La riproduzione di Procambarus clarkii (GIRARD, 1852) in Friuli Venezia Giulia: monitoraggio e studio del ruolo fisiologico dell'ormone gonado-inibitorio (GIH) per il contenimento numerico della specie

Procambarus clarkii è un crostaceo decapode proveniente dagli Stati Uniti meridionali, noto con i nomi di gambero rosso della Louisiana, o gambero killer. Questo crostaceo possiede una serie di caratteristiche biologiche che gli hanno permesso una diffusione incredibilmente rapida in quasi tutti i continenti, ad eccezione dell'Oceania e dell'Antartide. È una specie aliena invasiva che fu importata in Spagna dall'America nel 1972 per scopi di acquacoltura e da li ben presto si diffuse a tutta l'Europa, arrivando in Piemonte nel 1989 e in Friuli Venezia Giulia nel 2006. Questa specie rappresenta un problema serio per gli ecosistemi, in quanto è capace di alterarli in maniera significativa, causando un'importante riduzione della biodiversità sia animale che vegetale. In letteratura i principali lavori sulla biologia riproduttiva di questa specie sono stati fatti in massima parte in Spagna o Portogallo, mentre invece, in Italia, tale attività è stata svolta prevalentemente nella regione Toscana. Per approfondire in maniera diretta le conoscenze sulla riproduzione delle popolazioni che abitano i corsi d'acqua della regione FVG, tramite campionamenti in natura è stata avviata un'attività di monitoraggio che ha interessato uno specifico corso d'acqua durante il presunto periodo riproduttivo di questa specie. Dal monitoraggio è emerso come il periodo di riproduzione della popolazione in esame differisca rispetto alle popolazioni studiate in Toscana, Spagna e Portogallo e invece assomigli ad una popolazione studiata in Germania. Ciò costituisce una ulteriore prova della plasticità del ciclo biologico di questa specie originaria dei climi caldi della Louisiana, ma in grado di adattarsi anche a climi freddi, e rafforza la convinzione che sia necessario approfondire ulteriormente le conoscenze su questi organismi per poterne limitare la diffusione. Successivamente è stato investigato mediante tecniche di silenziamento genico indotto da RNA, l'RNAi, il ruolo fisiologico dell'Ormone Gonado-Inibitorio, GIH. Gli effetti fisiologici sono stati indagati a livello morfometrico, tramite l'uso degli indici gonado-somatico (GSI) ed epato-somatico (HSI), e tramite analisi di espressione genica impiegando la PCR quantitativa, RT-qPCR. A livello mormometrico negli animali trattati è stato osservato un aumento del peso degli ovari che ha raggiunto un valore doppio rispetto al peso delle gonadi delle femmine di controllo. A livello genico è stato osservato come, il silenziamento del GIH, sia in grado di promuovere la vitellogenesi esogena nell'epatopancreas di P. clarkii, aumentando l'espressione degli mRNA codificanti per le Vitellogenine. I risultati hanno confermato il ruolo di GIH come Ormone Gonado-Inibitorio in grado di agire in particolare sulla vitellogenesi secondaria di P. clarkii e lo propongono come possibile metodo innovativo di contenimento numerico di questa specie mediante l'uso di esche contenenti l'ormone di sintesi durante la stagione riproduttiva. La possibilità di veicolare attraverso l'apparato digerente anche piccole quantità di ormone attivo nell'emolinfa degli animali dovrebbe inibire la vitellogenesi esogena e la maturazione finale delle uova

Reproductive plasticity of a Procambarus clarkii population living 10\ub0C below its thermal optimum

In this study the annual reproductive biology of a Procambarus clarkii (Girard, 1852) population living in an atypical habitat with cold spring waters is investigated by monitoring Gonado-Somatic and Hepato-Somatic Indexes (GSI and HSI) and by performing cytology on ovaries. Despite its known preference for habitats with water temperature from 21 to 30 \ub0C, our results clearly confirm the adaptation of this population to the atypical thermal habitat, characterised by an annual mean water temperature value of 13.32 \ub10.08 \ub0C. Maximum gonadal development was reached in August, with maximum GSI median value of 0.64 (instead of reported values even 10 times higher for other populations), and ovigerous females were found in autumn, with mean realized fecundity of 35 \ub17 compared to 285\u2013995 reported from other habitats. Histological analysis was consistent with other studies and allowed us to follow ovarian development at cytological level. The importance of all these results is not to be underestimated: to our knowledge these findings are the first report of the coolest habitat successfully colonized by this species at the present time and so they have to be taken as a warning about the possible range expansion of P. clarkii also to northern and colder habitats that have few things in common with the native habitat of the species and, up to now, were considered \u201csafe\u201d from the invasion of the red swamp crayfish

