HyRAD-X Exome Capture Museomics Unravels Giant Ground Beetle Evolution.

Advances in phylogenomics contribute toward resolving long-standing evolutionary questions. Notwithstanding, genetic diversity contained within more than a billion biological specimens deposited in natural history museums remains recalcitrant to analysis owing to challenges posed by its intrinsically degraded nature. Yet that tantalizing resource could be critical in overcoming taxon sampling constraints hindering our ability to address major evolutionary questions. We addressed this impediment by developing phyloHyRAD, a new bioinformatic pipeline enabling locus recovery at a broad evolutionary scale from HyRAD-X exome capture of museum specimens of low DNA integrity using a benchtop RAD-derived exome-complexity-reduction probe set developed from high DNA integrity specimens. Our new pipeline can also successfully align raw RNAseq transcriptomic and ultraconserved element reads with the RAD-derived probe catalog. Using this method, we generated a robust timetree for Carabinae beetles, the lack of which had precluded study of macroevolutionary trends pertaining to their biogeography and wing-morphology evolution. We successfully recovered up to 2,945 loci with a mean of 1,788 loci across the exome of specimens of varying age. Coverage was not significantly linked to specimen age, demonstrating the wide exploitability of museum specimens. We also recovered fragmentary mitogenomes compatible with Sanger-sequenced mtDNA. Our phylogenomic timetree revealed a Lower Cretaceous origin for crown group Carabinae, with the extinct Aplothorax Waterhouse, 1841 nested within the genus Calosoma Weber, 1801 demonstrating the junior synonymy of Aplothorax syn. nov., resulting in the new combination Calosomaburchellii (Waterhouse, 1841) comb. nov. This study compellingly illustrates that HyRAD-X and phyloHyRAD efficiently provide genomic-level data sets informative at deep evolutionary scales.

A new species of cylindrical bark beetle (Zopheridae: Colydiinae) from Vanuatu establishes in Hawaii.

We describe Bulasconotus scaccarius sp. nov., a new species of cylindrical bark beetle (Zopheridae: Colydiinae: Synchitini), elevating the number of described species in its genus to two. Despite discovering this species in Hawaii, we suspect, based on collections and survey data, that it is endemic to the South Pacific archipelago of Vanuatu and has only recently arrived in Hawaii. Evidence of the diet of closely related genera indicates that the new species may be a predator of ambrosia beetles (Curculionidae: Scolytinae), conceivably posing a risk to those that are endemic to Hawaii.

One becomes two: second species of the Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) species complex is established on two Hawaiian Islands.

The cryptic species that make up the Euwallacea fornicatus species complex can be readily distinguished via their DNA sequences. Until recently, it was believed that the Hawaiian Islands had been invaded by only one of these cryptic species, E. perbrevis (tea shot hole borer; TSHB). However, following the 2016 deposition of a DNA sequence in the public repository GenBank, it became evident that another species, E. fornicatus (polyphagous shot hole borer; PSHB), had been detected in macadamia orchards on Hawai'i Island (the Big Island). We surveyed the two most-populous islands of Hawai'i, Big Island and O'ahu, and herein confirm that populations of TSHB and PSHB are established on both. Beetles were collected using a variety of techniques in macadamia orchards and natural areas. Individual specimens were identified to species using a high-resolution melt assay, described herein and validated by subsequent sequencing of specimens. It remains unclear how long each species has been present in the state, and while neither is currently recognized as causing serious economic or ecological damage in Hawai'i, the similarity of the newly-confirmed PSHB population to other damaging invasive PSHB populations around the world is discussed. Although the invasive PSHB populations in Hawai'i and California likely have different geographic origins within the beetle's native range, they share identical Fusarium and Graphium fungal symbionts, neither of which have been isolated from PSHB in that native range.

