NGS applications to understand invertebrate biodiversity of Antarctica and mechanisms of gene expression involved in climatic changes

Human activities, such as greenhouse emissions and pollution, are leading to global warming, environmental changes and biodiversity reduction. Pristine environments such as those of Antarctica are not immune to these phenomena, as is noticeable from the temperature shifts and ice-melting registered within the continent in recent decades. To date, many scientists focused on how marine species react to these changes but no molecular data are currently available for continental terrestrial invertebrates and in particular for Collembola (=springtails). Therefore, part of my PhD project was to study the transcriptomic response of the endemic Antarctic springtail Cryptopygus terranovus following a mid-term exposure of 20 days at 18°C. Expression data were compared with wild specimens sampled in native environment. Although individual plasticity in transcript modulation was recorded, several pathways appear to be differentially modulated: protein catabolism, fatty acid metabolism and a sexual response characterized by spermatid development were induced, while lipid catabolism was downregulated in treated samples. Moreover, the temperature experienced by these micro-invertebrates is a pivotal parameter to understand these animals' ecology and physiology. However, at present, detailed knowledge of microhabitat physical conditions in Antarctica is limited and biased towards sub-Antarctic and maritime Antarctic regions. To better understand these temperature conditions, it was analysed a year-round temperature data in ponds and soils in an area of the Victoria Land coast, comparing these measurements with air temperatures from the closest automatic weather station. Important difference in temperature dynamics between the air, soil and pond datasets was registered. Ponds were the warmest sites overall, mostly differing with the air temperatures due to their greater thermal capacity, which also influenced their patterns of freeze-thaw cycles and mean daily thermal excursion. Furthermore, to better understand the biodiversity of Collembola two new mitochondrial genomes of Antarctic springtails were sequenced and analysed. They were employed to revise the entire systematic of the class, its nucleotide composition and genome arrangement by comparing them to all the available sequences deposited in Genbank. In the phylogenetic analysis, with minor exceptions, it was confirmed the monophyly of Poduromorpha and Symphypleona sensu stricto (the latter recovered as the most basal group in the springtail phylogenetic tree), whereas monophyly of Neelipleona and Entomobryomorpha was only supported when some critical taxa in these two lineages were excluded. The genome arrangement review allowed to identify four new gene orders (one from the newly sequenced Tullbergia mixta), for a total of 16 models. Finally, nucleotide composition analyses confirmed the low AT bias in Collembola mitochondrial DNA respect to other Hexapoda, and that third codon position is inclined to mutation accumulation, especially in 4-fold amino-amino acids. To ease the process of mitochondrial genome analyses, it was created a web resource named EZmito, a free web server useful to analyse mitochondrial genomes. It is composed of five main tools: (i) EZsplit, useful to download and format sequences directly from the NCBI database; (ii) EZpipe, a pipeline designed to format mitochondrial sequences before the phylogenetic analysis; (iii) EZskew, which helps users to calculate nucleotide biases; (iv) EZcodon, a fast tool which calculates the Relative Synonym Codon Usage of different mtDNA species and (v) EZmix, which recognizes areas of inter molecular similarity indicative of the assembly of chimeric mitochondrial genomes. Interestingly, to date, the most used tool within the hub is EZcodon, followed by EZsplit and EZpipe

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 mitochon- drial DNAs sequenced so far. The gene content and order, as well as the nucleotide composition, con- form 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 mito- chondrial genomes to further investigate springtail systematics

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 Tenebriomolitor, and the super-worm Zophobasatratus (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

Molecular comparison among three Antarctic endemic springtail species and description of the mitochondrial genome of Friesea gretae (Hexapoda, Collembola)

Springtails and mites are the dominant groups of terrestrial arthropods in Antarctic terrestrial ecosystems. Their Antarctic diversity includes a limited number of species, which are frequently endemic to specific regions within the continent. Advances in molecular techniques, combined with the re-evaluation of morphological characters and the availability of new samples, have recently led to the identification of a number of new springtail species within previously named, but ill-defined, species entities described in the last century. One such species, the neanurid Friesea grisea, originally described from sub-Antarctic South Georgia, was for many years considered to be the only known springtail with a pan-Antarctic distribution. With the recent availability of new morphological and molecular data, it has now been firmly established that the different representatives previously referred to this taxon from the Antarctic Peninsula and Victoria Land (continental Antarctica) should no longer be considered as representing one and the same species, and three clearly distinct taxa have been recognized: F. antarctica, F. gretae and F. propria. In this study, the relationships among these three species are further explored through the sequencing of the complete mtDNA for F. gretae and the use of complete mitogenomic as well as cytochrome c oxidase I data. The data obtained provide further support that distinct species were originally hidden within the same taxon and that, despite the difficulties in obtaining reliable diagnostic morphological characters, F. gretae is genetically differentiated from F. propria (known to be present in different locations in Northern Victoria Land), as well as from F. antarctica (distributed in the Antarctic Peninsula)

