Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis.

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5'-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

Effects of the sulfated polysaccharide ulvan from Ulva ohnoi on the modulation of the immune response in Senegalese sole (Solea senegalensis).

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI, mhcII, psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp, inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan.

Anti-Bacterial and Immunostimulatory Properties of Ulvan-Loaded Chitosan Nanoparticles for Use in Aquaculture.

Alternative prophylactic strategies to limit farm animal infection are needed in order to avoid the use of antibiotics. Anti-bacterial and immunostimulatory properties of bioactive compounds are of great interest in aquaculture. Marine derived polysaccharides, such as chitosan and ulvan, together with nanotechnology, have become the focus of attention in the scientific community due to their wide range of biological properties. In this work, chitosan and ulvan-loaded chitosan nanoparticles (referred as CS-TPP NPs and CS-UL-TPP NPs, respectively), obtained by the ionotropic gelation method, had round shape, and the mean sizes were 137.00 ± 5.44 and 325.50 ± 4.95 nm, respectively. No study about the anti-bacterial activity of both types of NPs against Photobacterium damselae subsp. piscicida, an important fish pathogen, has been reported so far. Furthermore, the potential immunostimulatory effects of CS-UL-TPP NPs after oral administration in fish have not yet been evaluated. The percentage of bacterial inhibition against P. damselae subsp. piscicida was determined through in vitro assays, and it was significantly higher in CS-UL-TPP NPs than in CS-TPP NPs at concentrations below 0.03 mg mL-1. The effects on the immune system of CS-TPP and CS-UL-TPP NPs were evaluated in Solea senegalensis juveniles at 30 days after oral administration. Lysozyme activity as well as gene expression levels of il1b, il6, hamp1, tf and c3 was significantly higher in CS-UL-TPP NP-treated groups than in the controls, and no significant differences were observed in CS-TPP NP-treated groups. Thus, ulvan extracted from the macroalgae Ulva ohnoi could improve anti-bacterial and immunostimulant properties of CS-TPP NPs thereby making them suitable to be used as vaccine adjuvant or as immunostimulant.

Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup).

HSP90 proteins are chaperones that play a pivotal role in controlling multiple regulatory pathways such as stress defense, hormone signalling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, two cDNAs encoding for cytosolic HSP90, referred to as HSP90AA and HSP90AB, have been sequenced. Main features and sequence identities with other fish and mammals are described. Phylogenetic analysis grouped both genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, HSP90AB was constitutively expressed with lower transcript levels in skeletal muscle. In contrast, HSP90AA was mainly expressed in heart, skeletal muscle and skin. During metamorphosis, HSP90AB mRNA levels did not change whereas HSP90AA transcripts decreased significantly at the beginning of metamorphosis with the lowest mRNA levels at the metamorphosis climax. Due to the role of thyroid hormones (THs) on sole metamorphosis, the transcriptional regulation of HSP90 genes by THs was evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher HSP90AA mRNA levels than untreated control. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels with respect to the untreated control. Unlike HSP90AA, the transcript levels of HSP90AB did not vary under any treatments. The response of both HSP90 genes to thermal stress in post-metamorphic individuals was also studied. A heat shock treatment (+7.9 degrees C for 1 h) rapidly activated HSP90AA (but not HSP90AB) transcription, reaching a peak after 30 min and declining expression levels progressively in the following 24 h. No significant changes in HSP90AA or HSP90AB transcript levels after a cold shock (-10 degrees C for 1 h) were observed. Overall, these results demonstrate that HSP90AA transcription is down-regulated by THs and up-regulated after a heat shock in Senegalese sole.

Molecular characterization and gene expression of six trypsinogens in the flatfish Senegalese sole (Solea senegalensis Kaup) during larval development and in tissues.

