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The Black Soldier Fly (BSF) Hermetia illucens provides a promising strategy in the waste valorisation process and a sustainable alternative source of valuable nutrients, including lipids for food and feed. In the present study, the differences in growth performances and nutritional values of BSF V instar larvae and prepupae reared on vegetable waste were analyzed and compared focusing on fat content. V instar larvae showed higher capacity to bioconvert the substrate into biomass than prepupae. The nutritional composition and the fatty acid profiles were dependent on the developmental stage. The expression levels of acetyl-CoA carboxylase (acc), fatty acid synthase (fas), lipase (lip) and acyl-CoA dehydrogenase (acd) genes involved in the lipid metabolism pathway and herein characterized for the first time, were evaluated in order to understand the molecular basis underlying the observed differences in fatty acid profiles. Our results suggest that the different fatty acid profiles of BSF V instar larvae and prepupae may be related to the modulation of the lipid metabolism-related genes expression during larval development. Our study highlights substantial differences between H. illucens V instar larvae and prepupae giving important features regarding the opportunity to modulate the preferable fatty acid profile to meet the industrial requirements.

The black soldier fly (BSF) Hermetia illucens is receiving increasing attention as a sustainable fishmeal alternative protein source for aquaculture. To date, no studies have explored the effects of fishmeal replacement with BSF V instar larvae or prepupae meals due to their peculiar nutritional properties on fish performances. This study investigated the effects of 100% replacement of fishmeal (control diet) with defatted BSF meals (V instar larvae and prepupae meals, treatments) on growth performance and welfare of zebrafish (Danio rerio), from larvae to adults, in a 60-day feeding trial. Following the inclusion of BSF meals, the expression of key genes involved in growth (igf1, igf2, mstnb, myod1, myog, myf5), hydrolysis of chitin (chia.2, chia.3, chia.5), immune- (il1b, il6, tnfα), and stress- (hsp70 and nr3c1) responses, as assessed by qPCR, was modulated in all of the molecular pathways, except for the stress response. Overall, our findings showed that both BSF meals can totally replace fishmeal without adverse impacts on adult zebrafish growth parameters (final total and standard length, final body weight, weight gain, daily growth rate, specific growth rate) and welfare, with BSF prepupae meal inducing the most beneficial effects, thus suggesting their potential application to meet fish requirements in aquaculture.

Dietary supplements are commonly used by animals and humans and play key roles in diverse systems, such as the immune and reproductive systems, and in metabolism. Essential oils (EOs), which are natural substances, have potential for use in food supplementation; however, their effects on organisms remain to be elucidated. Here, we examine the effects of dietary Aloysia triphylla EO supplementation on zebrafish behavior, metabolism, stress response, and growth performance. We show that fish fed diets containing A. triphylla EO presented an anxiolytic response, with reduced exploratory activity and oxygen consumption; no changes were observed in neuroendocrine stress axis functioning and growth was not impaired. Taken together, these results suggest that the A. triphylla EO supplementation is a strong candidate for use in feed, since it ensures fish welfare (anxiolytic behavior) with decreased oxygen consumption. This makes it suitable for use in high-density production systems without causing damage to the neuroendocrine stress axis and without growth performance being impaired.

The genetic manipulation in many cyanobacterial strains is challenging yet. Thus, the development of new transformation protocols is desirable to facilitate the genetic engineering in cyanobacteria. Transformations using linear fragments yielded by PCR have advantages such as: less laborious methodology, faster procedure, low cost and unnecessary cloning steps. However, some strains presence extracellular nucleases, which reduce the efficiency in obtaining transformants. In this study, we demonstrate an improved protocol for genetic transformation in Synechococcus elongatus PCC 7942 using linear fragments employing EDTA-mediated inhibition of DNases. To conduct the transformation, linear PCR products containing the spectinomycin antibiotic resistance gene were employed. As result, 40 mM EDTA treatment increased the number of transformants obtained by eightfold in comparison to the conventional protocol using plasmid DNA. Thus, the application of exonuclease inhibitors can be considered a relevant improvement to manipulate cyanobacteria in a more efficient, faster way and as a low-cost alternative. This protocol must be helpful for other strains of cyanobacteria. Keywords: Cyanobacteria, EDTA, Exonuclease, PCR products