Structure of the Ambrosia Beetle (Coleoptera: Curculionidae) Mycangia Revealed Through Micro-Computed Tomography.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) rely on a symbiosis with fungi for their nutrition. Symbiotic fungi are preserved and transported in specialized storage structures called mycangia. Although pivotal in the symbiosis, mycangia have been notoriously difficult to study, given their minute size and membranous structure. We compared the application of novel visualization methods for the study of mycangia, namely micro-computed tomography (micro-CT) and laser ablation tomography (LATscan) with traditional paraffin sectioning. Micro-CT scanning has shown the greatest promise in new organ discovery, while sectioning remains the only method with sufficient resolution for cellular visualization. All three common types of mycangia (oral, mesonotal, and pronotal) were successfully visualized and presented for different species of ambrosia beetles: Ambrosiodmus minor (Stebbing) 1909, Euplatypus compositus (Say) 1823, Premnobius cavipennis Eichhoff 1878, Scolytoplatypus raja Blandford 1893, Xylosandrus crassiusculus (Motschulsky) 1866 and X. amputatus (Blandford) 1894. A reconstruction of the mycangium and the surrounding musculature in X. amputatus is also presented. The advantages of micro-CT compared to the previously commonly used microtome sectioning include the easy visualization and recording of three-dimensional structures, their position in reference to other internal structures, the ability to distinguish natural aberrations from technical artifacts, and the unprecedented visualizations of the anatomic context of mycangia enabled by the integrated software.

The contribution of mitochondrial metagenomics to large-scale data mining and phylogenetic analysis of Coleoptera.

A phylogenetic tree at the species level is still far off for highly diverse insect orders, including the Coleoptera, but the taxonomic breadth of public sequence databases is growing. In addition, new types of data may contribute to increasing taxon coverage, such as metagenomic shotgun sequencing for assembly of mitogenomes from bulk specimen samples. The current study explores the application of these techniques for large-scale efforts to build the tree of Coleoptera. We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of >3000 bp. These sequences were combined into a single dataset together with all mitochondrial data available at GenBank, in addition to nuclear markers widely used in molecular phylogenetics. The resulting matrix of nearly 16,000 species with two or more loci produced trees (RAxML) showing overall congruence with the Linnaean taxonomy at hierarchical levels from suborders to genera. We tested the role of full-length mitogenomes in stabilizing the tree from GenBank data, as mitogenomes might link terminals with non-overlapping gene representation. However, the mitogenome data were only partly useful in this respect, presumably because of the purely automated approach to assembly and gene delimitation, but improvements in future may be possible by using multiple assemblers and manual curation. In conclusion, the combination of data mining and metagenomic sequencing of bulk samples provided the largest phylogenetic tree of Coleoptera to date, which represents a summary of existing phylogenetic knowledge and a defensible tree of great utility, in particular for studies at the intra-familial level, despite some shortcomings for resolving basal nodes.

Metagenome Skimming of Insect Specimen Pools: Potential for Comparative Genomics.

Metagenomic analyses are challenging in metazoans, but high-copy number and repeat regions can be assembled from low-coverage sequencing by "genome skimming," which is applied here as a new way of characterizing metagenomes obtained in an ecological or taxonomic context. Illumina shotgun sequencing on two pools of Coleoptera (beetles) of approximately 200 species each were assembled into tens of thousands of scaffolds. Repeated low-coverage sequencing recovered similar scaffold sets consistently, although approximately 70% of scaffolds could not be identified against existing genome databases. Identifiable scaffolds included mitochondrial DNA, conserved sequences with hits to expressed sequence tag and protein databases, and known repeat elements of high and low complexity, including numerous copies of rRNA and histone genes. Assemblies of histones captured a diversity of gene order and primary sequence in Coleoptera. Scaffolds with similarity to multiple sites in available coleopteran genome sequences for Dendroctonus and Tribolium revealed high specificity of scaffolds to either of these genomes, in particular for high-copy number repeats. Numerous "clusters" of scaffolds mapped to the same genomic site revealed intra- and/or intergenomic variation within a metagenome pool. In addition to effect of taxonomic composition of the metagenomes, the number of mapped scaffolds also revealed structural differences between the two reference genomes, although the significance of this striking finding remains unclear. Finally, apparently exogenous sequences were recovered, including potential food plants, fungal pathogens, and bacterial symbionts. The "metagenome skimming" approach is useful for capturing the genomic diversity of poorly studied, species-rich lineages and opens new prospects in environmental genomics.

