Effect of Food Amounts on Larval Performance, Bacteriome and Molecular Immunologic Development during First-Feeding Culture of European Eel.

Production of European eel offspring has become a reality, but liquid diets during larval culture hold new challenges. This study focused on increasing food amounts without compromising well-being or healthy larvae-bacteria interactions. First-feeding larvae were fed two food amounts (Low = 0.5 mL food/L water vs. High = 1.5 mL food/L water) until 30 days post-hatch (dph). Results indicated that ~75% of larvae ingested the diet in both treatments, but upregulation of a stress/repair-related gene (hsp90) on 25 and 30 dph indicated nutritional inadequacy. Larvae fed a High amount of food were 3.68% bigger, while larvae in the Low-food group showed 45.2% lower gut fullness and upregulated expression of the gene encoding the "hunger hormone" ghrelin (ghrl), indicating signs of starvation. The High-food group larvae exhibited a healthier bacteriome with a higher abundance of potentially beneficial orders (Lactobacillales and Bacillales), whereas the Low-food group showed more potentially harmful orders (Vibrionales, Rhodobacterales, and Alteromonadales). While survival was initially lower in the High-food group, both treatments had comparable survival by the end of the experiment. In conclusion, feeding European eel larvae with High food amounts seemed beneficial, supported by increased gut fullness, reduced ghrl expression (no starvation), enhanced growth, and the presence of a healthier bacteriome.

Exploring bacterial community composition and immune gene expression of European eel larvae (Anguilla anguilla) in relation to first-feeding diets.

European eel (Anguilla anguilla) is a commercially important species for fisheries and aquaculture in Europe and the attempt to close the lifecycle in captivity is still at pioneering stage. The first feeding stage of this species is characterized by a critical period between 20 to 24 days post hatch (dph), which is associated with mortalities, indicating the point of no return. We hypothesized that this critical period might also be associated with larvae-bacterial interactions and the larval immune status. To test this, bacterial community composition and expression of immune and stress-related genes of hatchery-produced larvae were explored from the end of endogenous feeding (9 dph) until 28 dph, in response to three experimental first-feeding diets (Diet 1, Diet 2 and Diet 3). Changes in the water bacterial community composition were also followed. Results revealed that the larval stress/repair mechanism was activated during this critical period, marked by an upregulated expression of the hsp90 gene, independent of the diet fed. At the same time, a shift towards a potentially detrimental larval bacterial community was observed in all dietary groups. Here, a significant reduction in evenness of the larval bacterial community was observed, and several amplicon sequence variants belonging to potentially harmful bacterial genera were more abundant. This indicates that detrimental larvae-bacteria interactions were likely involved in the mortality observed. Beyond the critical period, the highest survival was registered for larvae fed Diet 3. Interestingly, genes encoding for pathogen recognition receptor TLR18 and complement component C1QC were upregulated in this group, potentially indicating a higher immunocompetency that facilitated a more successful handling of the harmful bacteria that dominated the bacterial community of larvae on 22 dph, ultimately leading to better survival, compared to the other two groups.

Exploring first-feeding diets for European eel larval culture: Insights at morphological, nutritional, and molecular levels.

Closing the life cycle of European eel (Anguilla anguilla) in captivity is targeted to provide a sustainable, year-round supply of juveniles for aquaculture. Present focus is on the nutritional requirements during the larval first-feeding period. In this study, three experimental diets were tested on hatchery-produced European eel larvae from the onset of the first-feeding stage commencing 10 days post hatch (dph) until 28 dph. Larval mortality was recorded daily, while sampling was conducted at regular intervals to record larval biometrics and analyze the expression of genes related to digestion, appetite, feed intake and growth. Two periods of high mortality were identified: the first appeared shortly after introduction of feeds (10-12 dph), while the second occurred 20-24 dph, indicating the "point of no return". This interpretation was supported at the molecular level by the expression of the gene encoding the "hunger hormone" ghrelin (ghrl) that peaked at 22 dph in all dietary trials, suggesting that most larvae were fasting. However, in larvae fed diet 3, ghrl expression was downregulated beyond 22 dph, which indicated that those larvae were no longer starving at this stage, while upregulation of genes encoding the major digestive enzymes (try, tgl, and amyl2a) advocated their healthy development. Moreover, for larvae fed diet 3, the expression of those genes as well as genes for feed intake (pomca) and growth (gh) continued to increase towards 28 dph. These results together with the registered highest survival, largest dry weight increase, and enhanced biometrics (length and body area) pointed to diet 3 as the best-performing. As a whole, this first-feeding study represents a landmark being the first to document European eel larval growth and survival beyond the point of no return, providing novel insights into the molecular development of digestive functions during the first feeding stage.

