MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects.IMPORTANCEWe created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Reducing the risk of rostral bending failure in Curculio Linnaeus, 1758.

With over 300 species worldwide, the genus Curculio Linnaeus, 1758 is a widespread, morphologically diverse lineage of weevils that mainly parasitize nuts. Females use the rostrum, an elongate cuticular extension of the head, to excavate oviposition sites. This process causes extreme bending and deformation of the rostrum, without apparent harm to the structure. The cuticle of the rostral apex exhibits substantial modifications to its composite structure that enhance the elasticity and resiliency of this structure. Here we develop finite element models of the head and rostrum for three Curculio species representing disparate North American clades and rostral morphotypes. The models were subjected to varying apical loads and to prescribed dislocation of the head capsule, with and without representing the cuticular modifications of the rostral apex. We found that the altered layer thicknesses and macrofiber orientation angles of the rostral apex fully explain the observed elasticity of the rostrum. These modifications have a synergistic effect that greatly enhances the flexibility of the rostral apex. Consequently, the cuticle composite profile of the rostral apex substantially mitigates the risk of fracture in dorso-apical flexion. Removing the cuticular modifications, in turn, causes a negative margin of safety for rostral bending, implying strong risk of catastrophic structural failure. The occipital sulci were identified as an important source of biomechanical constraint upon the elasticity of the rostrum, and exhibit the greatest risk of failure within this structure. The apical cuticle profile greatly reduced the maximum stresses and strain energy accumulated in the rostrum, thereby resulting in a positive margin of safety and reducing the risk of fracture. Our findings imply that the primary selective pressure influencing the evolution of the rostral cuticle was most likely negative selection of structural failure caused by bending. STATEMENT OF SIGNIFICANCE: Weevils are among the most diverse and evolutionarily successful animal lineages on Earth. Their success is driven in part by a structure called the rostrum, which gives weevil heads a characteristic "snout-like" appearance. Nut weevils in the genus Curculio use the rostrum to drill holes into developing fruits and nuts, into which they deposit their eggs. During oviposition this exceedingly slender structure is bent into a straightened configuration - in some species up to 90∘ - but does not suffer any damage during this process. Using finite element models of the rostra of three morphologically distinct species, we show that the Curculio rostrum is only able to withstand repeated, extreme bending because of modifications to the composite structure of the cuticle in the rostral apex. These modifications were shown previously to enhance the intrinsic toughness of the cuticle; in this study, we demonstrate that modification of the rostral cuticle also results in more evenly distributed bending stresses, further reducing the risk of fracture. This is the first time that the laminate profile, orthotropic behavior, and functional gradation of the cuticle have been incorporated into a three-dimensional finite element model of an insect cuticular structure. Our models highlight the significance of biomechanical constraint - i.e., avoidance of catastrophic structural failure - on the evolution of insect morphology.

Descriptions of four new species of Minyomerus Horn, 1876 sec. Jansen & Franz, 2018 (Coleoptera: Curculionidae), with notes on their distribution and phylogeny.

This contribution adopts the taxonomic concept approach, including the use of taxonomic concept labels (name sec. [according to] source) and region connection calculus-5 (RCC-5) articulations and alignments. Prior to this study, the broad-nosed weevil genus Minyomerus Horn, 1876 sec. Jansen & Franz, 2015 (Curculionidae [non-focal]: Entiminae [non-focal]: Tanymecini [non-focal]) contained 17 species distributed throughout the desert and plains regions of North America. In this review of Minyomerus sec. Jansen & Franz, 2018, we describe the following four species as new to science: Minyomerus ampullaceus sec. Jansen & Franz, 2018 (henceforth: [JF2018]), new species, Minyomerus franko [JF2018], new species, Minyomerus sculptilis [JF2018], new species, and Minyomerus tylotos [JF2018], new species. The four new species are added to, and integrated with, the preceding revision, and an updated key and phylogeny of Minyomerus [JF2018] are presented. A cladistic analysis using 52 morphological characters of 26 terminal taxa (5/21 outgroup/ingroup) yielded a single most-parsimonious cladogram (Length = 99 steps, consistency index = 60, retention index = 80). The analysis reaffirms the monophyly of Minyomerus [JF2018] with eight unreversed synapomorphies. The species-group placements, possible biogeographic origins, and natural history of the new species are discussed in detail.

Phylogenetic revision of Minyomerus Horn, 1876 sec. Jansen & Franz, 2015 (Coleoptera, Curculionidae) using taxonomic concept annotations and alignments.

This contribution adopts the taxonomic concept annotation and alignment approach. Accordingly, and where indicated, previous and newly inferred meanings of taxonomic names are individuated according to one specific source. Articulations among these concepts and pairwise, logically consistent alignments of original and revisionary classifications are also provided, in addition to conventional nomenclatural provenance information. A phylogenetic revision of the broad-nosed weevil genera Minyomerus Horn, 1876 sec. O'Brien & Wibmer (1982), and Piscatopus Sleeper, 1960 sec. O'Brien & Wibmer (1982) (Curculionidae [non-focal]: Entiminae [non-focal]: Tanymecini [non-focal]) is presented. Prior to this study, Minyomerus sec. O'Brien & Wibmer (1982) contained seven species, whereas the monotypic Piscatopus sec. O'Brien & Wibmer (1982) was comprised solely of Piscatopus griseus Sleeper, 1960 sec. O'Brien & Wibmer (1982). We thoroughly redescribe these recognized species-level entities and furthermore describe ten species as new to science: Minyomerus bulbifrons sec. Jansen & Franz (2015) (henceforth: [JF2015]), sp. n., Minyomerus aeriballux [JF2015], sp. n., Minyomerus cracens [JF2015], sp. n., Minyomerus gravivultus [JF2015], sp. n., Minyomerus imberbus [JF2015], sp. n., Minyomerus reburrus [JF2015], sp. n., Minyomerus politus [JF2015], sp. n., Minyomerus puticulatus [JF2015], sp. n., Minyomerus rutellirostris [JF2015], sp. n., and Minyomerus trisetosus [JF2015], sp. n. A cladistic analysis using 46 morphological characters of 22 terminal taxa (5/17 outgroup/ingroup) yielded a single most-parsimonious cladogram (L = 82, CI = 65, RI = 82). The analysis strongly supports the monophyly of Minyomerus [JF2015] with eight unreversed synapomorphies, and places Piscatopus griseus sec. O'Brien & Wibmer (1982) within the genus as sister to Minyomerus rutellirostris [JF2015]. Accordingly, Piscatopus sec. Sleeper (1960), syn. n. is changed to junior synonymy of Minyomerus [JF2015], and its sole member Piscatopus griseus sec. Sleeper (1960) is moved to Minyomerus [JF2015] as Minyomerus griseus [JF2015], comb. n. In addition, the formerly designated type Minyomerus innocuus Horn, 1876 sec. Pierce (1913), syn. n. is changed to junior synonymy of Minyomerus microps (Say, 1831) [JF2015] which has priority. The genus is widespread throughout western North America, ranging from Canada to Mexico and Baja California. Apparent patterns of interspecific diversity of exterior and genitalic morphology, varying host plant ranges, overlapping and widely extending species distributions, suggest an early origin for Minyomerus [JF2015], with a diversification that likely followed the development of North American desert biomes. Three species in the genus - i.e., Minyomerus languidus Horn, 1876 [JF2015], Minyomerus microps [JF2015], and Minyomerus trisetosus [JF2015] - are putatively considered parthenogenetic.