Variation in oocyte quality affects sustainability of finfish aquaculture products including Atlantic halibut (Hippoglossus hippoglossus L.). To have an insight into role of major egg yolk constituents in the oocyte quality, egg fatty acid (FA), amino acid (AA) and folate contents were related to normal blastomere symmetry, and fertilization and hatching success. Significant correlations were found between dihomo-c-linolenic acid (DGLA, 20:3n6), palmitic acid (PA, 16:00) and docosapentaenoic acid (DPA, 22:5n3), and normal blastomere symmetry and survival success. Egg concentrations of myristic acid (MA, 14:0), oleic acid (OA, 18:1n9), stearidonic acid (SA, 18:4n3) and eicosadienoic acid (EDA, 20:2n6) explained 56 % variation in fertilization success. OA and EDA explained 70 % of variation in blastomere symmetry. Palmitoleic acid (POA, 16:1n7) and linolenic acid (LNA, 18:3n3) concentrations explained 57% of variation in hatching success. Egg valine concentrations correlated with fertilization rates, and aspartic acid and leucine correlated with normal blastomere symmetry. Alanine and valine concentrations explained together 45% of variation in fertilization. Glutamic acid and tyrosine concentrations explained 68% of variation in normal blastomere symmetry while serine, arginine and valine explained 36% of variation in hatching. These FAs and AAa may be potential indicators of oocyte quality in Atlantic halibut.

Farming of Atlantic cod, Gadus morhua L., has been facing several challenges lately. Biological issues such as differences in egg quality and lack of high quality larvae are considered as prominent limiting factors. The aim of this study was to compare fertilization and hatching rates, incidence of malformed larvae at hatching, larval viability (high-salinity stress and starvation tolerance tests) and the biochemical profile (proteins, amino acids, lipids, fatty acids and minerals) between eggs produced from wild and farmed broodstocks. Twenty-five batches of eggs from farmed broodstock (FB) and 27 egg batches from wild broodstock (WB) were collected during the reproductive season. Fertilization and hatching rates were significantly higher in WB group (91 ± 3% and 86 ± 3%, respectively) than in FB (75 ± 3% and 66 ± 3%, respectively). In high-salinity stress test, WB larvae survived for a significantly longer time than FB larvae. No differences between egg batches from the two broodstocks were observed in starvation tolerance tests, incidence of malformed larvae at hatching, as well as in the total content of proteins, lipids, fatty acids, zinc, and calcium concentrations. Regarding fatty acids, FB eggs had significantly higher levels of C16:0, C18:0, C18:2n − 6, C18:3n − 3, C18:3n − 6, C20:4n − 6, C20n:5n − 3, C22:5n − 3 and C22:5n − 6, whereas WB eggs had significantly higher levels of C14:0, C16:1n − 7, C18:4n − 3, C20:4n − 3, C22:6n − 3 and C24:1n − 9. As for the amino acid profile, WB eggs had significantly higher levels of aspartic acid, serine, glycine, threonine, taurine and leucine, while FB eggs had significantly higher levels of proline, tryptophan, phenylalanine and arginine. The concentrations of minerals such as copper and phosphorus were significantly higher in WB eggs, whereas in FB eggs iron concentration was significantly higher. Overall, the biological parameters indicated that egg and larvae originating from WB were of superior quality than those from FB. The information on the biochemical profile may be useful in improving the quality of broodstock feeds for Atlantic cod.

The aim of this study was to evaluate the sperm quality of Brazilian flounder Paralichthys orbignyanus throughout its reproductive season. Sperm was collected at the beginning, middle and end of the breeding period. Spermatozoa density was maximum at the beginning (12.7 ± 0.92 × 109 cells mL−1) and at the end (11.8 ± 0.39 × 109 cells mL−1) of the breeding season (P

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.