An innovative index to incorporate transcriptomic data into weight of evidence approaches for environmental risk assessment

The sharp decrease in the cost of RNA-sequencing and the rapid improvement in computational analysis of eco-toxicogenomic data have brought new insights into the adverse effects of chemicals on aquatic organisms. Yet, transcriptomics is generally applied qualitatively in environmental risk assessments, hampering more effective exploitation of this evidence through multidisciplinary studies. In view of this limitation, a methodology is here presented to quantitatively elaborate transcriptional data in support to environmental risk assessment. The proposed methodology makes use of results from the application of Gene Set Enrichment Analysis to recent studies investigating the response of Mytilus galloprovincialis and Ruditapes philippinarum exposed to contaminants of emerging concern. The degree of changes in gene sets and the relevance of physiological reactions are integrated in the calculation of a hazard index. The outcome is then classified according to five hazard classes (from absent to severe), providing an evaluation of whole-transcriptome effects of chemical exposure. The application to experimental and simulated datasets proved that the method can effectively discriminate different levels of altered transcriptomic responses when compared to expert judgement (Spearman correlation coefficient of 0.96). A further application to data collected in two independent studies of Salmo trutta and Xenopus tropicalis exposed to contaminants confirmed the potential extension of the methodology to other aquatic species. This methodology can serve as a proof of concept for the integration of “genomic tools” in environmental risk assessment based on multidisciplinary investigations. To this end, the proposed transcriptomic hazard index can now be incorporated into quantitative Weight of Evidence approaches and weighed, with results from other types of analysis, to elucidate the role of chemicals in adverse ecological effects

Multi-tissue RNA-Seq Analysis and Long-read-based Genome Assembly Reveal Complex Sex-specific Gene Regulation and Molecular Evolution in the Manila Clam

The molecular factors and gene regulation involved in sex determination and gonad differentiation in bivalve molluscs are unknown. It has been suggested that doubly uniparental inheritance (DUI) of mitochondria may be involved in these processes in species such as the ubiquitous and commercially relevant Manila clam, Ruditapes philippinarum. We present the first long-read-based de novo genome assembly of a Manila clam, and a RNA-Seq multi-tissue analysis of 15 females and 15 males. The highly contiguous genome assembly was used as reference to investigate gene expression, alternative splicing, sequence evolution, tissue-specific co-expression networks, and sexual contrasting SNPs. Differential expression (DE) and differential splicing (DS) analyses revealed sex-specific transcriptional regulation in gonads, but not in somatic tissues. Co-expression networks revealed complex gene regulation in gonads, and genes in gonad-associated modules showed high tissue specificity. However, male gonad-associated modules showed contrasting patterns of sequence evolution and tissue specificity. One gene set was related to the structural organization of male gametes and presented slow sequence evolution but high pleiotropy, whereas another gene set was enriched in reproduction-related processes and characterized by fast sequence evolution and tissue specificity. Sexual contrasting SNPs were found in genes overrepresented in mitochondrial-related functions, providing new candidates for investigating the relationship between mitochondria and sex in DUI species. Together, these results increase our understanding of the role of DE, DS, and sequence evolution of sex-specific genes in an understudied taxon. We also provide resourceful genomic data for studies regarding sex diagnosis and breeding in bivalves

Transcriptome analysis reveals immune pathways underlying resistance in the common carp Cyprinus carpio against the oomycete Aphanomyces invadans

Infection with Aphanomyces invadans is a serious fish disease with major global impacts. Despite affecting over 160 fish species, some of the species like the common carp Cyprinus carpio are resistant to A. invadans infection. In the present study, we investigated the transcriptomes of head kidney of common carp experimentally infected with A. invadans. In time course analysis, 5288 genes were found to be differentially expressed (DEGs), of which 731 were involved in 21 immune pathways. The analysis of immune-related DEGs suggested that efficient processing and presentation of A. invadans antigens, enhanced phagocytosis, recognition of pathogen-associated molecular patterns, and increased recruitment of leukocytes to the sites of infection contribute to resistance of common carp against A. invadans. Herein, we provide a systematic understanding of the disease resistance mechanisms in common carp at molecular level as a valuable resource for developing disease management strategies for this devastating fish-pathogenic oomycete

Molecular insights into the mechanisms of susceptibility of Labeo rohita against oomycete Aphanomyces invadans

Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome, is one of the most destructive pathogens of freshwater fishes. To date, the disease has been reported from over 160 fish species in 20 countries and notably, this is the first non-salmonid disease that has resulted in major impacts globally. In particular, Indian major carps (IMCs) are highly susceptible to this disease. To increase our knowledge particularly with regards to host immune response against A. invadans infection in a susceptible host, the gene expression profile in head kidney of A. invadans-infected and control rohu, Labeo rohita was investigated using RNA sequencing. Time course analysis of RNA-Seq data revealed 5608 differentially expressed genes, involved among others in Antigen processing and presentation, Leukocyte transendothelial migration, IL-17 signaling, Chemokine signaling, C-type lectin receptor signaling and Toll-like receptor signaling pathways. In the affected pathways, a number of immune genes were found to be downregulated, suggesting an immune evasion strategy of A. invadans in establishing the infection. The information generated in this study offers first systematic mechanistic understanding of the host-pathogen interaction that might underpin the development of new management strategies for this economically devastating fish-pathogenic oomycete A. invadans

Contaminants from dredged sediments alter the transcriptome of Manila clam and induce shifts in microbiota composition

Background The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam’s microbial communities. Results Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments’ chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities. Conclusions Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon

The effects of daily cyclic hypoxia on the ecophysiology of the Atlantic ditch shrimp, Palaemon varians

Oxygen partial pressure (pO2) is not always constant in aquatic environments and can vary on different timescales, from hours to weeks. In many coastal environments, such as estuaries, lagoons or marshes, pO2 level scan vary on a daily base, resulting in daily cyclic hypoxia. By monitoring temperature and pO2 in the Lymington salt marshes (UK), I was able to quantify diel and seasonal pO2 variability in this coastal habitat:diel oscillations in pO2 were measured in winter, spring and summer, and the greatest pO2 oscillations were recorded in summer, when pO2 could fluctuate from ~42 kPa to ~ 1 kPa every 12 hours, causing diel cyclichypoxia. Even if cyclic hypoxia is common in numerous coastal areas around the world and affects many species, this phenomenon is less studied in comparison to acute or chronic hypoxia. The aim of this thesis was to characterize the short-term effects and the long-term consequences of daily cyclic hypoxia on the physiology of an important decapod crustacean, Palaemon varians. This species was found in the Lymington salt marshes (UK) and, in the laboratory, was subjected to a cyclic hypoxic regime that mimicked conditions measured in the field during summer.In the laboratory, a short 8-hour exposure to hypoxia (pO2 < critical oxygen pressure, pcrit) induced behavioural and metabolic changes and suppressed feeding and ammoniacal excretion. Long-term exposure to diel cyclic hypoxia induced changes in the transcriptome of the animals, prompted an acceleration of the moult cycle (validated at transcriptional and phenotypic level) and eventually resulted in morphological changes to the gills, which increased lamellar surface area. Further, long-term exposure to cyclic hypoxia impaired animal’s growth (in terms of body weight and length), reduced ammoniacal excretion and negatively influenced reproduction by reducing egg yolk content. Interestingly, long-term acclimation to cyclic hypoxia increased thermal tolerance and copper tolerance in comparison to control animals, probably as a consequence of the morphological changes to the gills induced by cyclic hypoxia. Overall, results underline that a short hypoxic exposure repeated daily was able to induce in P. varians alterations at multiple levels of biological organisation. In particular, the observed long-term consequences(i.e. growth reduction, reduced ammoniacal excretion and impaired reproduction) might have important ecological implications for the species and for its ecosystem

Acclimation to cyclic hypoxia improves thermal tolerance and copper survival in the caridean shrimp Palaemon varians

In response to the continuous variation of environmental parameters, species must be able to adjust their physiology to overcome stressful conditions, a process known as acclimatization. Numerous laboratory studies have been conducted to understand and describe the mechanisms of acclimation to one environmental stressor (e.g. cyclic hypoxia), but currently our understanding of how acclimation to one stressor can change tolerance to a subsequent stressor is limited. Here, in two different experiments, we used the shrimp Palaemon varians to test how, following 28-days acclimation to cyclic hypoxia (mimicking a cyclic hypoxic regime currently found in its natural habitat), critical thermal maximum (CT max) and sensitivity to copper (Cu 2+) exposure (30 mgL −1) changed in comparison to shrimp acclimated to normoxic conditions and then exposed to thermal stress or Cu 2+. Acclimation to cyclic hypoxia improved both CT max (~1 °C higher than controls) and survival to acute Cu 2+ exposure (~30% higher than controls) and induced significant gene expression changes (i.e. up-regulation of heat shock protein 70 – HSP70, hypoxia inducible factor – HIF, phosphoenolpyruvate carboxykinase – PEPCK, glucose 6-P transporter – G6Pt, metallothionein – Mt, and down-regulation of hemocyanin – Hem) in animals acclimated to cyclic hypoxia. Our results demonstrate how acclimation to cyclic hypoxia improved tolerance to subsequent stressors, highlighting the complexity of predicting organismal performance in variable (i.e. where multiple parameters can simultaneously change during the day) environments. </p