[Novel tropomyosin consensus B and T epitopes involved in cross-reactivity between 10 different species. An in silico study]. / Nuevos epítopes B y T, consenso de tropomiosina involucradas en reactividad cruzada entre diez especies diferentes. Un estudio in silico.

OBJECTIVE: This study aimed to identify by in silico methods tropomyosin consensus B and T epitopes of shrimp species, house dust mites, insects, and nematodes associated with allergic diseases in tropical countries. METHODS: In silico analysis included tropomyosin from mites (Der p 10, Der f 10, Blo t 10), insects (Aed a 10, Per a 7, Bla g 7), shrimp (Lit v 1, Pen m 1, Pen a 1), and nematode (Asc l 3) all sequences were taken from the UniProt database. Linear IgE epitopes were predicted with AlgPred 2.0 and validated with BepiPred 3.0. MHC-II binding T cell epitopes were predicted using the IEDB server, which implements nine predictive methods (consensus method, combinatorial library, NN-align-2.3, NN- align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1, and NetMHCIIpan 3.2) these predictions focused on 10 HLA-DR and 2 HLA-DQ alleles associated with allergic diseases. Subsequently, consensus B and T epitopes present in all species were identified. RESULTS: We identified 12 sequences that behaved as IgE-epitopes and B-cell epitopes, three of them: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN215, 251KEVDRLEDELV261 were consensus in all species. Eleven peptides (T-epitopes) showed strong binding (percentile rank ≤ 2.0) to HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401, HLA-DQA1*03:01/DQB1*03:02, and HLA- DQA1*05:01/DQB1*02:01. Only two T-epitopes were consensus in all species: 167RKLAMVEADLERAEERAEt GEsKIVELEEELRV199, and 218EEeY KQQIKT LTaKLKEAEARAEFAERSV246. Subsequently, we identified 2 B and T epitope sequences and reached a consensus between species 167RKLAMVEA174 and 192ELEEELRV199. CONCLUSIONS: These data describe three sequences that may explain the IgE cross-reactivity between the analyzed species. In addition, the consensus B and T epitopes can be used for further in vitro investigations and may help to design multiple-epitope protein-based immunotherapy for tropomyosin-related allergic diseases.

OBJETIVO: Este estudio tuvo como objetivo identificar mediante métodos in silico epítopes B y T consenso de tropomiosina de especies de camarón, ácaros del polvo doméstico, insectos y nematodos asociados a enfermedades alérgicas en países tropicales. MÉTODOS: El análisis in silico incluyó tropomiosina de ácaros (Der p 10, Der f 10, Blo t 10), insectos (Aed a 10, Per a 7, Bla g 7), camarones (Lit v 1, Pen m 1, Pen a 1), y nematodo (Asc l 3). Todas las secuencias se tomaron de la base de datos UniProt. Los epítopes IgE lineales se predijeron con AlgPred 2.0 y se validaron con BepiPred 3.0. Los epítopes de células T de unión a MHC-II se predijeron utilizando el servidor IEDB, que implementa nueve métodos predictivos (método de consenso, biblioteca combinatoria, NN-align-2.3, NN-align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1 y NetMHCIIpan 3.2). Estas predicciones se centraron en diez alelos HLA-DR y 2 HLA-DQ asociados con enfermedades alérgicas. Posteriormente, se identificaron epítopes consenso B y T presentes en todas las especies. RESULTADOS: Se identificaron 12 secuencias que se comportaron como epítopes de IgE y, también, como epítopes de células B. Tres de ellas: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN213 y 251KEVDRLEDELV261, fueron consenso en todas las especies. Once péptidos mostraron una fuerte unión (rango percentil ≤ 2,0) a HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401 y a HLA HLA-DQA1*03:01/DQB1*03:02, o HLA-DQA1*05:01/DQB1*02:01. Solo se encontraron dos secuencias: 167RKLAMVEADLERAEERAEtGEsKIVELEEELRV199 con fuerte afinidad por HLA-DQA1*03:01/DQB1*03:02, y HLA-DQA1*05:01/DQB1*02:01. Se identificaron dos secuencias que son epítopos B y T, y son consenso entre especies: 167RKLAMVEA174 y 192ELEEELRV199. CONCLUSIONES: Estos datos describen tres secuencias que pueden explicar la reactividad cruzada de IgE entre las especies analizadas. Además, los epítopos B y T consenso se pueden usar para investigaciones in vitro adicionales, y pueden ayudar a diseñar inmunoterapia basada en proteínas de múltiepítopes para enfermedades alérgicas relacionadas con la tropomiosina.

