De novo assembly and annotation of Popillia japonica's genome with initial clues to its potential as an invasive pest.

BACKGROUND: The spread of Popillia japonica in non-native areas (USA, Canada, the Azores islands, Italy and Switzerland) poses a significant threat to agriculture and horticulture, as well as to endemic floral biodiversity, entailing that appropriate control measures must be taken to reduce its density and limit its further spread. In this context, the availability of a high quality genomic sequence for the species is liable to foster basic research on the ecology and evolution of the species, as well as on possible biotechnologically-oriented and genetically-informed control measures. RESULTS: The genomic sequence presented and described here is an improvement with respect to the available draft sequence in terms of completeness and contiguity, and includes structural and functional annotations. A comparative analysis of gene families of interest, related to the species ecology and potential for polyphagy and adaptability, revealed a contraction of gustatory receptor genes and a paralogous expansion of some subgroups/subfamilies of odorant receptors, ionotropic receptors and cytochrome P450s. CONCLUSIONS: The new genomic sequence as well as the comparative analyses data may provide a clue to explain the staggering invasive potential of the species and may serve to identify targets for potential biotechnological applications aimed at its control.

The direction, timing and demography of Popillia japonica (Coleoptera) invasion reconstructed using complete mitochondrial genomes.

The Japanese beetle Popillia japonica is a pest insect that feeds on hundreds of species of wild and cultivated plants including important fruit, vegetable, and field crops. Native to Japan, the pest has invaded large areas of the USA, Canada, the Azores (Portugal), Italy, and Ticino (Switzerland), and it is considered a priority for control in the European Union. We determined the complete mitochondrial genome sequence in 86 individuals covering the entire distribution of the species. Phylogenetic analysis supports a major division between South Japan and Central/North Japan, with invasive samples coming from the latter. The origin of invasive USA samples is incompatible, in terms of the timing of the event, with a single introduction, with multiple Japanese lineages having been introduced and one accounting for most of the population expansion locally. The origin of the two invasive European populations is compatible with two different invasions followed by minimal differentiation locally. Population analyses provide the possibility to estimate the rate of sequence change from the data and to date major invasion events. Demographic analysis identifies a population expansion followed by a period of contraction prior to the invasion. The present study adds a time and demographic dimension to available reconstructions.

The mitogenome of the true bug Nysius cymoides (Insecta, Heteroptera) and the phylogeny of Lygaeoidea.

The complete mitochondrial genome of the true bug (Homoptera) Nysius cymoides (Spinola, 1837) is herein described and used for phylogenetic comparison with other species of Lygaeoidea. The mtDNA has a gene order and other molecular features typically observed in hexapods, and a long A + T-rich region, due to the occurrence of several repeat units. The phylogenetic analyses support the monophyly of all families except Rhyparochromidae.

The complete mitochondrial genome of Trissolcus japonicus (Hymenoptera: Scelionidae), the candidate for the biological control of Halyomorpha halys (Hemiptera: Pentatomidae).

The samurai wasp Trissolcus japonicus (Ashmead, 1904) is a parasitoid hymenopteran that came into the limelight as the natural enemy of Halyomorpha halys. Here, we present the complete sequence of the mitochondrial genome of the CREATJ laboratory strain, naturally recovered in Italy in 2018. The molecule conforms to the typical model of animal mitochondrial genomes. Gene order is identical to that of its congeneric Trissolcus basalis. Phylogenetic analysis confirms its placement within monophyletic Scelionidae and Telenominae as the sister group of T. basalis.

EZmito: a simple and fast tool for multiple mitogenome analyses.

Complete mitochondrial genome data are frequently applied to address phylogenetic/phylogeographic issues at different taxonomic levels in ecology and evolution. While sample preparation/sequencing is becoming more and more straightforward thanks to dropping costs for next-generation sequencing (NGS), data preparation and visualization remains a manually intensive step that may lead to errors if improperly conducted. We have elaborated, and here introduce, EZmito, a simple and intuitive, freely accessible Web Server aimed at automating some of these tasks. EZmito is divided into three main tools: EZpipe that assembles DNA matrices for phylo-mitogenomic analyses; EZskew that calculates genome, strand, and codon nucleotide compositional skews and EZcodon which computes Relative Synonymous Codon Usage statistics as well as amino acid usage frequency over multiple mitogenomes. Output is produced in tabular format as well as publication-quality graphics.

Evidence for strong environmental control on bacterial microbiomes of Antarctic springtails.

