Parasitological research in the molecular age.

New technological methods, such as rapidly developing molecular approaches, often provide new tools for scientific advances. However, these new tools are often not utilized equally across different research areas, possibly leading to disparities in progress between these areas. Here, we use empirical evidence from the scientific literature to test for potential discrepancies in the use of genetic tools to study parasitic vs non-parasitic organisms across three distinguishable molecular periods, the allozyme, nucleotide and genomics periods. Publications on parasites constitute only a fraction (<5%) of the total research output across all molecular periods and are dominated by medically relevant parasites (especially protists), particularly during the early phase of each period. Our analysis suggests an increasing complexity of topics and research questions being addressed with the development of more sophisticated molecular tools, with the research focus between the periods shifting from predominantly species discovery to broader theory-focused questions. We conclude that both new and older molecular methods offer powerful tools for research on parasites, including their diverse roles in ecosystems and their relevance as human pathogens. While older methods, such as barcoding approaches, will continue to feature in the molecular toolbox of parasitologists for years to come, we encourage parasitologists to be more responsive to new approaches that provide the tools to address broader questions.

Reproductive isolation and environmental adaptation shape the phylogeography of mountain pine beetle (Dendroctonus ponderosae).

Chromosomal rearrangement can be an important mechanism driving population differentiation and incipient speciation. In the mountain pine beetle (MPB, Dendroctonus ponderosae), deletions on the Y chromosome that are polymorphic among populations are associated with reproductive incompatibility. Here, we used RAD sequencing across the entire MPB range in western North America to reveal the extent of the phylogeographic differences between Y haplotypes compared to autosomal and X-linked loci. Clustering and geneflow analyses revealed three distinct Y haplogroups geographically positioned within and on either side of the Great Basin Desert. Despite close geographic proximity between populations on the boundaries of each Y haplogroup, there was extremely low Y haplogroup mixing among populations, and gene flow on the autosomes was reduced across Y haplogroup boundaries. These results are consistent with a previous study suggesting that independent degradation of a recently evolved neo-Y chromosome in previously isolated populations causes male sterility or inviability among Y haplotype lineages. Phylogeographic results supported historic contraction of MPB into three separate Pleistocene glacial refugia followed by postglacial range expansion and secondary contact. Distinct sets of SNPs were statistically associated with environmental data among the most genetically distinct sets of geographic populations. This finding suggests that the process of adaptation to local climatic conditions is influenced by population genetic structure, with evidence for largely independent evolution in the most genetically isolated Y haplogroup.

Morphological differentiation despite gene flow in an endangered grasshopper.

BACKGROUND: Gene flow is traditionally considered a limitation to speciation because selection is required to counter the homogenising effect of allele exchange. Here we report on two sympatric short-horned grasshoppers species in the South Island of New Zealand; one (Sigaus australis) widespread and the other (Sigaus childi) a narrow endemic. RESULTS: Of the 79 putatively neutral markers (mtDNA, microsatellite loci, ITS sequences and RAD-seq SNPs) all but one marker we examined showed extensive allele sharing, and similar or identical allele frequencies in the two species where they co-occur. We found no genetic evidence of deviation from random mating in the region of sympatry. However, analysis of morphological and geometric traits revealed no evidence of morphological introgression. CONCLUSIONS: Based on phenotype the two species are clearly distinct, but their genotypes thus far reveal no divergence. The best explanation for this is that some loci associated with the distinguishing morphological characters are under strong selection, but exchange of neutral loci is occurring freely between the two species. Although it is easier to define species as requiring a barrier between them, a dynamic model that accommodates gene flow is a biologically more reasonable explanation for these grasshoppers.

Fossil-calibrated phylogenies of Southern cave weta show dispersal and extinction confound biogeographic signal.

The biota of continents and islands are commonly considered to have a source-sink relationship, but small islands can harbour distinctive taxa. The distribution of four monotypic genera of Orthoptera on young subantarctic islands indicates a role for long-distance dispersal and extinction. Phylogenetic relationships were inferred from whole mtDNA genomes and nuclear sequences (45S cassette; four histones). We used a fossil and one palaeogeographic event to calibrate molecular clock analysis. We confirm that neither the Australian nor Aotearoa-New Zealand Rhaphidophoridae faunas are monophyletic. The radiation of Macropathinae may have begun in the late Jurassic, but trans-oceanic dispersal is required to explain the current distribution of some lineages within this subfamily. Dating the most recent common ancestor of seven island endemic species with their nearest mainland relative suggests that each existed long before their island home was available. Time estimates from our fossil-calibrated molecular clock analysis suggest several lineages have not been detected on mainland New Zealand, Australia, or elsewhere most probably due to their extinction, providing evidence that patterns of extinction, which are not consistently linked to range size or lineage age, confound biogeographic signal.

