Development of biomimetic in vitro models of large airways

Routinely adopted in vitro models of the respiratory tract do not reproduce the complexity associated in vivo with the cellular milieu, such as the presence of an extracellular matrix (ECM), the crosstalk between multiple cell types, and the inclusion of physiological mechanical stimuli. In this work, different strategies were assessed for the development of a biomimetic, high-throughput in vitro model of the human primary and secondary bronchi, replicating the complexity of the cellular environment as observed in vivo whilst maintaining cost-efficiency and reproducibility. A first strategy centred on the seeding of human bronchial epithelial cells (HBECs) on decellularised bronchial tissue. However, low cell adherence on the tissue-derived scaffolds was reported. A second approach was based on the use of a commercial decellularised lung-derived hydrogel termed “DLH”. Whilst HBECs adhered on the hydrogel, they did not reach confluency. A third strategy was centred on the use of commercial high-throughput microfluidic chips. In these platforms, HBECs adhered and proliferated. Additionally, the inclusion of fluid flow increased cell survival and proliferation. However, issues associated with the contraction of the hydrogel included in the platforms and cell death at late timepoints impeded the onset of mucociliary differentiation. Finally, a fourth strategy centred on the use of high-throughput screening (HTS), 96-well plate-format transwell® systems seeded with HBECs, in co-culture with human lung fibroblasts (HLFs) seeded on DLH. The DLH did not have any adverse paracrine effects on the mucociliary differentiation of HBECs. However, variable results were obtained with regards to the seeding of HLFs on the DLH, as the cells displayed a spindle-like or a circular shape in separate experiments. Whilst further optimisation is necessary, the combination of HBECs, HLFs and DLH on HTS transwell® systems proved to be a promising strategy for the development of a biomimetic bronchial in vitro model

Strength and Limitations of DNA Barcode under the Multidimensional Species Perspective

DNA barcoding aims at providing an efficient method for specieslevel separation using a partial sequence of the mitochondrial COI gene. The efficiency of the barcode in separating species is based on the amount of genetic distance among samples. While in many taxa the species can be efficiently identified through the barcode, other situations cannot be treated by this approach. The causes for such discrepancy appear to be mostly related to the nature of speciation events and to the different roles of the genetic system, natural selection and evolutionary time. Thus, DNA barcode represent just one important descriptor in the framework of the multidimensional species approach

Unveiling an ancient biological invasion: molecular analysis of an old European alien, the crested porcupine (Hystrix cristata)

<p>Abstract</p> <p>Background</p> <p>Biological invasions can be considered one of the main threats to biodiversity, and the recognition of common ecological and evolutionary features among invaders can help developing a predictive framework to control further invasions. In particular, the analysis of successful invasive species and of their autochthonous source populations by means of genetic, phylogeographic and demographic tools can provide novel insights into the study of biological invasion patterns. Today, long-term dynamics of biological invasions are still poorly understood and need further investigations. Moreover, distribution and molecular data on native populations could contribute to the recognition of common evolutionary features of successful aliens.</p> <p>Results</p> <p>We analyzed 2,195 mitochondrial base pairs, including Cytochrome <it>b</it>, Control Region and rRNA 12S, in 161 Italian and 27 African specimens and assessed the ancient invasive origin of Italian crested porcupine (<it>Hystrix cristata</it>) populations from Tunisia. Molecular coalescent-based Bayesian analyses proposed the Roman Age as a putative timeframe of introduction and suggested a retention of genetic diversity during the early phases of colonization. The characterization of the native African genetic background revealed the existence of two differentiated clades: a Mediterranean group and a Sub-Saharan one. Both standard population genetic and advanced molecular demography tools (Bayesian Skyline Plot) did not evidence a clear genetic signature of the expected increase in population size after introduction. Along with the genetic diversity retention during the bottlenecked steps of introduction, this finding could be better described by hypothesizing a multi-invasion event.</p> <p>Conclusion</p> <p>Evidences of the ancient anthropogenic invasive origin of the Italian <it>Hystrix cristata </it>populations were clearly shown and the native African genetic background was preliminary described. A more complex pattern than a simple demographic exponential growth from a single propagule seems to have characterized this long-term invasion.</p

Make it simple: mating behaviour of Euphydryas aurinia provincialis (Lepidoptera: Nymphalidae)

Courtship in butterflies can vary among species, from simple to elaborate displays, which are thought to promote conspecific mating and drive a female, at first unreceptive, to eventually mate. In this paper, we studied the courtship behaviour of a population of E. aurinia provincialis (Nymphalidae) of the Italian region Latium, finding clear differences in acceptance/refusal of females and in consequent departure/persistence of males. Despite the presence of a period of female unreceptivity after eclosion, virgin females usually do not show resistance, and accept to mate after a simple courtship. Once males meet a female, they immediately try to copulate clasping female genitalia, and proceed in mating performing long copulations. They also secrete a mating plug to prevent subsequent matings. On the contrary, already-mated females can strongly reject any approaching male and they always display Wide Wing fluttering, which is a behavioural pattern usually followed by male departure. In case of persistent males, prolonged and energy demanding sexual interactions, as Carrousels, can occur, with rare final copulation. Here, we analyse the effects of female refusal on male sexual behaviour and discuss potential selective pressures that might be acting on such behavioural traits

Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a 'time-dependent' mitochondrial substitution rate