[Frequency of sensitization to shrimp with house dust mite immunotherapy with polymerized extracts]. / Frecuencia de sensibilización a camarón con inmunoterapia a ácaros con extractos polimerizados.

OBJECTIVE: To evaluate the risk of IgE sensitization and symptoms to shrimp in a population that has received AIT with polymerized mite extract. METHODS: Patients with allergic rhinitis sensitized to dust mites (Dermatophogides spp) with an indication for mite AIT were included. Those patients who had not yet received AIT or had received less than 6 doses were included as controls and those who had received more than 24 doses of AIT were included as cases. Sensitization to shrimp was assessed by skin prick test with complete shrimp extract and/or shrimp-specific IgE. RESULTS: A total of 68 patients were included; 47 cases and 21 controls. When calculating the odds ratio of sensitization according to time with immunotherapy we observed that there were no differences between the group of cases and controls (OR 0.76 95% CI 0.26 to 2.22 p 0.7 by MacNemar technique). Factors such as consumption or not of shrimp and frequency of consumption do not seem to be related to the outcome. CONCLUSION: In contrast to what was reported with aqueous extracts, we observed that AIT with polymerized extracts is not a risk factor for shrimp sensitization. It is necessary to reproduce these results with a larger sample size to explore other factors.

OBJETIVO: Evaluar el riesgo de sensibilización IgE y síntomas a camarón en una población que ha recibido AIT con extracto polimerizado para ácaros. MÉTODOS: Se incluyeron pacientes con rinitis alérgica sensibilizados a ácaros del polvo (Dermatophogides spp) con indicación de AIT para ácaros. Aquellos pacientes que no habían aún recibido AIT o llevaban menos de seis dosis, fueron incluidos como controles, y aquellos que llevaban más de 24 dosis de AIT, fueron incluidos como casos. Se evaluó la sensibilización a camarón mediante prueba cutánea con extracto completo de camarón y/o IgE específica a camarón. RESULTADOS: En total, 68 pacientes fueron incluidos; 47 casos y 21 controles. Al calcular el odds ratio de la sensibilización de acuerdo al tiempo con la inmunoterapia, observamos que no había diferencias entre el grupo de casos y controles (OR 0,76 95% IC 0,26 a 2,22 p 0,7 por técnica de MacNemar). Factores como el consumo o no de camarón y la frecuencia de consumo, no parecen estar relacionados con el desenlace. CONCLUSIÓN: A diferencia de lo reportado con extractos acuosos, observamos de AIT con extractos polimerizados para no es un factor de riesgo para la sensibilización a camarón. Es necesario reproducir estos resultados con un mayor tamaño de muestra que permita explorar otros factores.

Clinical Relevance of Shrimp Sensitization in Patients with Allergic Rhinitis: Anti-Der p 10 IgE as Predictor.

INTRODUCTION: Cross-reactivity between shrimp and house dust mite (HDM) proteins has been widely documented. In tropical region, shrimp (5-15%) and mite sensitization (80-95%) is prevalent in allergic patients. However, the clinical relevance of shrimp sensitization in patients with allergic rhinitis (AR) has been poorly studied. The aim of this study was to determine the prevalence and the clinical relevance shrimp IgE sensitization in AR patients sensitized to Dermatophagoides pteronyssinus. METHODS: The study was conducted in Medellin (Colombia). A cross-sectional study in patients with AR sensitized to HDM was performed in 3 steps: (i) assessment of IgE sensitization frequency to shrimp Penaeus azteca, Litopenaeus vannamei, and tropomyosin homologous allergens rDer p 10, rPen a 1, and rLit v 1, (ii) evaluation of the clinical relevance of shrimp sensitization using oral challenge test (OCT) and (iii) identification of possible risk factors for positive-OCT results. Ethical committee approval was obtained. RESULTS: From 443 patients with AR, 86 (19.4%) were sensitized to shrimp and 23 of them (26.7%) had shrimp allergy diagnosis. Thirty-six of the patients sensitized to shrimp (41.2%) reported not previously consumed this food and eleven of them had a positive-OCT (30.5%). There was not statistically significant difference in total IgE or sIgE (D. pteronyssinus, P. azteca, L. vannamei, rPen a 1, and rLit v 1) between OCT groups (positive vs. negative results). Anti-Der p 10 IgE was associated with risk for a positive-OCT in different multivariable scenarios. DISCUSSION/CONCLUSION: Our results suggest that in patients with HDM-associated AR and shrimp IgE sensitization is necessary to evaluate the clinical relevance of shrimp IgE even if the patient has never consumed shrimp because of cross-reactivity. Anti-Der p 10 could be a possible biomarker of clinical relevance to shrimp sensitization and could reduce the need for OCTs.