Genome assembly and annotation of the mermithid nematode Mermis nigrescens.

Genetic studies of nematodes have been dominated by Caenorhabditis elegans as a model species. A lack of genomic resources has limited the expansion of genetic research to other groups of nematodes. Here, we report a draft genome assembly of a mermithid nematode, Mermis nigrescens. Mermithidae are insect parasitic nematodes with hosts including a wide range of terrestrial arthropods. We sequenced, assembled, and annotated the whole genome of M. nigrescens using nanopore long reads and 10X Chromium link reads. The assembly is 524 Mb in size consisting of 867 scaffolds. The N50 value is 2.42 Mb, and half of the assembly is in the 30 longest scaffolds. The assembly BUSCO score from the eukaryotic database (eukaryota_odb10) indicates that the genome is 86.7% complete and 5.1% partial. The genome has a high level of heterozygosity (6.6%) with a repeat content of 83.98%. mRNA-seq reads from different sized nematodes (≤2 cm, 3.5-7 cm, and >7 cm body length) representing different developmental stages were also generated and used for the genome annotation. Using ab initio and evidence-based gene model predictions, 12,313 protein-coding genes and 24,186 mRNAs were annotated. These genomic resources will help researchers investigate the various aspects of the biology and host-parasite interactions of mermithid nematodes.

Standard metabolic rate variation among New Zealand Orthoptera.

Standard metabolic rates (SMR) of ectotherms reflect the energetic cost of self-maintenance and thus provide important information about life-history strategies of organisms. We examined variation in SMR among fifteen species of New Zealand orthopteran. These species represent a heterogeneous group with a wide geographic distribution, differing morphologies and life histories. Gathering original data on morphological and physiological traits of individual species is a first step towards understanding existing variability. Individual metabolic rates of ectotherms are one of the first traits to respond to climate change. Baseline SMR datasets are valuable for modeling current species distributions and their responses to a changing climate. At higher latitudes, the average environmental temperature decreases. The pattern that cold-adapted ectotherms display higher SMR at colder temperatures and greater thermal sensitivity to compensate for lower temperatures and the shorter growing and reproductive seasons is predicted from the metabolic cold adaptation (MCA) hypothesis. We predict higher SMR for the orthopteran species found at higher latitudes. We further compared the index of thermal sensitivity Q10 per species. We used closed-system respirometry to measure SMR, at two test temperatures (4 °C and 14 °C), for the fifteen species acclimated to the same conditions. As expected, we found significant differences in SMR among species. The rate of oxygen consumption was positively correlated with body mass. Our findings do not support the MCA hypothesis. In fact, we found evidence of co-gradient variation in SMR, whereby insects from higher elevations and latitudes presented lower SMR. We discuss our findings in relation to life histories and ecology of each species. The novel physiological data presented will aid in understanding potential responses of these unusual species to changing climatic conditions in Aotearoa/New Zealand.

crabs-A software program to generate curated reference databases for metabarcoding sequencing data.

The measurement of biodiversity is an integral aspect of life science research. With the establishment of second- and third-generation sequencing technologies, an increasing amount of metabarcoding data is being generated as we seek to describe the extent and patterns of biodiversity in multiple contexts. The reliability and accuracy of taxonomically assigning metabarcoding sequencing data have been shown to be critically influenced by the quality and completeness of reference databases. Custom, curated, eukaryotic reference databases, however, are scarce, as are the software programs for generating them. Here, we present crabs (Creating Reference databases for Amplicon-Based Sequencing), a software package to create custom reference databases for metabarcoding studies. crabs includes tools to download sequences from multiple online repositories (i.e., NCBI, BOLD, EMBL, MitoFish), retrieve amplicon regions through in silico PCR analysis and pairwise global alignments, curate the database through multiple filtering parameters (e.g., dereplication, sequence length, sequence quality, unresolved taxonomy, inclusion/exclusion filter), export the reference database in multiple formats for immediate use in taxonomy assignment software, and investigate the reference database through implemented visualizations for diversity, primer efficiency, reference sequence length, database completeness and taxonomic resolution. crabs is a versatile tool for generating curated reference databases of user-specified genetic markers to aid taxonomy assignment from metabarcoding sequencing data. crabs can be installed via docker and is available for download as a conda package and via GitHub (https://github.com/gjeunen/reference_database_creator).

