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Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.

Abstract Background The amphipod crustacean Palmorchestia hypogaea occurs only in La Palma (Canary Islands) and is one of the few terrestrial amphipods in the world that have adapted to a strictly troglobitic life in volcanic cave habitats. A surface-dwelling closely related species (Palmorchestia epigaea) lives in the humid laurel forest on the same island. Previous studies have suggested that an ancestral littoral Orchestia species colonized the humid forests of La Palma and that subsequent drought episodes in the Canaries reduced the distribution of P. epigaea favouring the colonization of lava tubes through an adaptive shift. This was followed by dispersal via the hypogean crevicular system. Results P. hypogaea and P. epigaea did not form reciprocally monophyletic mitochondrial DNA clades. They showed geographically highly structured and genetically divergent populations with current gene flow limited to geographically close surface locations. Coalescence times using Bayesian estimations assuming a non-correlated relaxed clock with a normal prior distribution of the age of La Palma, together with the lack of association of habitat type with ancestral and recent haplotypes, suggest that their adaptation to cave life is relatively ancient. Conclusion The data gathered here provide evidence for multiple invasions of the volcanic cave systems that have acted as refuges. A re-evaluation of the taxonomic status of the extant species of Palmorchestia is needed, as the division of the two species by habitat and ecology is unnatural. The information obtained here, and that from previous studies on hypogean fauna, shows the importance of factors such as the uncoupling of morphological and genetic evolution, the role of climatic change and regressive evolution as key processes in leading to subterranean biodiversity.

Understanding patterns of community structure and the causes for their variation can be furthered by comparative biogeographic analyses of island biotas. We used woody plant data at the local scale to investigate variations in species rarity, alpha, beta, and gamma diversity within and between three islands from the oceanic archipelagoes of Azores, Canaries and Mascarene. We used standardized protocols to sample ten 50 m × 50 m forest plots in each of the three islands with contrasting climate and regional species pools: Terceira (Azores), Tenerife (Canaries), and Reunion (Mascarene Islands). Occupancy frequency distributions and species abundance distributions were used to investigate rarity. The partitioning of beta diversity in a distance-decay framework was used to test for spatial patterns of community composition. Rarity was much more pronounced in the highly diverse islands of Tenerife and Reunion than in the regionally poorer island of Terceira. The number of species rose faster with increasing sample area in both Tenerife and Reunion. The slope of the species rank abundance curve was steeper in Terceira whereas the richer island assemblages approached a lognormal model. Compositional changes according to spatial distance were mostly due to replacement of species in Terceira and Reunion. Our results point to important differences in the community structure of Terceira, which is the less diverse and temperate region in comparison to Tenerife and Reunion which are highly diverse.

A new blind weevil belonging to the genus Oromia Alonso-Zarazaga, 1987 is described, being found in the underground of the laurel forest of La Gomera (Canary Islands). Individuals were mainly collected in a colluvial mesocavernous shallow substratum, besides one specimen collected in the deep humic layer of soil. This new species has clear diagnostic differences from the other Oromia species. The number of taxa in this endemic Canarian genus increases to four species, easily identified using the key provided in this article. New data on other Canarian subterranean weevils are also provided.

Two new dipluran species of the family Campodeidae have been unearthed in the Canary Islands. Remycampa herbanica sp. nov. was found in a highly threatened lava tube on Fuerteventura island. It is related to the soil-dwelling northwest African Remycampa launeyi that also inhabits four of the Canary Islands. The two known Remycampa species are characterized by a torsion of the labial palps. They differ chiefly in the distribution of macrosetae and in the features of cave adaptation of R. herbanica, i.e. elongation of body and appendages, and a higher number of olfactory chemoreceptors with a coniform shape unique within campodeids. Spaniocampa relicta sp. nov. was collected in the mesovoid shallow substratum (MSS) and has been assigned to a formerly monotypic genus that includes the soil-dwelling Spaniocampa prima from the Republic of Guinea. The two species differ in the number of abdominal macrosetae. Females of S. relicta sp. nov. have small setae arranged in groups along the posterior border of the first urosternite. These structures of unknown function have never been described in other campodeid species. Sequencing the COI barcode region of R. herbanica has been produced but it proved insufficient to identify closest relatives. The two new hexapods from subterranean habitats raise the Canarian campodeid fauna to six species. Five of them are living in soil and/or MSS, whereas the cave-adapted R. herbanica is known only from a single, particularly endangered lava tube distant from other caves.