Addition of Aurantiochytrium sp. meal in the diet affects immunity and thermal shock resistance of the pacific white shrimp / Adição de farinha de Aurantiochytrium sp. na dieta afeta a imunidade e resistência ao choque térmico do camarão-branco-do-pacífico

This paper reports the evaluation of digestibility, immunological parameters, and resistance to thermal shock at low temperature in Litopenaeus vannamei fed diets with diferent Aurantiochytrium sp. meal additions (0; 0.5; 1 and 2%). First, the apparent digestibility coefficient of the ingredient was determined. The digestibility of the microalgae meal was high for protein (74.90%); around 60% for lipids, and for docosahexaenoic fatty acid (DHA) it was 55.61%. After, shrimp rearing with the feed additive was carried out in a clear water system, containing 25 shrimp (initial weight: 4.89 ± 0.27g) per 400 L tank. Feeding occurred four times a day. After a three-week period, immunological parameters were evaluated and thermal shock was performed. Animals fed 0.5% and 2% of the microalgae Aurantiochytrium sp. showed higher survival to thermal shock. In immunological analyses, the serum agglutiniting titer was higher (p <0.05) in the 0.5 and 2% additions of the microalgae meal, and the phenoloxidase activity (PO) was higher in the 1% addition (p <0.05). It is concluded that there is good utilization of the nutrients of Aurantiochytrium sp. meal by L. vannamei and its addition to the diet (0.5 and 1%) increased shrimp resistance to thermal shock.(AU)

Avaliou-se a digestibilidade, os parâmetros imunológicos e a resistência ao choque térmico a baixa temperatura em Litopenaeus vannamei alimentado com dietas contendo diferentes proporções de farinha da microalga Aurantiochytrium sp. (0; 0,5; 1 e 2%). Inicialmente, foi determinado o coeficiente de digestibilidade aparente do ingrediente. A digestibilidade da farinha da microalga foi alta para proteína (74,9%), e em torno no 60% para lipídeos em geral, sendo que para o ácido graxo docosahexaenóico (DHA) foi de 55,61%. Posteriormente, o cultivo com o aditivo alimentar foi realizado em sistema de água clara, contendo 25 camarões (peso inicial 4,89 ± 0,27 g) por tanque de 400 L, alimentados quatro vezes ao dia. Após o período de três semanas, foram avaliados os parâmetros imunológicos e realizado o choque térmico. Os animais alimentados com 0,5% e 1% da microalga na dieta apresentaram melhor sobrevivência ao choque térmico. Nas análises imunológicas, o título aglutinante do soro foi significativamente superior (p <0,05) nas adições de 0,5 e 2% da farinha da microalga, e a atividade da fonoloxidade (PO) na adição de 1% (p <0,05). Conclui-se que a farinha de Aurantiochytrium sp. tem bom aproveitamento de seus nutrientes por L. vannamei e sua adição na dieta (0,5 e 1%) aumentou a resistência dos camarões à variação térmica.(AU)

In vitro antimicrobial activity of carvacrol against shrimp pathogens and its use as feed additive for the pacific white shrimp / Atividade antimocrobiana in vitro do carbacrol contra patógenos de camarões e seu uso como aditivo alimentar na dieta do camarão-branco-do-pacífico