The proof is in the poo: Non-invasive method to detect endoparasitic infection.

Almost every animal trait is strongly associated with parasitic infection or the potential exposure to parasites. Despite this importance, one of the greatest challenges that researchers still face is to accurately determine the status and severity of the endoparasitic infection without killing and dissecting the host. Thus, the precise detection of infection with minimal handling of the individual will improve experimental designs in live animal research. Here, we quantified extracellular DNA from two species of endoparasitic worm that grow within the host body cavity, hairworms (phylum Nematomorpha) and mermithids (phylum Nematoda), from the frass of their insect host, a cave weta (Orthoptera: Rhaphidophoridae) and an earwig (Dermaptera: Forficulidae), respectively. Frass collection was done at two successive time periods, to test if parasitic growth correlated with relative DNA quantity in the frass. We developed and optimized two highly specific TaqMan assays, one for each parasite-specific DNA amplification. We were able to detect infection prevalence with 100% accuracy in individuals identified as infected through post-study dissections. An additional infection in earwigs was detected with the TaqMan assay alone, probably because some worms were either too small or degraded to observe during dissection. No difference in DNA quantity was detected between sampling periods, although future protocols could be refined to support such a trend. This study demonstrates that a noninvasive and minimally stressful method can be used to detect endoparasitic infection with greater accuracy than dissection alone, helping improve protocols for live animal studies.

Genome assembly and annotation of the European earwig Forficula auricularia (subspecies B).

The European earwig Forficula auricularia is an important model for studies of maternal care, sexual selection, sociality, and host-parasite interactions. However, detailed genetic investigations of this species are hindered by a lack of genomic resources. Here, we present a high-quality hybrid genome assembly for Forficula auricularia using Nanopore long-reads and 10× linked-reads. The final assembly is 1.06 Gb in length with 31.03% GC content. It consists of 919 scaffolds with an N50 of 12.55 Mb. Half of the genome is present in only 20 scaffolds. Benchmarking Universal Single-Copy Orthologs scores are ∼90% from 3 sets of single-copy orthologs (eukaryotic, insect, and arthropod). The total repeat elements in the genome are 64.62%. The MAKER2 pipeline annotated 12,876 protein-coding genes and 21,031 mRNAs. Phylogenetic analysis revealed the assembled genome as that of species B, one of the 2 known genetic subspecies of Forficula auricularia. The genome assembly, annotation, and associated resources will be of high value to a large and diverse group of researchers working on dermapterans.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

The Adaptiveness of Host Behavioural Manipulation Assessed Using Tinbergen's Four Questions.

Host organisms show altered phenotypic reactions when parasitised, some of which result from adaptive host manipulation, a phenomenon that has long been debated. Here, we provide an overview and discuss the rationale in distinguishing adaptive versus nonadaptive host behavioural manipulation. We discuss Poulin's criteria of adaptive host behavioural manipulation within the context of Tinbergen's four questions of ethology, while highlighting the importance of both the proximate and evolutionary explanations of such traits. We also provide guidelines for future studies exploring the adaptiveness of host behavioural manipulation. Through this article, we seek to encourage researchers to consider both the proximate and ultimate causes of host behavioural manipulation to infer on the adaptiveness of such traits.

The Apennines as a cryptic Pleistocene refugium of the bark beetle Pityogenes chalcographus (Coleoptera: Curculionidae).

The Apennine Mountains in Italy are an important biogeographical region and of particular interest in phylogeographical research, because they have been a refugium during Pleistocene glaciation events for numerous European species. We performed a genetic study on the Eurasian bark beetle Pityogenes chalcographus (Linnaeus, 1760), focusing on two Apennine (Italian) and two Central European (Austrian) locations to assess the influence of the Apennines in the evolutionary history of the beetle, particularly during the Pleistocene. We analysed a part of the mitochondrial COI gene and a set of 5470 informative genome-wide markers to understand its biogeography. We found 75 distinct mitochondrial haplotypes, which are structured in three main clades. In general, the Apennine locations harbour a higher number of mitochondrial clades than Central European sites, with one specific clade exclusively detected in the Apennines. Analysis of our genome-wide, multi-locus dataset reveals a clustering of P. chalcographus by geography, with Italian individuals clearly separated from Austrian samples. Our data highlight the significance of the Apennines for the genetic diversity of P. chalcographus and support the hypothesis that this area was an important refugium during unfavourable conditions in the Pleistocene. We discuss additional life-history traits and processes that shaped the evolution of this widespread beetle.