Bulk de novo mitogenome assembly from pooled total DNA elucidates the phylogeny of weevils (Coleoptera: Curculionoidea).

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverse animal groups. However, the relative difficulty and high cost associated with obtaining de novo full mitogenomes have frequently led to conspicuously low taxon sampling in ensuing studies. Here, we report the successful use of an economical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-generation sequencing of a single library made from pooled total DNA extracts of numerous target species. To avoid the use of separate indexed libraries for each specimen, and an associated increase in cost, we incorporate standard polymerase chain reaction-based "bait" sequences to identify the assembled mitogenomes. The method was applied to study the higher level phylogenetic relationships in the weevils (Coleoptera: Curculionoidea), producing 92 newly assembled mitogenomes obtained in a single Illumina MiSeq run. The analysis supported a separate origin of wood-boring behavior by the subfamilies Scolytinae, Platypodinae, and Cossoninae. This finding contradicts morphological hypotheses proposing a close relationship between the first two of these but is congruent with previous molecular studies, reinforcing the utility of mitogenomes in phylogeny reconstruction. Our methodology provides a technically simple procedure for generating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for which bait sequences are the only required prior genome knowledge.

Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation.

Stem cell transplantation (SCT) has achieved significant therapeutic success over the last 10 years, providing a viable treatment option for many previously incurable diseases. However, several inherent limitations of the procedure have restricted its widespread use. These include: lack of sufficient donors for all recipients, a period of bone marrow (BM) aplasia leading to severe, prolonged neutropenia and thrombocytopenia, and the potential for tumor contamination in autologous SCT. Umbilical cord blood (UCB) provides a unique, and potentially more successful, approach to alleviating these limitations. Ex vivo manipulation of hematopoietic stem (HSCs) and progenitor cells (HPCs) derived from UCB using a liquid culture system has revealed that the primitive HSCs from UCB are not identical to their BM counterparts. In fact, these cells may derive from a more primitive stem cell compartment. Ultimately, successful engraftment of UCB HSCs manipulated in an ex vivo environment may lead to a larger number of these life-saving procedures being performed and the full potential of SCT realized.

Treatment of posttransplant lymphoproliferative disease in the central nervous system of a lung transplant recipient using allogeneic leukocytes.

Posttransplant Epstein-Barr virus-related lymphoproliferative disease (PT-LPD) is a common and often fatal complication following solid organ and hematopoietic stem cell transplantation. PT-LPD following solid organ transplantation generally occurs in B cells of recipient origin in contrast to PT-LPD in marrow transplant recipients, which is exclusively of donor origin. The efficacy of adoptive immunotherapy using donor leukocytes to treat PT-LPD in bone marrow transplant recipients has recently been reported. Because PT-LPD in solid organ transplant recipients is generally of recipient origin, the potential application of adoptive immunotherapy of PT-LPD in solid organ recipients obligates the use of either autologous or allogeneic HLA identical leukocytes, with the attendant risk of organ rejection if cells mismatched with the transplanted organ are used. Nonirradiated allogeneic mononuclear cells from an Epstein-Barr virus (EBV)-seropositive, HLA-identical normal sibling were used to treat a monoclonal EBV lymphoma of recipient origin in the central nervous system of a child who had undergone an HLA-mismatched cadaveric lung transplant. The patient received three separate mononuclear cell infusions over a 9-month period, each containing 1 x 10(6) CD3+ mononuclear cells per kilogram. Complete clinical, radiological, and pathological remission was achieved with this treatment regimen. The response correlated with in vivo reconstitution of normal EBV-specific cytotoxic activity and cytotoxic T lymphocyte precursor frequency. Use of allogeneic HLA-compatible mononuclear cells may thus offer an additional mode of therapy for EBV-related lymphoproliferative disease in selected solid organ transplant recipients refractory to conventional therapies.

