Bisphenol A (BPA) is an emerging pollutant of global concern. Viviparous fish Goodea atripinnis is endemic to the Central Mexican Plateau where BPA was detected; however, few studies examined the influence of this chemical on native viviparous fish. The effects of BPA (sublethal dose) were determined on DNA integrity and Foxl2 expression in G. atripinnis gonads, and interactions of BPA with FOXL2 protein. Genotoxicity analysis revealed that % comets, at 14 and 28 days and comet tail length (at 14 days) were significantly higher in exposed compared to controls. In general, the % DNA tail was not markedly higher in BPA-treated fish; however, tail moment related to tail length exhibited significant increases in DNA damage. RT-qPCR assays showed Foxl2 overexpression after 14 and 28 days of exposure in females; while in males, Foxl2 was overexpressed after 28 days. In silico analysis demonstrated that BPA interacted with seven residues located in FOXL2 homeodomain. In summary, sublethal BPA doses induced DNA damage and changes in Foxl2 expression in gonadal cells of G. atripinnis, which may adversely affect reproduction in BPA-exposed wild populations. Foxl2 overexpression and BPA-FOXL2 interaction suggested alterations in processes involving Foxl2. Viviparous fish may thus serve as potential non-conventional models for assessing pollutants effects.

Dendroctonus bark beetles are the most destructive agents in coniferous forests. These beetles come into contact with the toxic compounds of their host's chemical defenses throughout their life cycle, some of which are also used by the insects as kairomones to select their host trees during the colonization process. However, little is known about the molecular mechanisms by which the insects counteract the toxicity of these compounds. Here, two sibling species of bark beetles, D. valens and D. rhizophagus, were stimulated with vapors of a blend of their main kairomones (α-pinene, β-pinene and 3-carene), in order to compare the transcriptional response of their gut. A total of 48 180 unigenes were identified in D. valens and 43 704 in D. rhizophagus, in response to kairomones blend. The analysis of differential gene expression showed a transcriptional response in D. valens (739 unigenes, 0.58–10.36 Log2FC) related to digestive process and in D. rhizophagus (322 unigenes 0.87–13.08 Log2FC) related to xenobiotics metabolism. The expression profiles of detoxification genes mainly evidenced the up-regulation of COEs and GSTs in D. valens, and the up-regulation of P450s in D. rhizophagus. Results suggest that terpenes metabolism comes accompanied by an integral hormetic response, result of compensatory mechanisms, including the activation of other metabolic pathways, to ensure the supply of energy and the survival of organisms which is specific for each species, according to its life history and ecological strategy.

Bark beetles commonly produce de novo terpenoid pheromones using precursors synthesized through the mevalonate pathway. This process is regulated by Juvenile Hormone III (JH III). In this work, the expression levels of mevalonate pathway genes were quantified after phloem feeding—to induce the endogenous synthesis of JH III—and after the topical application of a JH III solution. The mevalonate pathway genes from D. rhizophagus were cloned, molecularly characterized, and their expression levels were quantified. Also, the terpenoid compounds produced in the gut were identified and quantified by Gas Chromatography Mass Spectrometry (GC-MS). The feeding treatment produced an evident upregulation, mainly in acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IPPI) genes, and males reached higher expression levels compared to females. In contrast, the JH III treatment did not present a clear pattern of upregulation in any sex or time. Notably, the genes responsible for the synthesis of frontalin and ipsdienol precursors (geranyl diphosphate synthase/farnesyl diphosphate synthase (GPPS/FPPS) and geranylgeranyl diphosphate synthase (GGPPS)) were not clearly upregulated, nor were these compounds further identified. Furthermore, trans-verbenol and myrtenol were the most abundant compounds in the gut, which are derived from an α-pinene transformation rather than de novo synthesis. Hence, the expression of mevalonate pathway genes in D. rhizophagus gut is not directed to the production of terpenoid pheromones, regardless of their frequent occurrence in the genus Dendroctonus.

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Dendroctonus-bark beetles are natural components and key ecological agents of coniferous forests. They spend most of their lives under the bark, where they are exposed to highly toxic terpenes present in the oleoresin. Cytochrome P450 (CYP) is a multigene family involved in the detoxification of these compounds. It has been demonstrated that CYP6DE and CYP6DJ subfamilies hydroxylate monoterpenes, whose derivatives can act as pheromone synergist compounds or be pheromones themselves in these insects. Given the diversity and functional role of CYPs, we investigated whether these cytochromes have retained their function throughout the evolution of these insects. To test this hypothesis, we performed a Bayesian phylogenetic analysis to determine phylogenetic subgroups of cytochromes in these subfamilies. Subgroups were mapped and reconciled with the Dendroctonus phylogeny. Molecular docking analyses were performed with the cytochromes of each subgroup and enantiomers of α-pinene and β-pinene, (+)-3-carene, β-myrcene and R-(+)-limonene. In addition, functional divergence analysis was performed to identify critical amino acid sites that influence changes in catalytic site conformation and/or protein folding. Three and two phylogenetic subgroups were recovered for the CYP6DE and CYP6DJ subfamilies, respectively. Mapping and reconciliation analysis showed different gain and loss patterns for cytochromes of each subgroup. Functional predictions indicated that the cytochromes analyzed are able to hydroxylate all monoterpenes; however, they showed preferential affinities to different monoterpenes. Functional divergence analyses indicated that the CYP6DE subfamily has experimented type I and II divergence, whereas the CYP6DJ subfamily has evolved under strong functional constraints. Results suggest cytochromes of the CYP6DE subfamily evolve to reinforce their detoxifying capacity hydroxylating mainly α- and β-pinene to (+) and (−)-trans-verbenol, being the negative enantiomer used as a pheromone by several Dendroctonus species; whereas cytochromes of the CYP6DJ subfamily appear to retain their original function related to the detoxification of these compounds.