In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio) were produced using two transgenes, both comprised of the carp beta-actin promoter driving the expression of either the green fluorescent protein (GFP) reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.

Significant efforts have been made to elucidate factors affecting egg quality in fish. Recently, we have shown that eggs originating from wild broodstock (WB) of Atlantic cod (Gadus morhua L.) are of superior quality to those derived from farmed broodstock (FB), and this is associated with differences in the chemical composition of egg yolk. However, maternal transcripts, accumulated during oogenesis, have not been studied extensively in fish. The aim of the present study was to characterize putative maternal mRNA transcriptome in fertilized eggs of Atlantic cod and to compare transcript pools between WB and FB in order to investigate the relation between egg developmental potential and putative maternal mRNA deposits. We performed high-throughput 454 pyrosequencing. For each WB and FB group, five cDNA libraries were individually tagged and sequenced, resulting in 98,687 (WB) and 119,333 (FB) average reads per library. Sequencing reads were de novo assembled, annotated, and mapped. Out of 13,726 identified isotigs, 238 were differentially expressed between WB and FB, with 155 isotigs significantly upregulated in WB. The sequence reads were mapped to 11,340 different Atlantic cod transcripts and 158 sequences were differentially expressed between the 2 groups. Important transcripts involved in fructose metabolism, fatty acid metabolism, glycerophospholipid metabolism, and oxidative phosphorylation were differentially represented between the two broodstock groups, showing potential as biomarkers of egg quality in teleosts. Our findings contribute to the hypothesis that maternal mRNAs affect egg quality and, consequently, the early development of fish.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:53:11Z No. of bitstreams: 1 S0001-37652012000200017.pdf: 47432223 bytes, checksum: 0a33e748f2be8bd48b59f585b01dd7e9 (MD5) Made available in DSpace on 2013-08-22T18:53:11Z (GMT). No. of bitstreams: 1 S0001-37652012000200017.pdf: 47432223 bytes, checksum: 0a33e748f2be8bd48b59f585b01dd7e9 (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T19:44:42Z (GMT). No. of bitstreams: 2 S0001-37652012000200017.pdf: 47432223 bytes, checksum: 0a33e748f2be8bd48b59f585b01dd7e9 (MD5) S0001-37652012000200017.pdf.txt: 23124 bytes, checksum: 8a44dd874a9a9847502911fa926779bb (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:18Z No. of bitstreams: 2 S0001-37652012000200017.pdf: 47432223 bytes, checksum: 0a33e748f2be8bd48b59f585b01dd7e9 (MD5) S0001-37652012000200017.pdf.txt: 23124 bytes, checksum: 8a44dd874a9a9847502911fa926779bb (MD5) Made available in DSpace on 2014-05-20T13:50:18Z (GMT). No. of bitstreams: 2 S0001-37652012000200017.pdf: 47432223 bytes, checksum: 0a33e748f2be8bd48b59f585b01dd7e9 (MD5) S0001-37652012000200017.pdf.txt: 23124 bytes, checksum: 8a44dd874a9a9847502911fa926779bb (MD5) Previous issue date: 2012-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A presença de níveis mais elevados do hormônio de crescimento (GH) em animais transgênicos poderia levar a várias alterações fisiológicas. Uma linhagem transgênica de paulistinha (Danio rerio) para o GH foi comparada com amostras não transgênicas (NT) desta espécie, através de uma abordagem de DDRT-PCR, com o objetivo de identificar transcritos candidatos diferencialmente expressos em tecido cerebral que poderiam estar envolvidos na superexpressão de GH. Análises densitométricas de dois produtos de amplificação selecionados, p300 e ADCY2, apontaram uma expressão gênica significativamente menor nas amostras transgênicas de paulistinha (104.02 ± 57.71; 224.10 ± 91.73), quando comparadas com as amostras NT (249.75 ± 30.08; 342.95±65.19). Os presentes dados indicam que p300 e ADCY2 estão envolvidos em um sistema de regulação do GH, quando altos níveis circulantes desse hormônio são encontrados em paulistinha. The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes. UNESP Instituto de Biociências Departamento de Genética Universidade Federal do Rio Grande Instituto de Ciências Biológicas UNESP Instituto de Biociências Departamento de Genética