Collembola are a key component of the soil biota globally, playing an important role in community and ecosystem dynamics. Equally significant are their associated microbiomes, that can contribute to key metabolic functions. In the present study, we investigated the bacterial community composition of four Antarctic springtail species to assess if and how the extreme Antarctic environment has shaped the collembolans' microbiomes. Springtails were collected from two biogeographical regions, the maritime and the continental Antarctic. From each region, two endemic species, belonging to the genera Cryptopygus (Isotomidae, Entomobryomorpha) and Friesea (Neanuridae, Poduromorpha), were included. This experimental design allowed us to quantify the relative importance of ecological factors (different regions of occurrence) and/or phylogenetic divergence in the host (different Orders) in shaping the Collembola microbiome. The diversity and richness of springtail microbiomes was lower in the Antarctic taxa compared to published information from species from temperate regions. The microbiome composition was predominantly species-specific, with a limited core microbiome shared across the four species examined. While both geographic origin and host species influenced the associated microbiomes, the former was the prevalent driver, with closer similarity between springtails from the same bioregion than between those belonging to the same genus.

Characterization of the complete mitochondrial genome of Neoasterolepisma foreli (Insecta: Zygentoma: Lepismatidae) and the phylogeny of basal Ectognatha.

The silverfish Neoasterolepisma foreli belongs to the family Lepismatidae within Zygentoma and is well known for the peculiar habit of living in strict association with ant nests (myrmecophily). In this study, we describe its mitochondrial genome, a circular molecule of 15,398 bp including the canonical 13 PCGs, 22 tRNAs, 2 rRNAs, as well as a 403 bp AT-rich region. A phylomitogenomic analysis of the new sequence, alongside basal hexapod mtDNAs, confirmed the monophyly of all orders, with some uncertainty over the position of the enigmatic Tricholepidion gertschi that would make Zygentoma paraphyletic. Neoasterolepisma foreli is recovered in a basal position within family Lepismatidae, at odd with our current understanding of the group that would, in turn, suggest a closer relationship with the genus Lepisma (Mendes, 1991).

Evidence for Cryptic Diversity in the "Pan-Antarctic" Springtail Friesea antarctica and the Description of Two New Species.

The invertebrate terrestrial fauna of Antarctica is being investigated with increasing interest to discover how life interacts with the extreme polar environment and how millions of years of evolution have shaped their biodiversity. Classical taxonomic approaches, complemented by molecular tools, are improving our understanding of the systematic relationships of some species, changing the nomenclature of taxa and challenging the taxonomic status of others. The springtail Friesea grisea has previously been described as the only species with a "pan-Antarctic" distribution. However, recent genetic comparisons have pointed to another scenario. The latest morphological study has confined F. grisea to the sub-Antarctic island of South Georgia, from which it was originally described, and resurrected F. antarctica as a congeneric species occurring on the continental mainland. Molecular data demonstrate that populations of this taxon, ostensibly occurring across Maritime and Continental Antarctica, as well as on some offshore islands, are evolutionarily isolated and divergent and cannot be included within a single species. The present study, combining morphological with molecular data, attempts to validate this hypothesis and challenges the taxonomic status of F. antarctica, suggesting that two additional new species, described here as Friesea gretae sp. nov. and Friesea propria sp. nov., are present in Continental Antarctica.

The complete mitochondrial genome of the springtail Allacma fusca, the internal phylogenetic relationships and gene order of Symphypleona.

Symphypleona (sensu stricto) are a group of Collembola (=springtails) that, despite displaying some variation in gene order, have been poorly investigated under the phylomitogenomic perspective. How families and subfamilies of this taxon are evolutionary related is still partially unknown. For this reason we sequenced, and herein described, the complete mitochondrial genome sequence of Allacma fusca (Sminthuridae). This sequence, alongside others from the literature, is here used to study the phylogenetic relationships among Symphypleona.

Re-Evaluating the Internal Phylogenetic Relationships of Collembola by Means of Mitogenome Data.