Genetic data are currently providing a large amount of new information on past distribution of species and are contributing to a new vision of Pleistocene ice ages. Nonetheless, an increasing number of studies on the 'time dependency' of mutation rates suggest that date assessments for evolutionary events of the Pleistocene might be overestimated. We analysed mitochondrial (mt) DNA (COI) sequence variation in 225 Parnassius mnemosyne individuals sampled across central and eastern Europe in order to assess (i) the existence of genetic signatures of Pleistocene climate shifts; and (ii) the timescale of demographic and evolutionary events. Our analyses reveal a phylogeographical pattern markedly influenced by the Pleistocene/Holocene climate shifts. Eastern Alpine and Balkan populations display comparatively high mtDNA diversity, suggesting multiple glacial refugia. On the other hand, three widely distributed and spatially segregated lineages occupy most of northern and eastern Europe, indicating postglacial recolonization from different refugial areas. We show that a conventional 'phylogenetic' substitution rate cannot account for the present distribution of genetic variation in this species, and we combine phylogeographical pattern and palaeoecological information in order to determine a suitable intraspecific rate through a Bayesian coalescent approach. We argue that our calibrated 'time-dependent' rate (0.096 substitutions/million years), offers the most convincing time frame for the evolutionary events inferred from sequence data. When scaled by the new rate, estimates of divergence between Balkan and Alpine lineages point to c. 19 000 years before present (last glacial maximum), and parameters of demographic expansion for northern lineages are consistent with postglacial warming (5-11 000 years before present)

Genetic differentiation and hybridization in two naturally occurring sympatric trout Salmo spp. forms from a small karstic lake.

In this study, multiple molecular markers [genotyping of 12 nuclear microsatellite loci and the protein-coding gene ldh-c1 * plus sequencing of the mitochondrial DNA (mtDNA) control region] were employed to investigate the genetic structure of the two trout forms, Salmo cettii and Salmo fibreni , inhabiting Lake Posta Fibreno, central Italy. The two forms were found to share a unique mtDNA haplotype, belonging to a widespread Mediterranean haplogroup (AD). Bayesian clustering analyses showed that these two forms correspond to well-defined autochthonous gene pools. Genetic introgression between the two gene pools, however, was observed, whose frequency appears to correlate with the environmental features of the spawning sites. The interplay of selection for the spawning sites, philopatry and natural selection can be argued to maintain genetic differentiation despite the lack of complete reproductive isolation

A taxonomic revision of western Eupholidoptera bush crickets (Orthoptera: Tettigoniidae): testing the discrimination power of DNA barcode.

The genus Eupholidoptera includes 46 Mediterranean species distributed from Turkey to Greece, Italy and southern France. In the eastern part of its range, Eupholidoptera has been considered to consist of several distinct species, while in the Balkans and Italian peninsula only E. chabrieri has been recognized. However, the status of some Italian populations, confined to particular geographic areas, remains uncertain. To investigate the delimitation of the Italian taxa of Eupholidoptera, we performed both morphological and molecular analyses. Morphological analysis was carried out by considering diagnostic characters usually used to distinguish different taxa, such as the shape of titillators in males and the subgenital plate in females. Molecular analysis was performed by sequencing three mitochondrial genes: 12S rRNA, 16S rRNA, partially sequenced and the entire gene of cox1 . Molecular markers were used to infer phylogenetic relationships among the Italian Eupholidoptera species and to reconstruct the historical processes that shaped their current geographic distribution. Results from both morphological and molecular analyses were used to revise the taxonomic arrangement of species. On the whole we were able to distinguish nine lineages of Italian Eupholidoptera, of which E. tyrrhenica sp.n. from Corsica is described as a new species

Estimating colonies of Plebeia droryana (Friese, 1900) (Hymenoptera, Apidae, Meliponini): adults, brood and nest structure

Estimate of stingless bee colonies including nest structures and quantitative brood and adult individuals are scarce. Here, we describe a new approach to estimate colonial parameters from nest structure, adults and brood. We used five colonies of Plebeia droryana (Friese, 1900) to evaluate colony size and weight of adult and brood. Nest architecture in P. droryana is similar to the species of the same genus but differ to the other stingless bees. In this species, we counted a total of 9 to 12 brood combs and a total of 19 to 25 food pots in the nests. The number of individuals in the colonies is considered small and our estimate was based on individual and group weight. Our study approach may contribute to further detailed studies of the species nest and considering the stingless bees to the pollination of agricultural crops and native flora of tropical regions, it is important to add information about the biology of P. droryana

Long and short term changes in abundance and distribution of butterflies: hints from the Lazio database

The DB on the occurrence data of the butterflies (Papilionoidea) of Lazio, at 14th February 2022 consisted of 36244 records including 154 species istributed throughout a total of 6719 sites. The data set included geoeferenced and chrono-referenced data collected from the literature, specialist-validated occurrences from websites (Forum Natura Mediterraneo, iNaturalist, Ornitho), as well as an important amount of original observations included in the database of the Lazio Biodiversity Observatory. All observations were used to create distribution maps. In order to evaluate any change in observations over time for the various species, all records were divided into three different periods: before 1980 (4425 records), 1980-2000 (6498 records) and post 2000 (25321 records). A finer subdivision was then examined within the post-2000 period: 2001-2007 (11888 records), 2008-2014 (4977 records), 2014-2021 (8456 records). Further analyses were carried out to highlight differences in the distribution of species as a function of altitude and / or changes in land use that have occurred in the last decades. The results show that qualitatively the species present in the region before 1980 are all still present today, however the abundance of related observations in several cases has changed considerably. Observations of an important portion of the species have significantly decreased in recent years. This trend is observed in the majority of mountain species and various habitat-specialist butterflies regardless of altitude. In some other species, often the most common or habitat-generalist butterflies, an increase was observed. The causes of these trends can be identified in the human land use and climate change, without excluding, however, the differences in data recording over time that could favor the most common species