Project description:BackgroundThe chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway.ResultsTo facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways.ConclusionThese data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune-related genes that can be applied to population and systematic studies of Heliothis virescens and other Noctuidae.

Project description:There is a need to move from binary hazard assessment to more quantitative assessment of genotoxicity to better inform human health risk assessment and understand the relevance of positive in vitro genotoxicity findings. New approach methodologies (NAMs), including transcriptomic biomarkers combined with high-throughput technologies, enable the testing of a broad concentration range, which allows quantitative assessment of in vitro results. Initial work with the transcriptomic GENOMARK and TGx-DDI biomarkers demonstrate their potential use for hazard identification and chemical prioritization; however, no study has evaluated the concordance and complementarity of GENOMARK and TGx-DDI. The overall aim of this study is to examine if a combined approach of integrating both transcriptomic biomarkers for genotoxicity in human relevant HepaRGTM cells increases the certainty in hazard calls and potency rankings of chemicals. A sub-aim is to investigate whether GENOMARK is applicable to the TempO-Seq® high-throughput sequencing technology, to collect concentration-response data to rapidly perform hazard classification and potency ranking. Therefore, HepaRGTM cells were exposed to 10 chemicals (i.e. eight known in vivo genotoxicants and two in vivo non-genotoxicants) in increasing concentrations over 72h. TempO-Seq® was used to obtain concentration-response data for both biomarkers. Benchmark concentration (BMC) modelling of chemicals that were classified positive was conducted to obtain BMCs and transcriptomic points of departure (tPODs) for potency ranking. The results confirm that GENOMARK is applicable to TempO-Seq® since it achieved 100% predictive accuracy. In addition, a high concordance was observed in the hazard classifications and potency rankings between both biomarkers. Overall, our findings show that in vitro transcriptomic data can be used to rapidly and effectively identify genotoxic hazards while simultaneously providing additional insights on potency that is more informative in a modern hazard assessment paradigm. The results of this case study support the high value of integrating these NAMs in a weight of evidence evaluation of genotoxicity using the important human-liver cell line.

Project description:BackgroundMoths rely heavily on pheromone communication for mate finding. The pheromone components of most moths are modified from the products of normal fatty acid metabolism by a set of tissue-specific enzymes. The turnip moth, Agrotis segetum uses a series of homologous fatty-alcohol acetate esters ((Z)-5-decenyl, (Z)-7-dodecenyl, and (Z)-9 tetradecenyl acetate) as its sex pheromone components. The ratio of the components differs between populations, making this species an interesting subject for studies of the enzymes involved in the biosynthetic pathway and their influence on sex pheromone variation.ResultsIllumina sequencing and comparative analysis of the transcriptomes of the pheromone gland and abdominal epidermal tissue, enabled us to identify genes coding for putative key enzymes involved in the pheromone biosynthetic pathway, such as fatty acid synthase, β-oxidation enzymes, fatty-acyl desaturases (FAD), fatty-acyl reductases (FAR), and acetyltransferases. We functionally assayed the previously identified ∆11-desaturase [GenBank: ES583599, JX679209] and FAR [GenBank: JX679210] and candidate acetyltransferases (34 genes) by heterologous expression in yeast. The functional assay confirmed that the ∆11-desaturase interacts with palmitate and produces (Z)-11-hexadecenoate, which is the common unsaturated precursor of three homologous pheromone component acetates produced by subsequent chain-shortening, reduction and acetylation. Much lower, but still visible, activity on 14C and 12C saturated acids may account for minor pheromone compounds previously observed in the pheromone gland. The FAR characterized can operate on various unsaturated fatty acids that are the immediate acyl precursors of the different A. segetum pheromone components. None of the putative acetyltransferases that we expressed heterologously did acetylate any of the fatty alcohols tested as substrates.ConclusionsThe massive sequencing technology generates enormous amounts of candidate genes potentially involved in pheromone biosynthesis but testing their function by heterologous expression or gene silencing is a bottleneck. We confirmed the function of a previously identified desaturase gene and a fatty-acyl reductase gene by heterologous expression, but the acetyltransferase postulated to be involved in pheromone biosynthesis remains illusive, in spite of 34 candidates being assayed. We also generated lists of gene candidates that may be useful for characterizing the acetyl-CoA carboxylase, fatty acid synthetase and β-oxidation enzymes.

Project description:Common bottlenose dolphins (Tursiops truncatus), produce a wide variety of vocal emissions for communication and echolocation, of which the pulsed repertoire has been the most difficult to categorize. Packets of high repetition, broadband pulses are still largely reported under a general designation of burst-pulses, and traditional attempts to classify these emissions rely mainly in their aural characteristics and in graphical aspects of spectrograms. Here, we present a quantitative analysis of pulsed signals emitted by wild bottlenose dolphins, in the Sado estuary, Portugal (2011-2014), and test the reliability of a traditional classification approach. Acoustic parameters (minimum frequency, maximum frequency, peak frequency, duration, repetition rate and inter-click-interval) were extracted from 930 pulsed signals, previously categorized using a traditional approach. Discriminant function analysis revealed a high reliability of the traditional classification approach (93.5% of pulsed signals were consistently assigned to their aurally based categories). According to the discriminant function analysis (Wilk's Λ = 0.11, F3, 2.41 = 282.75, P < 0.001), repetition rate is the feature that best enables the discrimination of different pulsed signals (structure coefficient = 0.98). Classification using hierarchical cluster analysis led to a similar categorization pattern: two main signal types with distinct magnitudes of repetition rate were clustered into five groups. The pulsed signals, here described, present significant differences in their time-frequency features, especially repetition rate (P < 0.001), inter-click-interval (P < 0.001) and duration (P < 0.001). We document the occurrence of a distinct signal type-short burst-pulses, and highlight the existence of a diverse repertoire of pulsed vocalizations emitted in graded sequences. The use of quantitative analysis of pulsed signals is essential to improve classifications and to better assess the contexts of emission, geographic variation and the functional significance of pulsed signals.

Dataset Information

Qualitative and quantitative analysis of chemicals emitted from the pheromone gland of individual Heliothis subflexa females.

Publications

Qualitative and quantitative analysis of chemicals emitted from the pheromone gland of individual Heliothis subflexa females.

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