This study aimed to evaluate the in vitro effect of the phenolic compound carvacrol on different microorganisms of importance in shrimp farming, and it's in vivo effect on zootechnical, immunological and microbiological performance, and resistance of Litopenaeus vannamei challenged with Vibrio parahaemolyticus. In particular, the antimicrobial activity of carvacrol was evaluated in vitro by analysis of the minimum inhibitory concentration (MIC) and by agar diffusion with Gram-negative and Gram-positive bacteria. The in vivo experiment was conducted using different concentrations of carvacrol (1, 3, 4 and 6 mg mL-¹) added to shrimp feed, together with a control diet without carvacrol. After four weeks, zootechnical, immunological and microbiological parameters and resistance of animals challenged with V. parahaemolyticus were evaluated. The MIC of carvacrol was 0.078 mg mL-¹ for Vibrio alginolyticus and Vibrio harveyi, while for the other bacteria, it was 0.156 mg mL-¹ of carvacrol. The greatest halos of inhibition were observed in V. parahaemolyticus and V. harveyi with significant differences demonstrated for the other microorganisms, except Escherichia coli. The in vivo results showed no significant differences among treatments. In conclusion, the antimicrobial activity of carvacrol was confirmed with Gram-negative and Gram-positive bacteria, and it is suggested that its antimicrobial potential is more effective against Vibrio spp. However, the concentrations of carvacrol used in vivo did not affect the parameters evaluated.(AU)

O objetivo do presente estudo foi avaliar o efeito in vitro do carvacrol contra diferentes microrganismos de importância na carcinicultura e o seu efeito in vivo no desempenho zootécnico, imunológico, microbiológico e na resistência de Litopenaeus vannamei desafiados com Vibrio parahaemolyticus. A atividade antimicrobiana do carvacrol foi realizada in vitro pela análise da concentração inibitória mínima (CIM) e por difusão em ágar, com bactérias Gram-negativas e Gram-positivas. Para o experimento in vivo foram adicionadas diferentes concentrações do carvacrol (1, 3, 4 e 6 mg mL-¹) na alimentação dos camarões e uma dieta controle. Após quatro semanas, foram avaliados parâmetros zootécnicos, imunológicos, microbiológicos e a resistência dos animais desafiados com V. parahaemolyticus. A CIM do carvacrol para Vibrio alginolyticuse Vibrio harveyi foi de 0,078 mg mL-¹, enquanto nas demais bactérias foi de 0,156 mg mL-¹ de carvacrol. Os maiores halos de inibição foram observados em V. parahaemolyticus e V. harveyie demonstraram diferenças significativas em relação aos demais microrganismos, exceto Escherichia coli. Os resultados in vivo não demonstraram diferenças significativas entre os tratamentos. Em conclusão, a atividade antimicrobiana do carvacrol foi confirmada com bactérias Gram-negativas e Gram-positivas e sugere-se que seu potencial antimicrobiano seja mais eficaz contra Vibrio spp. No entanto, as concentrações de carvacrol utilizadas in vivo não afetaram os parâmetros avaliados.(AU)

In vitro antimicrobial activity of carvacrol against shrimp pathogens and its use as feed additive for the pacific white shrimp / Atividade antimocrobiana in vitro do carbacrol contra patógenos de camarões e seu uso como aditivo alimentar na dieta do camarão-branco-do-pacífico

This study aimed to evaluate the in vitro effect of the phenolic compound carvacrol on different microorganisms of importance in shrimp farming, and it's in vivo effect on zootechnical, immunological and microbiological performance, and resistance of Litopenaeus vannamei challenged with Vibrio parahaemolyticus. In particular, the antimicrobial activity of carvacrol was evaluated in vitro by analysis of the minimum inhibitory concentration (MIC) and by agar diffusion with Gram-negative and Gram-positive bacteria. The in vivo experiment was conducted using different concentrations of carvacrol (1, 3, 4 and 6 mg mL-¹) added to shrimp feed, together with a control diet without carvacrol. After four weeks, zootechnical, immunological and microbiological parameters and resistance of animals challenged with V. parahaemolyticus were evaluated. The MIC of carvacrol was 0.078 mg mL-¹ for Vibrio alginolyticus and Vibrio harveyi, while for the other bacteria, it was 0.156 mg mL-¹ of carvacrol. The greatest halos of inhibition were observed in V. parahaemolyticus and V. harveyi with significant differences demonstrated for the other microorganisms, except Escherichia coli. The in vivo results showed no significant differences among treatments. In conclusion, the antimicrobial activity of carvacrol was confirmed with Gram-negative and Gram-positive bacteria, and it is suggested that its antimicrobial potential is more effective against Vibrio spp. However, the concentrations of carvacrol used in vivo did not affect the parameters evaluated.