The aim of this study was to evaluate the effects of growth hormone (GH) overexpression on the gene expression profile of multiple components of the antioxidant defense system (ADS) of different genotypes of a GH-transgenic zebrafish (Danio rerio) model. Several ADS-related genes were analyzed by semiquantitative reverse transcription-PCR in the liver of hemizygous (HE) and homozygous (HO) transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotype-dependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse, which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

Homozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NT and HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO (p < 0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p < 0.01) and 4.3 times (p < 0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins.

Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.

Most biological actions of growth hormone (GH) are mediated by the insulin-like growth factor I (IGF-I) that is produced after the interaction of the hormone with a specific cell surface receptor, the GH receptor (GHR). Even though the GH excess on fish metabolism is poorly known, several species have been genetically engineered for this hormone in order to improve growth for aquaculture. In some GH-transgenic fish growth has been dramatically increased, while in others high levels of transgene expression have shown inhibition of the growth response. In this study, we used for the first time different genotypes (hemizygous and homozygous) of a GH-transgenic zebrafish (Danio rerio) lineage as a model for studying the GH resistance induced by different GH transgene expression levels. The results obtained here demonstrated that homozygous fish did not grow as expected and have a lower condition factor, which implies a catabolic state. These findings are explained by a decreased IGF-I and GHR gene expression as a consequence of GH resistance. Together, our results demonstrated that homozygous GH-transgenic fish showed similar characteristics to the starvation-induced fish and could be an interesting model for studying the regulation of the GH/GHR/IGF-I axis in fish.

Background Microalgae are becoming a viable model for the expression of recombinant proteins. However, suitable levels of expression for commercial production have not yet been obtained. One way to increase the heterologous protein production is the codon optimization of the target gene so that it presents the preferential codons used by the host organism according to its own pool of transfer RNA (tRNA). For codon optimization the Codon Usage Database (CUD; http://www.kazusa.or.jp/codon) has been used. The database provides codon frequency tables based on gene transcripts, regardless of their level of expression. Therefore, the aim of this study was to determine if there are changes in the frequency of codons used when taking into account the level of expression of genes, considering the 50 most expressed genes and 50 least expressed genes from the microalgae Chlamydomonas reinhardtii.

We investigated the profiles of 25 genes involved in apoptosis (bcl-x2, casp3, casp8, ccar1, mcl1, and tpt1), immunity (bty, cathl, ifng, il1b, il6, il8, il10, lyzg, and tfa), oxidative stress (cat, gpx4, gsh-px, hsp70, hsp90a, and sod1), and stress axis (crh, pomc, grl1, and mlr) during Atlantic cod development and compared the mRNA transcript levels between samples from farmed (FB) and wild broodstock (WB) using real-time polymerase chain reaction. The suitability of nine endogenous housekeeping genes and an external standard (luciferase) as reference genes was also evaluated. The cycle threshold values of all housekeeping genes differed significantly throughout Atlantic cod development. Fertilization and hatching rates were significantly higher in WB group (95 ± 1.8% and 89 ± 2.8%, respectively) compared with FB (75 ± 3.4% and 66 ± 3.2%, respectively). Eleven target genes, namely, ccar1, casp3, bcl-x2, mcl-1, cat, gsh-px, hsp70, sod1, lyzg, il8, and grl were expressed in both groups at fertilization stage, indicating their maternal transfer. Among them, transcripts of gsh-px were more abundant in WB eggs, while the expression of hsp70 was significantly higher in FB eggs. In FB larvae, expression of cat, hsp70, hsp90a, pomc, mlr, grl1, bclx2, and il6 was significantly higher at hatching and the expression of cat, gpx4, casp3 and ccar1 was significantly higher at first feeding stages, than in WB group. These findings give an insight into the expressional changes in certain category of genes involved in the embryonic development of Atlantic cod, which may eventually determine the ultimate quality of the larvae.