Nanoscale transient polarization gratings

We present the generation of transient polarization gratings at the nanoscale, achieved using a tailored accelerator configuration of the FERMI free electron laser. We demonstrate the capabilities of such a transient polarization grating by comparing its induced dynamics with the ones triggered by a more conventional intensity grating on a thin film ferrimagnetic alloy. While the signal of the intensity grating is dominated by the thermoelastic response of the system, such a contribution is suppressed in the case of the polarization grating. This exposes helicity-dependent magnetization dynamics that have so-far remained hidden under the large thermally driven response. We anticipate nanoscale transient polarization gratings to become useful for the study of any physical, chemical and biological systems possessing chiral symmetry

A glimpse into the past : phylogenesis and protein domain analysis of the group XIV of C-type lectins in vertebrates

Background The group XIV of C-type lectin domain-containing proteins (CTLDcps) is one of the seventeen groups of CTLDcps discovered in mammals and composed by four members: CD93, Clec14A, CD248 and Thrombomodulin, which have shown to be important players in cancer and vascular biology. Although these proteins belong to the same family, their phylogenetic relationship has never been dissected. To resolve their evolution and characterize their protein domain composition we investigated CTLDcp genes in gnathostomes and cyclostomes and, by means of phylogenetic approaches as well as synteny analyses, we inferred an evolutionary scheme that attempts to unravel their evolution in modern vertebrates. Results Here, we evidenced the paralogy of the group XIV of CTLDcps in gnathostomes and discovered that a gene loss of CD248 and Clec14A occurred in different vertebrate groups, with CD248 being lost due to chromosome disruption in birds, while Clec14A loss in monotremes and marsupials did not involve chromosome rearrangements. Moreover, employing genome annotations of different lampreys as well as one hagfish species, we investigated the origin and evolution of modern group XIV of CTLDcps. Furthermore, we carefully retrieved and annotated gnathostome CTLDcp domains, pointed out important differences in domain composition between gnathostome classes, and assessed codon substitution rate of each domain by analyzing nonsynonymous (Ka) over synonymous (Ks) substitutions using one representative species per gnathostome order. Conclusions CTLDcps appeared with the advent of early vertebrates after a whole genome duplication followed by a sporadic tandem duplication. These duplication events gave rise to three CTLDcps in the ancestral vertebrate that underwent further duplications caused by the independent polyploidizations that characterized the evolution of cyclostomes and gnathostomes. Importantly, our analyses of CTLDcps in gnathostomes revealed critical inter-class differences in both extracellular and intracellular domains, which might help the interpretation of experimental results and the understanding of differences between animal models

Polystyrene shaping effect on the enriched bacterial community from the plastic‑eating Alphitobius diaperinus (Insecta: Coleoptera)

Plastic pollution has become a serious issue of global concern, and biodegradation of plastic wastes is representing one attractive environment-friendly alternative to traditional disposal paths. It is known that insects are involved in the plastic polymer degradation process, with reported evidence of tenebrionid beetle larvae capable to degrade polystyrene (PS), one of the most used plastics worldwide. Recently, a ribosomal RNA based survey on the insect gut microbiota of the lesser mealworm Alphitobius diaperinus has revealed diferentially abundant microbial taxa between PS-fed larvae and control group. Following these fndings, an enrichment bacterial culture was set up in liquid carbon-free basal medium with PS flm as sole carbon source using PS-fed larvae of A. diaperinus as inoculum. After two-months the culture was analysed both by molecular and culture-based methods. Isolated bacteria which had become prevalent under the selective enrichment conditions resulted ascribable to three taxonomic groups: Klebsiella, Pseudomonas, and Stenothrophomonas. The predominance of these groups in PS-fed larvae was confrmed by using bacterial 16S rRNA gene amplicon sequencing, and it was consistent with the results of previous reports. Isolated bacteria were able to attach to PS surfaces and SEM observations showed the presence of thin fbrillar structures connecting the bacterial cells to the abiotic surface

Bacterial and fungal diversity in the gut of polystyrene-fed Alphitobius diaperinus (Insecta: Coleoptera)

The use of plastics burgeoned in the last decades to become an essential component of our society. An envi-ronment friendly method to dispose of plastic waste is not available yet, to the outcome that these accumulate in landfills or are scattered as microplastics. New researches reported that some coleopteran species are able to destroy plastics thanks to their chewing mouthparts and the metabolic activity of their gut microbiota. This study shows that the lesser mealworm Alphitobius diaperinus is capable of feeding on, and apparently degrading, polystyrene. The gut microbiota of polystyrene-fed larvae was characterized using an NGS metagenomic approach, targeting both bacteria and fungi. Several microbe taxa emerged as differentially abundant between treatment and control groups (Cronobacter, Kocuria and Pseudomonas as bacteria, Aspergillus, Hyphodermella, Trichoderma as fungi). Some of them have been found in association with plastic compounds and/or have been proposed to be capable of plastic degradation. This research supports the notion that, although synthetic mol-ecules, unlike most natural compounds, do not generally enter the natural food chain to be degraded by the environmental microbiota, some microbial communities may be able to decompose plastics. We speculate that, once identified, such communities may open to the possibility of devising bioreactors for plastic degradation

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 (¼springtails) 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 Symphypleon