The application of large-scale genomics to Senegalese sole (Solea senegalensis) has allowed for the identification of six different trypsinogen genes. The catalytic triad (His-57, Asp-102, and Ser-195) and other residues required for trypsin functionality were conserved across all trypsinogens. Sequence identities, charges and phylogenetic analysis allowed them to be classified into three groups: group I or anionic trypsinogens (ssetryp1a, ssetryp1b and ssetryp1c), group II or cationic trypsinogen (ssetryp2) and group III or psychrophilic trypsinogens (ssetryp3 and ssetrypY). The expression profiles of these genes were studied in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, trypsinogens were expressed mainly in the intestine. Transcripts of ssetryp1c were the highest in all tissues except in brain where those of ssetryp2 were the most abundant. During larval development, ssetryp1 variants and ssetryp2 transcript levels increased from 2 to 6 days after hatching, and decreased thereafter. In contrast, transcripts of group III trypsinogens increased slightly or not significantly in premetamorphosis and decreased at metamorphosis. The expression levels ssetryp3 and ssetrypY were the lowest in larvae (from 172- to 1391-fold lower than ssetryp1 and ssetryp2). In contrast, they were expressed at a similar level as ssetryp2, although lower than ssetryp1, in juvenile tissues.

Molecular characterization and transcriptional regulation of the sodium-dependent vitamin C transporter genes (slc23a1 and slc23a2) in a teleost fish, the Senegalese sole (Solea senegalensis).

Vitamin C (ascorbic acid, AA) is an antioxidant that acts as a free radical scavenger and cofactor for several important enzymatic reactions, thus being important for normal cellular functions, growth and development. Accumulation of AA in cells depends on two types of sodium-dependent vitamin C transporters (SVCTs), designed as SVCT1 and SVCT2. In human, they are the products of SLC23A1 and SLC23A2 genes, respectively. In the present work, the molecular cloning of the cDNAs corresponding to slc23a1 and slc23a2 in a teleost fish, the Senegalese sole (Solea senegalensis Kaup, 1858) is first described. Sequence analysis of the predicted polypeptides revealed a conserved topology with those of mammals with important motifs involved in structure and function, being also present in svct1 and svct2. Phylogenetic analyses including a range of vertebrate SVCTs suggest that both transporters are the result of an ancient gene duplication event that occurred prior to the divergence of tetrapods and teleosts, which took place 450 million years ago. Expression profiles in juvenile tissues and during larval development were analyzed using a real-time PCR approach. In juvenile fish, slc23a1 was strongly expressed in intestine, whereas slc23a2 exhibited a widespread distribution in tissues. Transcripts of both genes were detected at early developmental stages, probably representing mRNAs of maternal origin. A possible regulation by their own substrate was detected after first uptakes of AA from diet in both genes. During metamorphosis, both slc23a1 and slc23a2 were down-regulated, the former in a thyroid hormone (TH) dependent way. This pattern coincided with a significant reduction in the AA content of larvae during metamorphosis. These results are interpreted in a physiological context of general reduction in the metabolism of metamorphic larvae. Data presented here provide the first step toward a better understanding of the physiological role of SVCTs in teleost fish.

Isolation and characterization of polymorphic microsatellites for the bastard sole (Microchirus azevia).

The bastard sole (Microchirus azevia) is a species of commercial interest in Spain. Nevertheless, little information is currently available about the genetic characteristics of wild populations. In this survey, we have developed eight new microsatellites using an enriched genome library protocol. Primers were screened on a total of 54 individuals from two wild populations (Mediterranean and Atlantic) from the south coast of Spain, revealing six to 18 alleles per locus with expected heterozygosities ranging from 0.51 to 0.94. These markers can potentially be useful tools for use in population genetic studies.

Phylogenetic differentiation between Atlantic Scomber colias and Pacific Scomber japonicus based on nuclear DNA sequences.

In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34 specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S. australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.

Molecular characterization and chromosomal mapping of the 5S rRNA gene in Solea senegalensis: a new linkage to the U1, U2, and U5 small nuclear RNA genes.

Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227- and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.