Comparative metabarcoding and biodiversity of gut-associated fungal assemblages of Dendroctonus species (Curculionidae: Scolytinae).

The genus Dendroctonus is a Holarctic taxon composed of 21 nominal species; some of these species are well known in the world as disturbance agents of forest ecosystems. Under the bark of the host tree, these insects are involved in complex and dynamic associations with phoretic ectosymbiotic and endosymbiotic communities. Unlike filamentous fungi and bacteria, the ecological role of yeasts in the bark beetle holobiont is poorly understood, though yeasts were the first group to be recorded as microbial symbionts of these beetles. Our aim was characterize and compare the gut fungal assemblages associated to 14 species of Dendroctonus using the internal transcribed spacer 2 (ITS2) region. A total of 615,542 sequences were recovered yielding 248 fungal amplicon sequence variants (ASVs). The fungal diversity was represented by 4 phyla, 16 classes, 34 orders, 54 families, and 71 genera with different relative abundances among Dendroctonus species. The α-diversity consisted of 32 genera of yeasts and 39 genera of filamentous fungi. An analysis of ß-diversity indicated differences in the composition of the gut fungal assemblages among bark beetle species, with differences in species and phylogenetic diversity. A common core mycobiome was recognized at the genus level, integrated mainly by Candida present in all bark beetles, Nakazawaea, Cladosporium, Ogataea, and Yamadazyma. The bipartite networks confirmed that these fungal genera showed a strong association between beetle species and dominant fungi, which are key to maintaining the structure and stability of the fungal community. The functional variation in the trophic structure was identified among libraries and species, with pathotroph-saprotroph-symbiotroph represented at the highest frequency, followed by saprotroph-symbiotroph, and saprotroph only. The overall network suggested that yeast and fungal ASVs in the gut of these beetles showed positive and negative associations among them. This study outlines a mycobiome associated with Dendroctonus nutrition and provides a starting point for future in vitro and omics approaches addressing potential ecological functions and interactions among fungal assemblages and beetle hosts.

Comparison of Clinical Decision Support Tools to Improve Pediatric Lipid Screening.

OBJECTIVE: To test whether different clinical decision support tools increase clinician orders and patient completions relative to standard practice and each other. STUDY DESIGN: A pragmatic, patient-randomized clinical trial in the electronic health record was conducted between October 2019 and April 2020 at Geisinger Health System in Pennsylvania, with 4 arms: care gap-a passive listing recommending screening; alert-a panel promoting and enabling lipid screen orders; both; and a standard practice-no guideline-based notification-control arm. Data were analyzed for 13 346 9- to 11-year-old patients seen within Geisinger primary care, cardiology, urgent care, or nutrition clinics, or who had an endocrinology visit. Principal outcomes were lipid screening orders by clinicians and completions by patients within 1 week of orders. RESULTS: Active (care gap and/or alert) vs control arm patients were significantly more likely (P < .05) to have lipid screening tests ordered and completed, with ORs ranging from 1.67 (95% CI 1.28-2.19) to 5.73 (95% CI 4.46-7.36) for orders and 1.54 (95% CI 1.04-2.27) to 2.90 (95% CI 2.02-4.15) for completions. Alerts, with or without care gaps listed, outperformed care gaps alone on orders, with odds ratios ranging from 2.92 (95% CI 2.32-3.66) to 3.43 (95% CI 2.73-4.29). CONCLUSIONS: Electronic alerts can increase lipid screening orders and completions, suggesting clinical decision support can improve guideline-concordant screening. The study also highlights electronic record-based patient randomization as a way to determine relative effectiveness of support tools. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04118348.

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.

Effects of Site Thermal Variation and Physiography on Flight Synchrony and Phenology of the North American Spruce Beetle (Coleoptera: Curculionidae, Scolytinae) and Associated Species in Colorado.

Spruce beetle, Dendroctonus rufipennis Kirby, is associated with forest mortality in Colorado and across western North America, yet it is not well understood how thermal variability affects basic population processes such as flight phenology. However, phenology-temperature relationships are important for understanding patterns of ecosystem disturbance, especially under projected climate warming. Here, we use a multiyear trapping study to test the hypothesis that spruce beetle flight synchrony, timing, and fitness traits (body size) are affected by variation in regional temperature and physiography. Large quantities of co-colonizing scolytines (Polygraphus convexifrons) (Coleoptera: Curculionidae, Scolytinae) and predatory beetles (Thanasimus undulatus) (Coleoptera: Cleridae) that may affect D. rufipennis populations also responded to spruce beetle synthetic pheromone lures. Relationships between flight patterns and environmental conditions were also analyzed for these species. The winter of 2018 was warmer and drier than winter 2017 and was associated with earlier flight for both scolytine species across most sites. The most important environmental factor driving D. rufipennis flight phenology was accumulated growing degree-days, with delayed flight cessation under warmer conditions and larger beetles following a warm winter. Flight was consistently more synchronous under colder growing season conditions for all species, but synchrony was not associated with winter temperatures. Warmer-than-average years promoted earlier flight of D. rufipennis and associated species, and less synchronous, prolonged flight across the region. Consequently, climate warming may be associated with earlier and potentially extended biotic pressure for spruce trees in the Rocky Mountain region, and flight phenology of multiple scolytines is plastic in response to thermal conditions.

