Metabarcoding of mycetangia from the Dendroctonus frontalis species complex (Curculionidae: Scolytinae) reveals diverse and functionally redundant fungal assemblages.

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.

Seasonal changes in the bacterial community structure of three eutrophicated urban lakes in Mexico city, with emphasis on Microcystis spp.

Artificial urban lakes commonly have physicochemical conditions that contribute to rapid anthropogenic eutrophication and development of cyanobacterial blooms. Microcystis is the dominat genus in most freshwater bodies and is one of the main producter of microcystins. Using 454-pyrosequencing we characterized the bacterial community, with special emphasis on Microcystis, in three recreational urban lakes from Mexico City in both wet and dry seasons. We also evaluated some physicochemical parameters that might influence the presence of Microcystis blooms, and we associated the relative abundance of heterotrophic and autotrophic bacterial communities with their possible metabolic capacities. A total of 14 phyla, 18 classes, 39 orders, 53 families and 48 bacterial genera were identified in both seasons in the three urban lakes. Cyanobacteria had the highest relative abundance followed by Proteobacteria and Actinobacteria. Microcystis was the dominant taxon followed by Arthrospira, Planktothrix and Synechococcus. We also found heterotrophic bacteria associated with the blooms, such as Rhodobacter, Pseudomonas, Sphingomonas and, Porphyrobacter. The highest richness, diversity and dominance were registered in the bacterial community of the Virgilio Uribe Olympic Rowing-Canoeing Track in both seasons, and the lowest values were found in the Chapultepec Lake. The canonical correspondence analysis showed that dissolved oxygen and NO3-N concentrations might explain the presence of Microcystis blooms. The metabolic prediction indicated that these communities are involved in photosynthesis, oxidative phosphorylation, methane metabolism, carbon fixation, and nitrogen and sulfur metabolism. The lakes studied had a high prevalence of Microcystis, but average values of microcystins did not exceed the maximum permissible level established by the United States Environmental Protection Agency for recreational and cultural activities. The presence of cyanobacteria and microcystins at low to moderate concentrations in the three lakes could result in ecosystem disruption and increase animal and human health risks.

Time-Course of CYP450 Genes Expression From Dendroctonus rhizophagus (Curculionidae: Scolytinae) During Early Hours of Drilling Bark and Settling Into the Host Tree.

Dendroctonus bark beetles (Scolytinae) are one of the most important disturbance agents of coniferous forests in North and Central America. These beetles spend their lives almost entirely under the tree bark, and their survival and reproductive success depend on their ability to overcome the toxic effect of the trees' oleoresin. The cytochromes P450 (CYPs) are associated with the detoxification process of xenobiotics, as well as other physiological processes. Different cytochromes (families 4, 6, and 9) in the Dendroctonus species have been expressed under several experimental conditions; nevertheless, the expression time-course of these genes is unknown. To explore the induction speed of CYPs, we evaluated the relative expression of the CYP6BW5, CYP6DG1, CYP6DJ2, CYP9Z18, and CYP9Z20 genes at the early hours of drilling and settling into a tree (1, 2, 4, 6, 8, 12, 18 h) both in females and males, solitary or paired, of the bark beetle Dendroctonus rhizophagus Thomas and Bright. Our findings show that the five genes were rapidly overexpressed in the early hours (1 to 6 h) in both sexes and in solitary and paired conditions, suggesting their participation in the detoxification process. Additionally, the CYPs expression shows up- and down-regulation patterns through these short times, suggesting their probable participation in other physiological processes as the biosynthesis of hormones, pheromones or compounds related to reproduction.

Changes in the Microbial Community of Pinus arizonica Saplings After Being Colonized by the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae).

The death of trees is an ecological process that promotes regeneration, organic matter recycling, and the structure of communities. However, diverse biotic and abiotic factors can disturb this process. Dendroctonus bark beetles (Curculionidae: Scolytinae) are natural inhabitants of pine forests, some of which produce periodic outbreaks, killing thousands of trees in the process. These insects spend almost their entire life cycle under tree bark, where they reproduce and feed on phloem. Tunneling and feeding of the beetles result in the death of the tree and an alteration of the resident microbiota as well as the introduction of microbes that the beetles vector. To understand how microbial communities in subcortical tissues of pines change after they are colonized by the bark beetle Dendroctonus rhizophagus, we compare both the bacterial and fungal community structures in two colonization stages of Pinus arizonica (Arizona pine) employing Illumina MiSeq. Our findings showed significant differences in diversity and the dominance of bacterial community in the two colonization stages with Shannon (P = 0.004) and Simpson (P = 0.0006) indices, respectively, but not in species richness with Chao1 (P = 0.19). In contrast, fungal communities in both stages showed significant differences in species richness with Chao1 (P = 0.0003) and a diversity with Shannon index (P = 0.038), but not in the dominance with the Simpson index (P = 0.12). The ß-diversity also showed significant changes in the structure of bacterial and fungal communities along the colonization stages, maintaining the dominant members in both cases. Our results suggest that microbial communities present in the Arizona pine at the tree early colonization stage by bark beetle change predictably over time.