Effect of parental origin on early life history traits of European eel.

Establishment of European eel (Anguilla anguilla) hatchery production will rely on selectively bred individuals that produce progeny with the best traits in successive generations. As such, this study used a quantitative genetic breeding design, between four females and nine males (four wild-caught and five cultured), to investigate the effect of paternal origin (wild-caught vs. cultured) and quantify the relative importance of parental effects, including genetic compatibility, on early life history (ELH) performance traits (i.e. fertilization success, embryonic survival at 32 hr post-fertilization, hatch success and larval deformities at 2 days post-hatch) of European eel. Wild-caught males had higher (56%) spermatocrit values than cultured males (45%), while fertilization success, embryonic survival, hatch success and larval deformities were not significantly impacted by paternal origin. This demonstrates that short-term domestication of male eels does not negatively affect offspring quality and enables the consideration of cultured male broodstock in future breeding programmes. Moreover, paternity significantly explained 9.5% of the variability in embryonic survival, providing further evidence that paternal effects need to be taken into consideration in assisted reproduction protocols. Furthermore, maternity significantly explained 54.8% of the variation for fertilization success, 61.7% for embryonic survival, 88.1% for hatching success and 62.8% for larval deformities, validating that maternity is a major factor influencing these "critical" ELH traits. At last, the parental interaction explained 12.8% of the variation for fertilization success, 8.3% for embryonic survival, 4.5% for hatch success and 20.5% for larval deformities. Thus, we conclude that eggs of one female can develop more successfully when crossed with a compatible male, highlighting the importance of mate choice for successful propagation of high-quality offspring. Together, this knowledge will improve early offspring performance, leading to future breeding programmes for this critically endangered and economically important species.

Labeling of fatty acid ligands with the strong electrophilic metal fragment [99mTc(N)(PNP)]2+ (PNP=diphosphane ligand).

The electrophilic metal fragment [(99m)Tc(N)(PNP)](2+) (PNP=diphosphane ligand) has been employed for the labeling of fatty acid chains of different lengths. To provide a site-specific group for the attachment of the metallic moiety, the fatty acid derivatives were functionalized by appending a bis-mercapto or, alternatively, a dithiocarbamato pi-donor chelating systems to one terminus of the carbon chain to yield both dianionic and monoanionic bifunctional ligands (L). The resulting complexes, [(99m)Tc(N)(PNP)(L)] (0/+), exhibited the usual asymmetrical structure in which a Tc(triple bond)N group was surrounded by two different bidentate chelating ligands. Dianionic ligands gave rise to neutral complexes, while monoanionic ligands afforded monocationic species. Biodistribution studies were carried out in rats. An isolated perfused rat heart model was employed to assess how structural changes in the radiolabeled fatty acid compound affect the myocardial first pass extraction. Results showed that only monocationic complexes accumulated in myocardium to a significant extent. Conversely, neutral complexes were not efficiently retained into the heart region and rapidly washed out. In isolated perfused rat heart experiments, monocationic complexes exhibited a behavior similar to that of the monocationic flow tracers (99m)Tc-MIBI and (99m)Tc-DBODC with almost identical extraction values, a result that could be attributed to the presence of the monopositive charge. Instead, a slightly lower myocardial extraction was found for neutral complexes. Comparison of the observed kinetic behavior of neutral complexes in the isolated perfused rat heart model with that of the myocardial metabolic tracer [(123)I]IPPA revealed that the introduction of the metallic moiety partially hampers recognition of the labeled fatty acids by cardiac enzymes, and consequently, their behavior did not completely reflect myocardial metabolism.

From symmetrical to asymmetrical nitrido phosphino-thiol complexes: a new class of neutral mixed-ligand (99m)Tc compounds as potential brain imaging agents.

A general procedure is presented for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core that could be used to design either essential or target specific imaging agents. This procedure is based on the chemical properties of a new monosubstituted [Tc(N)(R2PS)Cl(PPh3)] species composed of a TcN multiple bond and an ancillary phosphine thiol ligand (R2PSH). This intermediate readily reacts with bidentate mononegative ligands (S--Y) containing soft pi-donor coordinating atoms to give neutral pentacoordinate asymmetrical complexes of the type [Tc(N)(R2PS)(S--Y)]. The ability of several bidentate ligands containing different combination of heteroatoms (S, N, O) to form complexes with the [Tc(N)(R2PS)]+ building block was investigated. It was found that mononegative dithiocarbamate (DTC) or cysteine carboxyl derivate ligands promptly react with the monosubstituted species to form the final mixed compound in high yield. Preliminary biodistribution data in rats of some representative [Tc(N)(R2PS)(DTC)] compounds revealed an interesting initial brain uptake (in the range 0.20 +/- 0.01% ID/g and 0.91 +/- 0.06% ID/g), indicating their ability to cross in and out of the intact BBB. In these complexes the dithiocarbamate, or more generally the bidentate ligand (S--Y), can be designed to carry a functional group or a bioactive molecule, which could be involved in a trapping mechanism to increase brain retention for longer time intervals. These results could be conveniently utilized to devise a new procedure for the production of a novel class of brain perfusion and/or brain receptor imaging agents.

