysis of market products

Surveys of commercial markets combined with molecular taxonomy (i.e. molecular monitoring) provide a means to detect products from illegal, unregulated and/or unreported (IUU) exploitation, including the sale of fisheries bycatch and wild meat (bushmeat). Capture-recapture analyses of market products using DNA profiling have the potential to estimate the total number of individuals entering the market. However, these analyses are not directly analogous to those of living individuals because a ‘market individual’ does not die suddenly but, instead, remains available for a time in decreasing quantities, rather like the exponential decay of a radioactive isotope. Here we use mitochondrial DNA (mtDNA) sequences and microsatellite genotypes to individually identify products from North Pacific minke whales (Balaenoptera acutorostrata ssp.) purchased in 12 surveys of markets in the Republic of (South) Korea from 1999 to 2003. By applying a novel capture-recapture model with a decay rate parameter to the 205 unique DNA profiles found among 289 products, we estimated that the total number of whales entering trade across the five-year survey period was 827 (SE, 164; CV, 0.20) and that the average ‘half-life’ of products from an individual whale on the market was 1.82 months (SE, 0.24; CV, 0.13). Our estimate of whales in trade (reflecting the true numbers killed) was significantly greater than the officially reported bycatch of 458 whales for this period. This unregulated exploitation has serious implications for the survival of this genetically distinct coastal population. Although our capture-recapture model was developed for specific application to the Korean whale-meat markets, the exponential decay function could be modified to improve the estimates of trade in other wildmeat or fisheries markets or abundance of living populations by noninvasive genotyping

Likelihood ratio tests between concatenated sequences.

<p>model A assumes complete homogeneity among genes, while model B assumes different substitution rates but the same pattern of nucleotide substitution for each gene.</p