Pleistocene climate cycling and host plant association shaped the demographic history of the bark beetle Pityogenes chalcographus.

Historical climatic oscillations and co-evolutionary dependencies were key evolutionary drivers shaping the current population structure of numerous organisms. Here, we present a genome-wide study on the biogeography of the bark beetle Pityogenes chalcographus, a common and widespread insect in Eurasia. Using Restriction Associated DNA Sequencing, we studied the population structure of this beetle across a wide part of its western Palaearctic range with the goal of elucidating the role of Pleistocene glacial-interglacial cycling and its close relationship to its main host plant Norway spruce. Genetic distance among geographic sites was generally low, but clustering analysis revealed three genetically distinct groups, that is, southern, central/south-eastern, and north-eastern locations. Thus, three key P. chalcographus glacial refugia were identified: in the Italian-Dinaric region, the Carpathians, and the Russian plain, shared with its main host. The current phylogeographic signal was affected by genetic divergence among geographically isolated refugia during glacial periods and postglacial re-establishment of genetic exchange through secondary contact, reflected by admixture among genetic groups. Additionally, certain life history traits, like the beetle's dispersal and reproductive behaviour, considerably influenced its demographic history. Our results will help to understand the biogeography of other scolytine beetles, especially species with similar life history traits.

Targeted gene enrichment and high-throughput sequencing for environmental biomonitoring: a case study using freshwater macroinvertebrates.

Recent studies have advocated biomonitoring using DNA techniques. In this study, two high-throughput sequencing (HTS)-based methods were evaluated: amplicon metabarcoding of the cytochrome C oxidase subunit I (COI) mitochondrial gene and gene enrichment using MYbaits (targeting nine different genes including COI). The gene-enrichment method does not require PCR amplification and thus avoids biases associated with universal primers. Macroinvertebrate samples were collected from 12 New Zealand rivers. Macroinvertebrates were morphologically identified and enumerated, and their biomass determined. DNA was extracted from all macroinvertebrate samples and HTS undertaken using the illumina miseq platform. Macroinvertebrate communities were characterized from sequence data using either six genes (three of the original nine were not used) or just the COI gene in isolation. The gene-enrichment method (all genes) detected the highest number of taxa and obtained the strongest Spearman rank correlations between the number of sequence reads, abundance and biomass in 67% of the samples. Median detection rates across rare (<1% of the total abundance or biomass), moderately abundant (1-5%) and highly abundant (>5%) taxa were highest using the gene-enrichment method (all genes). Our data indicated primer biases occurred during amplicon metabarcoding with greater than 80% of sequence reads originating from one taxon in several samples. The accuracy and sensitivity of both HTS methods would be improved with more comprehensive reference sequence databases. The data from this study illustrate the challenges of using PCR amplification-based methods for biomonitoring and highlight the potential benefits of using approaches, such as gene enrichment, which circumvent the need for an initial PCR step.

Assessing the effects of salmon farming seabed enrichment using bacterial community diversity and high-throughput sequencing.

Aquaculture is an extremely valuable and rapidly expanding sector of the seafood industry. The sediment below active aquaculture farms receives inputs of organic matter from uneaten food and faecal material and this has led to concerns related to environmental sustainability. The impacts of organic enrichment on macrobenthic infauna are well characterized; however, much less is known about effect on bacterial communities. In this study, sediment, macrobenthic infauna samples and environmental data were collected along an enrichment gradient radiating out from a Chinook salmon (Oncorhynchus tshawytscha) farm (Marlborough Sounds; New Zealand). DNA and RNA were extracted and 16S rRNA metabarcodes from bacterial communities characterized using high-throughput sequencing. Desulfobacterales dominated at the cage (DNA and RNA), and at sites 50 m (DNA and RNA) and 150 m (RNA) from the farm. In contrast, unclassified bacteria from the class Gammaproteobacteria were the most abundant taxa at control sites (625 and 4000 m). Pronounced differences among DNA and RNA samples occurred at the cage site where Desulfobacterales abundance was markedly higher in RNA samples. There were strong correlations between shifts in bacterial communities and total organic matter and redox. This suggests that bacterial composition is strongly influenced by organic enrichment, a trait that may make them useful for assessing impacts associated with aquaculture farms.