Gut Bacterial Communities of Dendroctonus valens and D. mexicanus (Curculionidae: Scolytinae): A Metagenomic Analysis across Different Geographical Locations in Mexico.

Dendroctonus bark beetles are a worldwide significant pest of conifers. This genus comprises 20 species found in North and Central America, and Eurasia. Several studies have documented the microbiota associated with these bark beetles, but little is known regarding how the gut bacterial communities change across host range distribution. We use pyrosequencing to characterize the gut bacterial communities associated with six populations of Dendroctonus valens and D. mexicanus each across Mexico, determine the core bacteriome of both insects and infer the metabolic pathways of these communities with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to evaluate whether these routes are conserved across geographical locations. Our results show that the ß-diversity with UniFrac unweighted varies among locations of both bark beetles mainly due to absence/presence of some rare taxa. No association is found between the pairwise phylogenetic distance of bacterial communities and geographic distance. A strict intraspecific core bacteriome is determined for each bark beetle species, but these cores are different in composition and abundance. However, both bark beetles share the interspecific core bacteriome recorded previously for the Dendroctonus genus consisting of Enterobacter, Pantoea, Providencia, Pseudomonas, Rahnella, and Serratia. The predictions of metabolic pathways are the same in the different localities, suggesting that they are conserved through the geographical locations.

Bacteriome from Pinus arizonica and P. durangensis: Diversity, Comparison of Assemblages, and Overlapping Degree with the Gut Bacterial Community of a Bark Beetle That Kills Pines.

Symbioses between plants and microorganims have been fundamental in the evolution of both groups. The endophytic bacteria associated with conifers have been poorly studied in terms of diversity, ecology, and function. Coniferous trees of the genera Larix, Pseudotsugae, Picea and mainly Pinus, are hosts of many insects, including bark beetles and especially the Dendroctonus species. These insects colonize and kill these trees during their life cycle. Several bacteria detected in the gut and cuticle of these insects have been identified as endophytes in conifers. In this study, we characterized and compared the endophytic bacterial diversity in roots, phloem and bark of non-attacked saplings of Pinus arizonica and P. durangensis using 16S rRNA gene pyrosequencing. In addition, we evaluated the degree of taxonomic relatedness, and the association of metabolic function profiles of communities of endophytic bacteria and previously reported gut bacterial communities of D. rhizophagus; a specialized bark beetle that colonizes and kills saplings of these pine species. Our results showed that both pine species share a similar endophytic community. A total of seven bacterial phyla, 14 classes, 26 orders, 43 families, and 51 genera were identified. Enterobacteriaceae was the most abundant family across all samples, followed by Acetobacteraceae and Acidobacteriaceae, which agree with previous studies performed in other pines and conifers. Endophytic communities and that of the insect gut were significantly different, however, the taxonomic relatedness of certain bacterial genera of pines and insect assemblages suggested that some bacteria from pine tissues might be the same as those in the insect gut. Lastly, the metabolic profile using PICRUSt showed there to be a positive association between communities of both pines and insect gut. This study represents the baseline into the knowledge of the endophytic bacterial communities of two of the major hosts affected by D. rhizophagus.

Structure and dynamics of the gut bacterial microbiota of the bark beetle, Dendroctonus rhizophagus (Curculionidae: Scolytinae) across their life stages.

Bark beetles play an important role as agents of natural renovation and regeneration in coniferous forests. Several studies have documented the metabolic capacity of bacteria associated with the gut, body surface, and oral secretions of these insects; however, little is known about how the bacterial community structure changes during the life cycle of the beetles. This study represents the first comprehensive analysis of the bacterial community of the gut of the bark beetle D. rhizophagus during the insect's life cycle using 454 pyrosequencing. A total of 4 bacterial phyla, 7 classes, 15 families and 23 genera were identified. The α-diversity was low, as demonstrated in previous studies. The dominant bacterial taxa belonged to the Enterobacteriaceae and Pseudomonadaceae families. This low α-diversity can be attributed to the presence of defensive chemical compounds in conifers or due to different morpho-physiological factors in the gut of these insects acting as strong selective factors. Members of the genera Rahnella, Serratia, Pseudomonas and Propionibacterium were found at all life stages, and the first three genera, particularly Rahnella, were predominant suggesting the presence of a core microbiome in the gut. Significant differences in ß-diversity were observed, mainly due to bacterial taxa present at low frequencies and only in certain life stages. The predictive functional profiling indicated metabolic pathways related to metabolism of amino acids and carbohydrates, and membrane transport as the most significant in the community. These differences in the community structure might be due to several selective factors, such as gut compartmentalization, physicochemical conditions, and microbial interactions.