Laboratory and Field Evaluation of the Entomopathogenic Fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes) for Population Management of Spruce Beetle, Dendroctonus rufipennis (Coleoptera: Scolytinae), in Felled Trees and Factors Limiting Pathogen Success.

An isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) was tested for its ability to reduce survival and reproduction of spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), under laboratory and field conditions. Conidial suspension applied directly to adults or to filter papers that adults contacted had a median survival time of 3-4 d in laboratory assays and beetles died more rapidly when exposed to conidial suspension than when treated with surfactant solution only. In the field, conidial suspension was applied to the surface of felled and pheromone-baited Engelmann spruce (Picea engelmannii) trees using a backpack sprayer. Mortality of colonizing parent beetles (F0), reproduction (abundance of F1 offspring in logs), and emergence of F1 beetles from logs was compared between treated and nontreated logs. Application of spore suspension increased mortality of F0 adults by 36% on average. Total F1 reproduction was reduced by 17% and emergence from logs was reduced by 13% in treated logs, but considerable variability in reproduction and emergence was observed. Viable spores were re-isolated from treated logs up to 90 d after application, indicating that spores are capable of long-term persistence on the tree bole microhabitat. Subsequent in vitro tests revealed that temperatures below 15°C and exposure to spruce monoterpenes likely limit performance of B. bassiana under field conditions, but exposure to low-intensity light or interactions with spruce beetle symbiotic fungi were not strongly inhibitory. It is concluded that matching environmental tolerances of biocontrol fungi to field conditions can likely improve their usefulness for control of spruce beetle in windthrown trees.

XTHs from Fragaria vesca: genomic structure and transcriptomic analysis in ripening fruit and other tissues.

BACKGROUND: Fragaria vesca or 'woodland strawberry' has emerged as an attractive model for the study of ripening of non-climacteric fruit. It has several advantages, such as its small genome and its diploidy. The recent availability of the complete sequence of its genome opens the possibility for further analysis and its use as a reference species. Fruit softening is a physiological event and involves many biochemical changes that take place at the final stages of fruit development; among them, the remodeling of cell walls by the action of a set of enzymes. Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell wall-associated enzyme, which is encoded by a multigene family. Its action modifies the structure of xyloglucans, a diverse group of polysaccharides that crosslink with cellulose microfibrills, affecting therefore the functional structure of the cell wall. The aim of this work is to identify the XTH-encoding genes present in F. vesca and to determine its transcription level in ripening fruit. RESULTS: The search resulted in identification of 26 XTH-encoding genes named as FvXTHs. Genetic structure and phylogenetic analyses were performed allowing the classification of FvXTH genes into three phylogenetic groups: 17 in group I/II, 2 in group IIIA and 4 in group IIIB. Two sequences were included into the ancestral group. Through a comparative analysis, characteristic structural protein domains were found in FvXTH protein sequences. In complement, expression analyses of FvXTHs by qPCR were performed in fruit at different developmental and ripening stages, as well as, in other tissues. The results showed a diverse expression pattern of FvXTHs in several tissues, although most of them are highly expressed in roots. Their expression patterns are not related to their respective phylogenetic groups. In addition, most FvXTHs are expressed in ripe fruit, and interestingly, some of them (FvXTH 18 and 20, belonging to phylogenic group I/II, and FvXTH 25 and 26 to group IIIB) display an increasing expression pattern as the fruit ripens. CONCLUSION: A discrete group of FvXTHs (18, 20, 25 and 26) increases their expression during softening of F. vesca fruit, and could take part in cell wall remodeling required for softening in collaboration with other cell wall degrading enzymes.

Risk factors for diarrhea in children under five years of age residing in peri-urban communities in Cochabamba, Bolivia.

This study examined the relationship between childhood diarrhea prevalence and caregiver knowledge of the causes and prevention of diarrhea in a prospective cohort of 952 children < 5 years of age in Cochabamba, Bolivia. The survey of caregiver knowledge found that more than 80% of caregivers were unaware that hand washing with soap could prevent childhood diarrhea. Furthermore, when asked how to keep food safe for children to eat only 17% of caregivers reported hand washing before cooking and feeding a child. Lack of caregiver awareness of the importance of practices related to hygiene and sanitation for diarrhea prevention were significant risk factors for diarrheal disease in this cohort. The knowledge findings from this study suggest that health promotion in these communities should put further emphasis on increasing knowledge of how water treatment, hand washing with soap, proper disposal of child feces, and food preparation relate to childhood diarrhea prevention.

A cluster randomized controlled trial to reduce childhood diarrhea using hollow fiber water filter and/or hygiene-sanitation educational interventions.

Safe domestic potable water supplies are urgently needed to reduce childhood diarrheal disease. In periurban neighborhoods in Cochabamba, Bolivia, we conducted a cluster randomized controlled trial to evaluate the efficacy of a household-level hollow fiber filter and/or behavior change communication (BCC) on water, sanitation, and hygiene (WASH) to reduce the diarrheal disease in children less than 5 years of age. In total, 952 households were followed for a period of 12 weeks post-distribution of the study interventions. Households using Sawyer PointONE filters had significantly less diarrheal disease compared with the control arm during the intervention period, which was shown by diarrheal prevalence ratios of 0.21 (95% confidence interval [95% CI] = 0.15-0.30) for the filter arm and 0.27 (95% CI = 0.22-0.34) for the filter and WASH BCC arm. A non-significant reduction in diarrhea prevalence was reported in the WASH BCC study arm households (0.71, 95% CI = 0.59-0.86).