Effectiveness of functional ingredients to enhance gill disease in Atlantic salmon (Salmo salar, L.).

The development and application of functional feed ingredients represents a great opportunity to advance fish growth and health, boost the immune system, and induce physiological benefits beyond those provided by traditional feeds. In the present study, we looked at the feasibility of in vitro methods for screening the qualities of functional feed ingredients using the fish cell line RTgill-W1, which has never been used in fish nutrition, and the culture of Paramoeba perurans. Five functional feed ingredients (arginine, ß-glucan, vitamin C, and two phytogenic feed additives) were selected to investigate their effects on cell viability and reactive oxygen species production. Three of the selected ingredients (arginine and two phytogenic feed additives) were additionally tested to assess their potential amoebicidal activity. As these functional ingredients are the core of a commercially available feed (Protec Gill, Skretting AS), their beneficial effects were further assessed in a field trial in fish affected by complex gill disease. Here, the analyzed parameters included the evaluation of macroscopic and histopathological gill conditions, pathogen detections, and analyses of plasma parameters. RTgill-W1 cell line assays were a good tool for screening functional ingredients and provided information about the optimal ingredient concentration ranges, which can be helpful for adjusting the concentrations in future feed diets. Through the culture of P. perurans, the tested ingredients showed a clear amoebicidal activity, suggesting that their inclusions in dietary supplements could be a viable way to prevent microbial infections. A three-week period of feeding Protec Gill slowed the disease progression, by reducing the pathogen load and significantly improving gill tissue conditions, as revealed by histological evaluation. The use of diets containing selected functional ingredients may be a feasible strategy for preventing or mitigating the increasingly common gill diseases, particularly in cases of complex gill disease, as documented in this study.

Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes.

The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes.

A multidisciplinary integrated approach using Pachygrapsus marmoratus to assess the impact of port activities on mediterranean marine protected areas.

The establishment of marine protected areas is considered the main global strategy to halt the loss of marine biodiversity. Since most of marine areas are open systems, this form of habitat protection cannot prevent their contamination due to human activities performed outside of their borders. Innovative approaches to assess the health status of protected marine habitats are therefore needed. Here we developed a multidisciplinary approach that combines ecological characteristics, bioaccumulation of inorganic and organic pollutants, cell damage (micronuclei frequency, nuclear alterations and LPO) and enzymatic (AChE, CAT, IDH, LDH, GST and CAT) markers focused on an intertidal brachyuran crab, Pachygrapsus marmoratus, to assess the impacts of contaminant exposure on Mediterranean coastal habitats. As study sites we selected two protected areas and two sites within industrial ports of the Ligurian Sea. Our results showed that the selected crab species is an excellent bioindicator. Individuals collected in sites with the highest levels of heavy metal pollution showed the highest signals of stress responses at both cellular and enzymatic levels, coupled with a high incidence of the parasite Sacculina carcini, a signal of impairment of their standard development and reproduction cycle. We could also prove that one of the selected marine protected areas showed the same intensity of impact as its adjacent port site. Our multidisciplinary approach proved to be a valuable tool to assess the environmental quality and health of protected and disturbed Mediterranean coastal environments and to inform efficient management and protection schemes for such habitats.

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.