Cueva del Viento and Cueva de Felipe Reventón are lava tubes located in Tenerife, Canary Islands, and are considered the volcanic caves with the greatest cave-dwelling diversity in the world. Geological aspects of the island relevant to the formation of these caves are discussed, and their most outstanding internal geomorphological structures are described. An analysis of the environmental parameters relevant to animal communities is made, and an updated list of the cave-adapted species and their way of life into the caves is provided. Some paleontological data and comments on the conservation status of these tubes are included.

The study of subterranean Oniscidea in Portugal has been neglected for nearly 70 years, but recent investigations have revealed high diversity. All the terrestrial isopods from caves of mainland Portugal, including data from the literature and new material, are listed, revealing new biodiversity patterns. Twenty-seven species, belonging to 15 genera and six families, are known, of which 16 species are considered to be exclusively from subterranean ecosystems, i.e., troglobionts. Trichoniscidae is the most diverse family. Seven species in the family Trichoniscidae and one species in the family Styloniscidae are described as new (all with Reboleira & Taiti as authors): Trichoniscoides bellesi sp. nov. from the Montejunto Massif; T. sicoensis sp. nov. from the Sicó Massif; Metatrichoniscoides salirensis sp. nov. from the typhonic valley of Caldas da Rainha; Troglonethes olissipoensis sp. nov. from the Lisbon Peninsula; T. arrabidaensis sp. nov. from the Arrábida Massif; Miktoniscus longispina sp. nov. from the Sicó Massif and Cesaredas Plateau; Moserius inexpectatus sp. nov. from the Estremenho Massif; and Cordioniscus lusitanicus sp. nov. from Alentejo and Algarve, the southernmost provinces of Portugal. The subgenus Trogleluma Vandel, 1946 (Armadillidiidae) is raised to genus level. In this geographic region terrestrial isopods are the richest group of cave-adapted animals.

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h of reaction at 40 °C in [BMIM][PF6].

Temperature affects the duration of embryonic and larval periods in amphibians. Plasticity in time to metamorphosis is especially important in amphibian populations of Mediterranean semi-arid zones where temperatures are high and precipitation is low, increasing the rate of pond desiccation. In order to test the influence of water temperature on the larval development and growth of the natterjack toad (Bufo calamita), we collected two spawns in a semi¿arid zone at Balaguer (Lleida, NE Iberian peninsula). Approximately 50 (+/-10) eggs (stage 14-16) were raised in the lab at different temperature conditions: 10, 15, 20, 22.5 and 25ºC with 12:12 photoperiod. The results show a lengthening of development time with decreasing temperatures and a better survival performance of B. calamita to high temperatures. However, mean size at metamorphosis was not different across treatments, thus, suggesting that this population of B. calamita requires a minimum size to complete the metamorphosis. This study is the first approach to examine the effects that climatic factors have on the growth and development of B. calamita in semi-arid zones.

An overview of the obligate hypogean fauna in Portugal (including Azores and Madeira archipelagos) is provided, with a list of obligated cave-dwelling species and subspecies, and a general perspective about its conservation. All the available literature on subterranean Biology of Portugal since the first written record in 1870 until today has been revised. A total of 43 troglobiont and 67 stygobiont species and subspecies from 12 orders have been described so far in these areas, included in the so-called Mediterranean hotspot of biodiversity. The subterranean fauna in Portugal has been considered moderately poor with some endemic relicts and it remains to be demonstrated if this fact is still true after investing in standard surveys in cave environments. The major problems related to the conservation of cave fauna are discussed, but it is clear that the protection of this specialized fauna implies an adequate management of surface habitats.