Collembola are an ancient and early diverging lineage of basal hexapods that occur in virtually all terrestrial habitats on Earth. Phylogenetic relationships between the different orders of Collembola are fiercely debated. Despite a range of studies and the application of both morphological and genetic approaches (singly or in combination) to assess the evolutionary relationships of major lineages in the group, no consensus has been reached. Several mitogenome sequences have been published for key taxa of the class (and their number is increasing rapidly). Here, we describe two new Antarctic Collembola mitogenomes and compare all complete or semi-complete springtail mitogenome sequences available on GenBank in terms of both gene order and DNA sequence analyses in a genome evolution and molecular phylogenetic framework. With minor exceptions, we confirm the monophyly of Poduromorpha and Symphypleona sensu stricto (the latter placed at the most basal position in the springtail phylogenetic tree), whereas monophyly of Neelipleona and Entomobryomorpha is only supported when a handful of critical taxa in these two lineages are excluded. Finally, we review gene order models observed in the class, as well as the overall mitochondrial nucleotide composition.

Going Deeper into High and Low Phylogenetic Relationships of Protura.

Proturans are small, wingless, soil-dwelling arthropods, generally associated with the early diversification of Hexapoda. Their bizarre morphology, together with conflicting results of molecular studies, has nevertheless made their classification ambiguous. Furthermore, their limited dispersal capability (due to the primarily absence of wings) and their euedaphic lifestyle have greatly complicated species-level identification. Mitochondrial and nuclear markers have been applied herein to investigate and summarize proturan systematics at different hierarchical levels. Two new mitochondrial genomes are described and included in a phylum-level phylogenetic analysis, but the position of Protura could not be resolved with confidence due to an accelerated rate of substitution and extensive gene rearrangements. Mitochondrial and nuclear loci were also applied in order to revise the intra-class systematics, recovering three proturan orders and most of the families/subfamilies included as monophyletic, with the exception of the subfamily Acerentominae. At the species level, most morphologically described species were confirmed using molecular markers, with some exceptions, and the advantages of including nuclear, as well as mitochondrial, markers and morphology are discussed. At all levels, an enlarged taxon sampling and the integration of data from different sources may be of significant help in solving open questions that still persist on the evolutionary history of Protura.

The mitogenome of the jumping bristletail Trigoniophthalmus alternatus (Insecta, Microcoryphia) and the phylogeny of insect early-divergent lineages.

The complete mitochondrial genome of the machilid Trigoniophthalmus alternatus (Silvestri 1904) is herein described and applied to phylogenetic analyses, inclusive of the most early-divergent lineages of hexapods. Both gene content and order generally conform with the organization of the arthropods' mitochondrial genome. One gene translocation involving trnA is the autapomorphic character observed in this species. Another peculiar molecular feature is the long size of the A + T-rich region, due to the occurrence of repeat units. The phylogenetic analyses support the typical placement, along the hexapods' tree, of Ectognatha, Monocondylia and Dicondylia, with Diplura as the adelphotaxon of all true insects.

The mitochondrial genome of the springtail Bourletiella arvalis (Symphypleona, Collembola).

The complete mitochondrial genome of the springtail Bourletiella arvalis (Fitch, 1863) is herein described and applied to a Bayesian phylogenetic analysis, inclusive of all the Collembola mitochondrial DNAs sequenced so far. The gene content and order, as well as the nucleotide composition, conform with the well-known features of hexapods' mitochondrial genomes. The phylogenetic analysis supports the monophyly of Collembola, Poduromorpha, Entomobryomorpha and Symphypleona. However, no mtDNA from Neelipleona is available to date, therefore limiting the application of mitochondrial genomes to further investigate springtail systematics.

Cryptic Diversity Hidden within the Leafminer Genus Liriomyza (Diptera: Agromyzidae).

Leafminer insects of the genus Liriomyza are small flies whose larvae feed on the internal tissue of some of the most important crop plants for the human diet. Several of these pest species are highly uniform from the morphological point of view, meaning molecular data represents the only reliable taxonomic tool useful to define cryptic boundaries. In this study, both mitochondrial and nuclear molecular markers have been applied to investigate the population genetics of some Tunisian populations of the polyphagous species Liriomyza cicerina, one of the most important pest of chickpea cultivars in the whole Mediterranean region. Molecular data have been collected on larvae isolated from chickpea, faba bean, and lentil leaves, and used for population genetics, phylogenetics, and species delimitation analyses. Results point toward high differentiation levels between specimens collected on the three different legume crops, which, according to the species delimitation methods, are also sufficient to define incipient species differentiation and cryptic species occurrence, apparently tied up with host choice. Genetic data have also been applied for a phylogenetic comparison among Liriomyza species, further confirming their decisive role in the systematic studies of the genus.

Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut.