O objetivo do presente estudo foi avaliar o efeito in vitro do carvacrol contra diferentes microrganismos de importância na carcinicultura e o seu efeito in vivo no desempenho zootécnico, imunológico, microbiológico e na resistência de Litopenaeus vannamei desafiados com Vibrio parahaemolyticus. A atividade antimicrobiana do carvacrol foi realizada in vitro pela análise da concentração inibitória mínima (CIM) e por difusão em ágar, com bactérias Gram-negativas e Gram-positivas. Para o experimento in vivo foram adicionadas diferentes concentrações do carvacrol (1, 3, 4 e 6 mg mL-¹) na alimentação dos camarões e uma dieta controle. Após quatro semanas, foram avaliados parâmetros zootécnicos, imunológicos, microbiológicos e a resistência dos animais desafiados com V. parahaemolyticus. A CIM do carvacrol para Vibrio alginolyticuse Vibrio harveyi foi de 0,078 mg mL-¹, enquanto nas demais bactérias foi de 0,156 mg mL-¹ de carvacrol. Os maiores halos de inibição foram observados em V. parahaemolyticus e V. harveyie demonstraram diferenças significativas em relação aos demais microrganismos, exceto Escherichia coli. Os resultados in vivo não demonstraram diferenças significativas entre os tratamentos. Em conclusão, a atividade antimicrobiana do carvacrol foi confirmada com bactérias Gram-negativas e Gram-positivas e sugere-se que seu potencial antimicrobiano seja mais eficaz contra Vibrio spp. No entanto, as concentrações de carvacrol utilizadas in vivo não afetaram os parâmetros avaliados.

Addition of Aurantiochytrium sp. meal in the diet affects immunity and thermal shock resistance of the pacific white shrimp / Adição de farinha de Aurantiochytrium sp. na dieta afeta a imunidade e resistência ao choque térmico do camarão-branco-do-pacífico

This paper reports the evaluation of digestibility, immunological parameters, and resistance to thermal shock at low temperature in Litopenaeus vannamei fed diets with diferent Aurantiochytrium sp. meal additions (0; 0.5; 1 and 2%). First, the apparent digestibility coefficient of the ingredient was determined. The digestibility of the microalgae meal was high for protein (74.90%); around 60% for lipids, and for docosahexaenoic fatty acid (DHA) it was 55.61%. After, shrimp rearing with the feed additive was carried out in a clear water system, containing 25 shrimp (initial weight: 4.89 ± 0.27g) per 400 L tank. Feeding occurred four times a day. After a three-week period, immunological parameters were evaluated and thermal shock was performed. Animals fed 0.5% and 2% of the microalgae Aurantiochytrium sp. showed higher survival to thermal shock. In immunological analyses, the serum agglutiniting titer was higher (p <0.05) in the 0.5 and 2% additions of the microalgae meal, and the phenoloxidase activity (PO) was higher in the 1% addition (p <0.05). It is concluded that there is good utilization of the nutrients of Aurantiochytrium sp. meal by L. vannamei and its addition to the diet (0.5 and 1%) increased shrimp resistance to thermal shock.