Characterisation via electrospray ionisation multistage mass spectrometry of three related series of nitrido technetium complexes containing phosphinothiolate and dithiocarbamate ligands.

Nine nitrido technetium compounds comprising bis-substituted Tc(N)(PS)(2) (1-4) (PS = bidentate phosphinothiolate ligands) and Tc(N)(dtc)(2) (5, 6) derivatives (dtc = bidentate dithiocarbamate), and mixed-ligand Tc(N)(PS)(dtc) (7-9) species, were subjected to electrospray ionisation mass spectrometry and MS(n) experiments. Bis-substituted phosphinothiolato complexes 1-4 lead to the straightforward formation of dinuclear species reasonably originating from proton bound dimers. These dinuclear species do not show, under collisionally induced fragmentation processes, the formation of monomeric units but cleavages related to the ligand framework, thereby proving the high stability of the [Tc--H(+)--Tc] bond. Bis-dithiocarbamate compounds 5 and 6 show, instead, abundant [M+H](+), [M+Na](+) and [2M+Na](+) ions, and their collisionally induced fragmentations are highly favoured with cleavages related to the C--N and C--S bonds. During these processes, the coordination of a water molecule to [MH-L](+) product ions is observed, as proved by the collisionally induced H(2)O loss detected for this species. Mixed-ligand compounds 7 and 8 show the protonated molecules and Na(+)-cationised ions with fragmentation processes related to the dithiocarbamate moiety. This behaviour indicates that coordination of ether- and ester-substituted dithiocarbamates to the [Tc [triple chemical bond] N] group is weaker than that of phosphinothiolates. Conversely, diethyldithiocarbamate inserted in mixed complex 9 enhances both C--N and Tc--S bonds, and fragmentation processes suggest that metal-phosphinothiolate and metal-dithiocarbamate show comparable strength.

Synthesis, characterization, and biological evaluation of neutral nitrido technetium(V) mixed ligand complexes containing dithiolates and aminodiphosphines. A novel system for linking technetium to biomolecules.

A new biomolecule labeling method that utilizes the [(99m)Tc(N)(PNP)](2+) metal fragment is presented. Thus, a series of nitrido mixed-ligand M(V) complexes (M = (99m)Tc, (99g)Tc, Re), [M(N)(Ln)(PNP)], where Ln is the dianionic form of a dithiolate or substituted-dithiolate ligand and PNP is an aminodiphosphine, is described. (99m)Tc complexes can be prepared using either a two-step or a three-step procedure starting from generator-eluted pertechnetate through a prereduced mixture of [(99m)Tc(N)]-containing species, followed by sequential or contemporary addition of the relevant dithiolate and aminodiphosphine. The reactions of 2,3-dimercaptopropionic acid (H(2)L1) with [Tc(N)(PNP)](2+) were investigated in detail. It was found that this bidentate ligand coordinated the metal fragment through the [S(-),S(-)] donor atom pair, to yield neutral mixed-ligand complexes [(99m)Tc(N)(L1)(PNP)] in high specific activity. The additional carboxylic functional group was not involved in metal coordination, thus remaining available for conjugation to target-specific molecules. Dithiolates incorporating pendant functional group(s) gave rise to a 1:1 diastereoisomeric mixture of syn-[M(N)(Ln)(PNP)] and anti-[M(N)(Ln)(PNP)] derivatives, depending on the relative orientation of the dithiolate substituent(s) with respect to the terminal nitrido group, and no isomeric conversion was detected. (99m)Tc species had been proven to be identical with the (99g)Tc complexes prepared at the macroscopic level by comparison of the corresponding radiometric and UV/vis HPLC profiles. Challenge experiments with cysteine or glutathione indicated that these physiological agents had no effect on the stability of this class of mixed-ligand (99m)Tc-complexes. Biodistribution studies in rats of selected (99m)Tc-complexes showed a rapid clearance from the blood and tissues after 60 min pi.