Protozoa, of the taxon Gregarinasina, are a heterogeneous group of Apicomplexa that includes ~1600 species. They are parasites of a large variety of both marine and terrestrial invertebrates, mainly annelids, arthropods and mollusks. Unlike coccidians and heamosporidians, gregarines have not proven to have a negative effect on human welfare; thus, they have been poorly investigated. This study focuses on the molecular identification and phylogeny of the gregarine species found in the midgut of two insect species that are considered as an alternative source of animal proteins for the human diet: the mealworm Tenebrio molitor, and the super-worm Zophobas atratus (Coleoptera: Tenebrionidae). Gregarine specimens were isolated from the gut of both larval and adult stages of T. molitor specimens, as well as from Z. atratus larvae. The morphological analyses were restricted to the identification of the different parasite morpho-types, likely corresponding either to different life-cycle stages or to alternative gregarine species. The samples were also used for the DNA extraction necessary for their genetic characterization. Finally, the efficiency of different molecular markers (i.e., 18S rDNA gene alone or combined with the Internal Transcribed Spacer 1) was assessed when applied either to gregarine species identification and to phylogenetic inference.

Redescription and neotype designation of the Antarctic springtail Folsomotoma octooculata (Collembola: Isotomidae).

The growing interest in Antarctic collembolan species has led us to re-evaluate both the morphological and molecular aspects of different species adapted to the extreme Antarctic environment. The genus Folsomotoma includes nine species mainly distributed in Antarctic and subantarctic areas and a few additionally from Australia and South America. We herein designate the neotype of Folsomotoma octooculata (Willem 1901) and redescribe its morphological characters with particular emphasis on the sensory and ordinary chaetotaxy. Furthermore, based on analyses of both our new and previously published morphological data for F. octooculata, we propose a systematic key of the species for the genus.

A new cave-dwelling species of Deuteraphorura from northern Italy (Collembola, Onychiuridae).

A new cave-dwelling species, Deuteraphorura pierolucciisp. n., is described from northern Italy. The size of adult specimens, number, and distribution of dorsal and ventral pseudocelli, and number of compound vesicles of the post antennal organ were used to distinguish it among other congeneric species.

First record of gregarines (Apicomplexa) in seminal vesicle of insect.

Gregarines (Apicomplexa) are a diverse group of protozoan parasites, which infects gut and other body cavities of invertebrate hosts. In reproductive system of insects, gregarine has been reported only in the accessory glands and spermathecae of females; therefore, this is the first report of a gregarine species in seminal vesicles of insects. Different developmental stages, including sporozoytes, oocysts and trophozoites were described from morphological descriptions using light and electron transmission microscopy. The parasites were described in seminal vesicles of the beetle Tribolium castaneum a model organism and an important insect pest. DNA sequence analysis suggests that the protozoan parasite was an Ascogregarina sp.

MtPAN(3): site-class specific amino acid replacement matrices for mitochondrial proteins of Pancrustacea and Collembola.

Phylogenetic analyses of Pancrustacea have generally relied on empirical models of amino acid substitution estimated from large reference datasets and applied to the entire alignment. More recently, following the observation that different sites, or groups of sites, may evolve under different evolutionary constraints, methods have been developed to deal with site or site-class specific models. A set of three matrices has been here developed based on an alignment of complete mitochondrial pancrustacean genomes partitioned using an unsupervised clustering procedure acting over per-site physiochemical properties. The performance of the proposed matrix set - named MtPAN(3) - was compared to relevant single matrix models (MtZOA, MtART, MtPAN) under ML and BI. While the application of the new model does not solve some of the topological problems frequently encountered with pancrustacean mitogenomic phylogenetic analyses, MtPAN(3) largely outperforms its competitors based on AIC and Bayes factors, indicating a significantly improved fit to the empirical data. The applicability of the new model, as well as of multiple matrix models in general, is discussed and an R/BioPerl script that implements the procedure is provided.

Repeated regions in mitochondrial genomes: distribution, origin and evolutionary significance.

All complete or nearly complete mitochondrial genomes of Metazoa (2819) have been subject to bioinformatic analysis to investigate the distribution and features of repeated and palindromic sequences. Repeats are ubiquitous, with 29.9% of genomes containing at least one and 1.95% of total genome length being repeated. Repeat boundaries were tested for the presence of secondary structure motifs, consensus sequences or small repeats, features generally reported as associated with duplications. No significant relationship was detected, suggesting the non ubiquitousness of such features. A mechanism related to gene conversion is proposed to explain the origin of small interspersed repeats.