Avaliou-se a digestibilidade, os parâmetros imunológicos e a resistência ao choque térmico a baixa temperatura em Litopenaeus vannamei alimentado com dietas contendo diferentes proporções de farinha da microalga Aurantiochytrium sp. (0; 0,5; 1 e 2%). Inicialmente, foi determinado o coeficiente de digestibilidade aparente do ingrediente. A digestibilidade da farinha da microalga foi alta para proteína (74,9%), e em torno no 60% para lipídeos em geral, sendo que para o ácido graxo docosahexaenóico (DHA) foi de 55,61%. Posteriormente, o cultivo com o aditivo alimentar foi realizado em sistema de água clara, contendo 25 camarões (peso inicial 4,89 ± 0,27 g) por tanque de 400 L, alimentados quatro vezes ao dia. Após o período de três semanas, foram avaliados os parâmetros imunológicos e realizado o choque térmico. Os animais alimentados com 0,5% e 1% da microalga na dieta apresentaram melhor sobrevivência ao choque térmico. Nas análises imunológicas, o título aglutinante do soro foi significativamente superior (p <0,05) nas adições de 0,5 e 2% da farinha da microalga, e a atividade da fonoloxidade (PO) na adição de 1% (p <0,05). Conclui-se que a farinha de Aurantiochytrium sp. tem bom aproveitamento de seus nutrientes por L. vannamei e sua adição na dieta (0,5 e 1%) aumentou a resistência dos camarões à variação térmica.

Transcriptional response of immune-related genes in Litopenaeus vannamei cultured in recirculating aquaculture systems with elevated CO2

This short-term study evaluated the effect of non-lethal high CO2 concentration on the transcriptional response of immune-related genes of Pacific white shrimp (Litopenaeus vannamei) cultured in recirculating aquaculture systems (RAS). Two experimental groups were created: high CO2 (47.67±2.04 mg L−1) and low CO2 (2.0±1.93 mg L−1). Shrimp of 8.85±1.20 g were placed randomly at a density equivalent to 100 individuals m−3 and were monitored at 6, 12, 18, and 24 h. The transcriptional response of immune-related genes was analyzed by qPCR. Gene expression of hemocyanin, prophenoloxidase, and heat shock protein 60 was downregulated at 24 h, suggesting affectations on oxygen transportation, melanization, and protein functioning of L. vannamei under high CO2 concentrations. Also, gene up-regulation of lipopolysaccharide- and ß-glucan-binding protein and cytosolic manganese superoxide dismutase can impair the bacterial recognition and antioxidant defense of shrimp exposed to high CO2 concentrations. These results suggest that concentration at about 47 mg L−1 of CO2 can significantly influence the transcriptional response modulation of immune-related genes.

Shedding the Light on Litopenaeus vannamei Differential Muscle and Hepatopancreas Immune Responses in White Spot Syndrome Virus (WSSV) Exposure.

White Spot Syndrome Virus (WSSV) is one of the main threats to farming Litopenaeus vannamei, the most important crustacean commercialized in aquaculture worldwide. Here, we performed RNA-seq analyses in hepatopancreas and muscle from WSSV-negative (healthy) and WSSV-positive (unhealthy) L. vannamei, previously exposed to the virus, to obtain new insights about the molecular basis of resistance to WSSV. We detected 71% of our reads mapped against the recently described L. vannamei genome. This is the first report mapping RNA-seq transcripts from shrimps exposed to WSSV against the species reference genome. Differentially expressed gene (DEG) analyses were performed for four independent comparisons, and 13,338 DEGs were identified. When the redundancies and isoforms were disregarded, we observed 8351 and 6514 DEGs, respectively. Interestingly, after crossing the data, we detected a common set of DEGs for hepatopancreas and healthy shrimps, as well as another one for muscle and unhealthy shrimps. Our findings indicate that genes related to apoptosis, melanization, and the Imd pathway are likely to be involved in response to WSSV, offering knowledge about WSSV defense in shrimps exposed to the virus but not infected. These data present potential to be applied in further genetic studies in penaeids and other farmed shrimp species.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

Modulation of nodularin toxicity in shrimp Litopenaeus vannamei (BOONE, 1931) fed with dietary açai (Euterpe oleracea) inclusion.

