RESUMO

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 ß-pinene, (+)-3-carene, ß-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 ß-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.

RESUMO

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

RESUMO

Dendroctonus-bark beetles are associated with microbes that can detoxify terpenes, degrade complex molecules, supplement and recycle nutrients, fix nitrogen, produce semiochemicals, and regulate ecological interactions between microbes. Females of some Dendroctonus species harbor microbes in specialized organs called mycetangia; yet little is known about the microbial diversity contained in these structures. Here, we use metabarcoding to characterize mycetangial fungi from beetle species in the Dendroctonus frontalis complex, and analyze variation in biodiversity of microbial assemblages between beetle species. Overall fungal diversity was represented by 4 phyla, 13 classes, 25 orders, 39 families, and 48 genera, including 33 filamentous fungi, and 15 yeasts. The most abundant genera were Entomocorticium, Candida, Ophiostoma-Sporothrix, Ogataea, Nakazawaea, Yamadazyma, Ceratocystiopsis, Grosmannia-Leptographium, Absidia, and Cyberlindnera. Analysis of α-diversity indicated that fungal assemblages of D. vitei showed the highest richness and diversity, whereas those associated with D. brevicomis and D. barberi had the lowest richness and diversity, respectively. Analysis of ß-diversity showed clear differentiation in the assemblages associated with D. adjunctus, D. barberi, and D. brevicomis, but not between closely related species, including D. frontalis and D. mesoamericanus and D. mexicanus and D. vitei. A core mycobiome was not statistically identified; however, the genus Ceratocystiopsis was shared among seven beetle species. Interpretation of a tanglegram suggests evolutionary congruence between fungal assemblages and species of the D. frontalis complex. The presence of different amplicon sequence variants (ASVs) of the same genus in assemblages from species of the D. frontalis complex outlines the complexity of molecular networks, with the most complex assemblages identified from D. vitei, D. mesoamericanus, D. adjunctus, and D. frontalis. Analysis of functional variation of fungal assemblages indicated multiple trophic groupings, symbiotroph/saprotroph guilds represented with the highest frequency (∼31% of identified genera). These findings improve our knowledge about the diversity of mycetangial communities in species of the D. frontalis complex and suggest that minimal apparently specific assemblages are maintained and regulated within mycetangia.

RESUMO

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, ß-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.

RESUMO

Effectiveness of pyrethroid insecticides against pests including bark beetles (Dendroctonus spp.) is well known, but little is known about their (a) residuality and persistence in forests after application to control an outbreak and (b) ability to bioaccumulate in insects, which could cause adverse effects on the trophic chains. The 24 h lethal toxicity, bioaccumulation factors, and lethal body burdens of the pyrethroid insecticides bifenthrin, deltamethrin, and cypermethrin were evaluated in the bark beetle Dendroctonus mexicanus. The residuality and persistence of the insecticide bifenthrin in the forest ecosystem (soil, water, and sediment) was also monitored after its application in a region infested by bark beetles. We determined whether contamination of soil, water, and sediments occurred near the sanitation site. For D. mexicanus, the most toxic insecticide for adults was bifenthrin for topical application (LC50 = 0.94 mg/L) and cypermethrin for bark application (LC50 = 5.04 mg/L). The insecticide that bioaccumulated the most in the insect body was deltamethrin (622.41 µg g-1 dw) and the insecticide that bioaccumulated the least amount was cypermethrin (183.09 µg g-1 dw). However, cypermethrin was the active substance that presented the lowest body burdens, and therefore required a lower dose to cause death of D. mexicanus adults. Our results demonstrate that prolonged presence of bifenthrin in the forest ecosystem could lead to bioaccumulation in soil, sediment, and insects.

Assuntos

RESUMO

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 Dendroctonusrhizophagus 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.

Assuntos

RESUMO

The western pine beetle (WPB), Dendroctonus brevicomis LeConte, is a major mortality agent of pines in North America. A total of 706 adults of WPB from 81 geographical sites were analyzed with traditional and geometric morphometric methods to evaluate the variation of discrete and quantitative morphological characters with particular attention to the antenna, spermatheca, and seminal rod. Principal coordinates and canonical variate analyses supported three geographical groups in WPB: (1) West, from British Columbia to southern California along the Pacific coast, Idaho, and Montana; (2) East-SMOC, including Nevada, Utah, Colorado, Arizona, New Mexico, Texas, Chihuahua, and Durango; and (3) SMOR, including Coahuila, Nuevo Leon, and Tamaulipas. The pubescence length on the elytral declivity was a robust character for separating West specimens from the other groups. Additionally, the genitalia shape both female and male in dorsal view was a reliable character for discriminating among groups. Based on these results, which agree with genetic and chemical ecology evidence, we herein reinstate Dendroctonus barberi Hopkins (East-SMOC group) and remove it from synonymy with D. brevicomis (West group). Differences in the spermatheca and seminal rod shape of SMOR specimens suggest that these populations might be a different species from D. barberi and D. brevicomis.

RESUMO

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.

Assuntos

RESUMO

The distribution range of the western pine beetle Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae) is supported only by scattered records in the northern parts of Mexico, suggesting that its populations may be marginal and rare in this region. In this study, we review the geographical distribution of D. brevicomis in northern Mexico and perform a geometric morphometric analysis of seminal rod shape to evaluate its reliability for identifying this species with respect to other members of the Dendroctonus frontalis Zimmermann (Coleoptera: Curculionidae) complex. Our results provide 30 new records, with 26 distributed in the Sierra Madre Occidental and 4 in the Sierra Madre Oriental. These records extend the known distribution range of D. brevicomis to Durango and Tamaulipas states in northern Mexico. Furthermore, we find high geographic variation in size and shape of the seminal rod, with conspicous differences among individuals from different geographical regions, namely west and east of the Great Basin and between mountain systems in Mexico.

Assuntos