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Phenotypic plasticity, the ability of one genotype to produce multiple phenotypes, is crucial to an organism’s capacity to acclimate to changing environments. Acclimation by means of phenotypic plasticity may allow species to cope with fast-changing environments. Phenotypic plasticity can manifest either as acute, developmental, or transgenerational. In this dissertation, I explored the molecular processes underlying these three plasticity types using non-model teleost species, in an attempt to shed light into key aspects of plasticity. In Chapter I, I describe the transcriptomic changes involved in the acute phenotypic plasticity of a euryhaline fish, the Arabian pupfish, in response to an increase in water salinity over a timeline. Osmoregulation and immune system were found to be crucial for long-term seawater adaptation, while in the short-term, cellular stress response mechanisms were activated. Tissue remodeling processes were also transiently elicited, equipping the fish with modifications necessary for acclimation to the new environment. Chapter II and Chapter III explore the effects of developmental and transgenerational phenotypic plasticity in a coral reef fish, the spiny damselfish, exposed to simulated ocean warming. In Chapter II, I tackle the fundamental question of the importance of timing in the transmission of transgenerational effects. I discovered that a developmental exposure of parents to warming might contribute to adaptive transgenerational plasticity, by adjustment of energy production pathways and gene expression regulation. Conversely, the exposure to warming during reproduction has no beneficial effects, but rather causes stress response mechanisms in the parents themselves and in their offspring. Finally, in Chapter III I disentangle the individual paternal and maternal contributions to the transmission of transgenerational plasticity. Fathers and mothers were found to elicit common but also parent-specific effects. Parent-specific transgenerational mechanisms involved in acclimation to warming included maternally influenced changes in lipid metabolism and paternally inherited epigenetic stress-response mechanisms. However, potentially maladaptive parental condition-transfer signatures were also observed, especially when the parental thermal histories did not coincide. Taken together, this dissertation provides novel information regarding the molecular mechanisms underlying phenotypic plasticity. A deeper understanding of these processes is critical to make predictions on species acclimation potential in a rapid-changing world.

The oral administration of substances in crustaceans is a growing field of research, and is considered the most effective way to convey elements in their body instead injections or other invasive techniques. It presents huge potentialities, in fact the production of emulsions that preserves the substance to be conveyed until it reaches the target organ finds application in the development of new vaccines, in the convey of substances for aquaculture purposes and in the administration of stressors to contrast the spreading of invasive crustacean species. The red swamp crayfish ( Procambarus clarkii , Girard 1852) is one of the most spread invasive species worldwide, but it is also considered among the most economically important freshwater crustacean species. For these reasons it is considered as a paradigmatic species. The chemical synthesis of the D-Crustacean Hyperglycemic Hormone isoform (D-Phe-CHH) from P. clarkii is here presented, with its species-specificity, verified through the injection of D-Phe-CHH in both the red swamp crayfish and the threatened European species Austropotamobius italicus (Faxon, 1984). The D-Phe-CHH has been successfully oral-delivered in P. clarkii by using a water/oil/water microemulsion entrapped in a jellified matrix. After the administration of CHH, the glycaemia levels significantly increased in eyestalk-less animals at hour 6, and in intact crayfish at hour 5, from the ingestion. These encouraging findings pave the way for the use of the double emulsion method to oral administer different peptides to both handle physiological aspects of P. clarkii life cycle for aquaculture purposes and to create species-specific peptides-based mix to hamper the spreading of invasive crayfish populations. Statement of relevance Through the injection of the de novo synthetized D-Phe-Crustacean Hyperglycaemic Hormone isoform in the haemolymphatic circulation of P. clarkii , we evaluated its action in triggering hyperglycaemia. The peptide has been then successfully orally delivered in P. clarkii by w/o/w microemulsion and its bioactivity has been demonstrated. The formulated emulsion to convey peptides in crustaceans represents a very promising technique to modulate growth and reproduction in aquaculture plans.

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 °C, 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 ±0.08 °C. 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 ±7 compared to 285–995 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 “safe” from the invasion of the red swamp crayfish.

Recent studies have addressed to the taxonomical characterization of Italian pike as a new species named Esox cisalpinus, which differs from the European Esox lucius. A taxonomical study of the pike features in the Friuli Venezia Giulia region (Northeast Italy) is here presented, since previous studies did not consider this area which could represent an overlapping zone for the two species. Main meristic characters were investigated, and genetic analyses were carried out using molecular marjers. Our results conform the genetic separation from the E. lucius, while among meristic characters only the number of lateral-line scales differs between the species. E. cisalpinus was observed in most of the investigated sites, except for an isolated lentic habitat where E. lucius was found. As E. cisalpinus is likely autochthonous in friuli venezia Giulia as in other Italian regions, we highlight the requirement of furtheranalyses in order to clarify if hybridization can occur and to plan appropriate management safeguards for native populations.

RNA interference has frequently been applied to modulate gene function in organisms. With the aim of creating new autocidal methods based on neuro-endocrine disruptors for invasive populations of Procambarus clarkii, we silenced the Crustacean Hyperglycemic Hormone (CHH) by injecting the corresponding dsRNA. CHH is a pleiotropic hormone that primarily regulates the mobilization of energy reserves and plays a pivotal role in stress responses. Here, we describe two experiments aimed at testing whether CHH silencing significantly alters important physiological aspects. The first experiment investigates the effects of CHH silencing at the glycemic and transcriptomic level in the eyestalk. The second experiment explores the long-term effects of CHH silencing and the effects on mortality and moulting rates. Osmotic deficits and mortality were recorded in specimens injected with CHH dsRNA, whilst controls were injected with GFP dsRNA. After 20 days, despite still silenced for CHH, individuals that survived recovered a strong hyperglycemic response after serotonin injection due to the compensatory effect of two peptides belonging to the crustacean neurohormone CHH protein family.