Conrad P. D. T. Gillett

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.

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.

Metagenomic sequencing of dung beetle intestinal contents directly detects and identifies mammalian fauna

Cost, time, and expertise constraints limit traditional observation-based comprehensive biodiversity assessment. Therefore, surrogate focal taxa representative of wider biodiversity are commonly used as an imperfect ‘proxy’. Contemporary biodiversity assessments are also increasingly benefiting from the combination of high-throughput sequencing and metagenomic methodologies that enable identification of environmental DNA samples. However, there is a need for empirical studies combining the use of surrogate taxa with metagenomic approaches, that promise rapid and efficient biodiversity assessment. We here tested for the first time the possibility of using the intestinal contents of wild-collected dung beetles (Scarabaeidae) as a source of mammalian DNA, in a metagenomics proof-of-concept approach to directly detect and identify mammals from an area of savanna-scrub in southern Africa. Dung beetles have been purveyed as an indirect proxy measure of mammalian diversity, owing to their dependence upon vertebrate dung as a food source, and the ease with which they can be comprehensively sampled using simple and repeatable trapping protocols, achievable much faster than vertebrate surveys. Following shotgun sequencing of gut content DNA extractions from ten dung beetle species, we used in silico filters to identify mammals by searching the resulting reads against known mammalian mitochondrial DNA from online sequence repositories, matching 546 paired reads to known mitogenomes held in GenBank, and 634 reads to known mammal barcode sequences held in BOLD. Identified mammalian sequences were consistent with wild and domesticated ungulates known from the sampling site, and included blue wildebeest, plains zebra, and domestic cattle and goat. Four dung beetle samples yielded sufficient sequence data to successfully assemble the near-complete mitogenome of blue wildebeest at up to 21 X mean coverage, despite low initial DNA concentrations, unambiguously corroborating identification. It is conceptually and practically possible to rapidly and economically apply metagenomic techniques in dung beetle gut sequencing to detect the presence of mammals upon whose dung the beetles have fed. Since the approach can be readily scaled up, it may prove to be of practical use as a complement to traditional biodiversity assessment methods, and should be tested in usefulness for detecting rare, endangered or cryptic mammal species.

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

Abstract 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.

New Observations on Three Species of Eucraniine Dung Beetles (Coleoptera: Scarabaeidae: Scarabaeinae) in Mendoza and San Juan Provinces, Argentina

Eucraniini is a small monophyletic tribe of scarabaeine dung beetles found exclusively in the Chacoan and Monte biogeographical regions of Argentina (Philips et al. 2002). This endemic Argentinian tribe comprises four genera: Ennearabdus van Lansberge (one species); Eucranium Brullé (six species); Anomiopsoides Blackwelder (four species) and Glyphoderus Westwood (three species) (Ocampo 2004, 2005, 2007, 2010a, b). These beetles all occur in arid zones and inhabit thorny deserts, typically with sand dunes and incomplete vegetation cover, comprising of a variety of flowering herbs, thorny bushes, and grasses. In this environment, these dung beetles display bizarre behavior in comparison to other dung beetles. Some aspects of this are well described in the literature and include unusual diurnal foraging. Their locomotion during foraging is interesting, as during midmornings and late afternoons, they run rapidly over sand dunes, in a zig-zag fashion upon only their middle and hindlegs, whilst holding their forelegs parallel to and above the ground surface. Most reports indicate that dry dung pellets are collected, but also fragments of dried horse dung (Zunino et al. 1989; Ocampo 2004, 2005, 2010b). The dung pellets are grasped by the forelegs as they are carried forwards rather than being formed into a ball and rolled backwards as by, for example, species of Scarabaeus Linnaeus (personal observations). The pellets are taken into burrows preconstructed at an angle into the sand. Eucraniines are reportedly not attracted to pitfall traps baited with fresh dung (Zunino et al. 1989; Monteresino et al. 2003). Only Ennearabdus lobocephalus (Harold, 1868), the sole fully-winged species in the tribe, is reported as being attracted to fresh dung of large mammals, but it still transports pellets of dry dung to its burrow (Ocampo 2010a). In December 2016, we visited the Mendoza and San Juan provinces of western Argentina for two weeks. Six days were spent in the foothills of the Andes around Potrerillos and Uspallata (Mendoza), where surprisingly, despite extensive searching and trapping, no scarab dung beetles, other than two dead specimens (one each of Bolbites onitoides Harold, 1868 and a species of Malagoniella Martı́nez) were found. From the mountains, we moved further north to the Lavalle Department of Mendoza and to Santa Lucı́a in San Juan Province. These areas proved to be more rewarding with regard to dung beetles, and three sites were visited where diurnal activity of eucraniines was observed. Some of the observations made are at variance with, or add additional information to, those already published. They are given below for each of the three species encountered.