Clinical application of a new glucose analyzer in the neonatal intensive care unit: comparison with other methods.

We evaluated the operation of the Yellow Springs Instrument Co. (YSI) glucose analyzer (model 23A) by clinical nurses for the measurement of blood glucose in the intensive care nursery. In vitro performance was determined with the use of aqueous standards; with a 2-point calibration of 0.0 and 200 mg/dl, a precision of better than 1.0% of each standard (25, 50, 100, 200 mg/dl) was achieved, and the linearity was excellent (Y = 0.99X - 0.49, r = 0.99). The YSI correlated well with a manual spectrophotometric glucose oxidase method (r = 0.99) and the Kodak Ektachem analyzer (r = 0.98) using human umbilical cord blood samples. Five trained clinical nurses performed all YSI and glucose reagent strip analyses, including all in vitro and patient samples. Four reagent strip methods were compared with the YSI from 104 neonatal heel-stick blood samples: Glucometer II with memory (r = 0.73), Glucostix (r = 0.74), Dextrostix (r = 0.70), and Chemstrip bG (r = 0.83). We conclude that clinical nurses can and do learn to use the YSI with excellent precision and that the YSI represents an improved method for measuring glucose concentrations in the newborn intensive care nursery.

Safety of newborn discharge in less than 36 hours in an indigent population.

The safety of a moderately early newborn discharge program and the ability to follow up within 48 hours of release was evaluated in an indigent population from our institution. A retrospective chart review of 2000 consecutive admissions to our normal newborn nursery was conducted to determine the following: (1) length of nursery stay; (2) reason for extension of nursery stay beyond 36 hours; (3) documentation of outpatient follow-up within 48 hours of discharge for infants released within 36 hours of birth; and (4) incidence of and reason for readmission to the hospital within one week of initial discharge. A total of 1091 infants (54.6%) were discharged within 24 to 36 hours of birth, with documentation of outpatient follow-up in 994 (91.1%). Twenty-five (2.3%) of the early discharges required readmission within seven days of initial discharge compared with a 0.89% incidence among infants hospitalized greater than 48 hours. Twenty-four of the 25 readmissions did have outpatient follow-up, and no serious complications occurred. The data demonstrate that moderately early neonatal discharge can be safely accomplished in an indigent population with the aid of a successful outpatient follow-up program.

Probable graft-vs-graft reaction in an infant after exchange transfusion and marrow transplantation.

A newborn with graft-vs-host (GVH) disease following an exchange transfusion was treated by attempting to eradicate the incompatible graft and to reconstitute the child hematologically and immunologically with a bone marrow transplant. The patient was a female term infant (blood group B, Rh+ Coombs test positive) who received a one-unit group O, Rh- exchange transfusion from an unrelated female donor for hyperbilirubinemia due to ABO incompatibility on day 2. Signs of acute GVH disease began on day 8 and the clinical diagnosis was supported by skin biopsy. With antithymocyte globulin and high dose dexamethasone, the GVH reaction improved somewhat. Cyclophosphamide, 200 mg/kg total dose, was given over four days followed by a marrow graft from a brother who was HLA-A, B identical, and probably also D locus compatible in mixed lymphocyte culture. All signs of GVH resolved with cyclophosphamide treatment and hematologic reconstitution was evident by 14 days after transplant. Two weeks later the GVH reaction and aplastic anemia recurred and Y chromatin was detected in only 6% of marrow cells. The infant died on day 80. Autopsy showed disseminated candidiasis, disseminated cytomegalovirus infection, thymic dysplasia, hypoplastic marrow, and other histopathologic changes consistent with GVH disease. The persistence of female cells in blood and bone marrow and the destruction of the reconstituted marrow suggest that the original incompatible transfusion-derived graft was not eliminated and that it ultimately rejected the histocompatible marrow graft.