This study evaluated the effect of dietary inclusion of lyophilized açaí Euterpe oleracea (LEO) on redox status of shrimp Litopenaeus vannamei (initial weight 1.5 ± 0.39 g) upon exposure to cyanotoxin nodularin (NOD) in bioflocs system. Three hundred juvenile shrimps were randomly divided into two groups and fed twice a day with two diets: one containing 0.00 (control diet) and the other 10.0% LEO (w/w) for 30-days. After the feeding period, both shrimp groups were submitted to three treatments (14 L; 7 shrimp/tank) with different concentrations of cyanotoxin NOD (0.00; 0.25; and 1.00 µg/L) dissolved in water with 96 h of exposure. Then, the shrimps were sampled (n = 15/treatment) for the determination of reduced glutathione (GSH), the activity of glutathione-S-transferase (GST), sulfhydryl groups associated to proteins (P-SH), and lipid peroxidation (TBARS) in the hepatopancreas, gills and muscle. The NOD accumulation was measured in the muscle. The results revealed that dietary LEO significantly increased GSH levels in the hepatopancreas and gills of the shrimps exposed to NOD. Toxin exposure did not modify GST activity in all organs. Muscle TBARS levels were lower in the shrimp fed with the LEO diet and exposed to NOD. The NOD toxin did not accumulate in the muscle but notably was detected in the control groups fed or not with dietary LEO. Açaí was able to induce the antioxidant system of L. vannamei, as well as lowered the oxidative damage in shrimps exposed to NOD, suggesting its use as a chemoprotectant against cyanotoxins.

Environmental rearing conditions are key determinants of changes in immune gene expression patterns in shrimp midgut.

The super-intensive BioFloc Technology (BFT) system has been highlighted as a promising eco-friendly alternative to the traditional shrimp rearing systems. To gain insight into the impact of environmental rearing conditions on shrimp intestinal immunity, we assessed the expression profile of key immunological genes in the midgut of Litopenaeus vannamei shrimp reared in two contrasting culture systems: the indoor super-intensive BFT and the outdoor intensive Green-Water System (GWS). From the 30 analyzed genes, the expression levels of 25 genes were higher in the midgut of shrimp reared in BFT than in GWS. The main functional categories represented in BFT-shrimp were the prophenoloxidase-activating system, immune signaling, antimicrobial peptides, and RNA interference pathway. Comparatively, only the RNAi pathway gene Dicer-1 (LvDcr1) was more expressed in animals from the GWS group. However, despite the differences in gene expression, the total midgut bacterial abundance was similar between the experimental groups. Altogether, our results suggest that the microbial-rich environment offered by the BFT system can be acting as an immunostimulant by altering the immune expression profile of the midgut. The gene expression level found in GWS animals could be related to the chronic presence of the IMNV in the Brazilian Northeast. Knowing the effects of environmental stress factors on the intestinal immune defenses can provide an in-depth understanding of the relationship between cultivated shrimp and the major pathogens affecting the shrimp industry.

Induction of innate immune response in whiteleg shrimp (Litopenaeus vannamei) embryos.

The immune response of commercially relevant marine invertebrates has been extensively studied, in search of new disease-control strategies. Immune training is considered a novel approach that could help improve resistance to different pathogens. Here, we stimulated the white shrimp (Litopenaeus vannamei) during embryo development by exposure to heat-killed bacteria and evaluated their effect on hatching, larval development, and the expression of immune-related genes. In addition, we evaluated its impact on the response of shrimp nauplii during a challenge with Vibrio parahaemolyticus. We observed that the percentage of hatching and the resistance to bacterial infection increased due to the treatment of embryos with heat-killed cells of Vibrio and Bacillus. Apparently different stimuli could generate a differential pattern of gene expression, e.g., Vibrio induced a strong effector immune response whereas Bacillus elicited a protective immune profile. In addition, each response was triggered by molecular patterns detected in the environment. The results obtained in this study provide new insights for immune training to improve shrimp farming.

Immunological response of white shrimp (Litopenaeus vannamei) to sublethal concentrations of malathion and endosulfan, and their mixture.

Cellular and humoral responses were evaluated in Litopenaeus vannamei juveniles when exposed to malathion, endosulfan, and their mixture. Each experiment was performed in the hemolymph collected at each exposure time (5 and 96 h) in duplicate; total hemocyte count, coagulation time, hemocyanin concentration, phenoloxidase (PO) and superoxide dismutase (SOD) activities were quantified. Survival was not affected by pesticides applied individually and mixed. Clotting time did not show significant differences concerning increase of concentration percentage of the pesticides tested. In organisms exposed to the pesticide mixture, hemocyanin decreased at 5 h of exposure as the concentration increased. Only in the malathion experiment did exposed shrimp to 10 and 50% of the LC50-96 h show significantly (p < 0.05) higher hemocyte contents. For malathion, significantly (p < 0.05) lower PO values in shrimp exposed to higher concentrations (10 and 50% of the LC50-96 h) were determined. While for the mixture treatment, high SOD value was determined at high exposure time and concentration. Malathion was the pesticide that showed an effect on some variables even at sublethal concentrations. The Continuous Concentration Criteria of the United States Environmental Protection Agency did not represent effects on the variables when they were compared with the averages of the control group.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations.

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

Molecular cloning and modeling of the Tp53-induced glycolysis and apoptotic regulator (TIGAR) from the Pacific white shrimp Litopenaeus vannamei and its expression in response to hypoxia.

Hypoxia is a common stressor for aquaculture species. The Pacific white shrimp Litopenaeus vannamei survives low dissolved oxygen (DO) conditions by adjusting its energy metabolism. In vertebrates, the transcription factor p53 regulates glucose metabolism under stress through diverse target genes like the Tp53-induced glycolysis and apoptotic regulator (TIGAR), a protein similar to fructose-2,6-bisphosphatase that has a pro-survival role in cells participating in the defense against oxidative damage. Until now, TIGAR has been not reported in any invertebrate species, including crustaceans. In this work, we report the molecular cloning of the white shrimp TIGAR. The cDNA sequence is 765 bp encoding a 254 amino acid protein. Bioinformatics analyses predicted that although the overall sequence identities of L. vannamei TIGAR and vertebrate proteins are not very high (33.61%-35.34%), they have a remarkable predicted structural similarity with full conservation of catalytic residues, secondary and three-dimensional structures. Gene expression analysis by RT-qPCR revealed that the mRNA abundance of TIGAR in white shrimp is tissue-specific under normal oxygen conditions, with higher expression in gills than hepatopancreas and muscle. Also, gene expression in gills and hepatopancreas is modified by environmental hypoxia, suggesting that TIGAR participates in the cellular tolerance of L. vannamei to this stressor.

The white spot syndrome virus hijacks the expression of the Penaeus vannamei Toll signaling pathway to evade host immunity and facilitate its replication.

The white spot syndrome virus (WSSV), the most lethal pathogen of shrimp, is a dsDNA virus with approximately a 300,000 base pairs and contains approximately 180-500 predicted open reading frames (ORFs), of which only 6% show homology to any known protein from other viruses or organisms. Although most of its ORFs encode enzymes for nucleotide metabolism, DNA replication, and protein modification, the WSSV uses some of its encoded proteins successfully to take control of the metabolism of the host and avoid immune responses. The contribution of the shrimp innate immune response to prevent viral invasions is recognized but yet not fully understood. Thus, the role of several components of Toll pathway of the shrimp Penaeus vannamei against WSSV has been previously described, and the consequential effects occurring through the cascade remain unknown. In the current study the effects of WSSV over various components of the shrimp Toll pathway were studied. The gene expression of Spätzle, Toll, Tube, Cactus and Dorsal was altered after 6-12 h post inoculation. The expression of LvToll3, LvCactus, LvDorsal, decreased ~4.4-, ~3.7- and ~7.3-fold at 48, 24 and 48 hpi, respectively. Furthermore, a remarkable reduction (~18-fold) in the expression of the gene encoding LvCactus in WSSV infected specimens was observed at 6 hpi. This may be a sophisticated strategy exploited by WSSV to evade the Toll-mediated immune action, and to promote its replication, thereby contributing to viral fitness.

Potential immunomodulatory and protective effects of the Arthrospira-based dietary supplement on shrimp intestinal immune defenses.

Herein, we evaluated the immunomodulatory and the antiviral protective properties of a cyanobacteria-enriched diet on the immune responses of the Pacific white shrimp Litopenaeus vannamei challenged with the White spot syndrome virus (WSSV). Shrimp were fed with an Arthrospira platensis supplemented feed during 20 days, and its effects were examined by evaluating well-known standardized shrimp immune parameters (total hemocyte counts, total protein concentration, phenoloxidase activity, and serum agglutination titer). Additionally, we assessed the expression of crucial genes involved in both hemolymph- and gut-based immunities related to the shrimp capacity to circumvent viral and microbial infections. Dietary supplementation improved shrimp survival rates after challenge with a median lethal dose of WSSV. From all immune parameters tested, only the serum agglutination titer was higher in treated animals. On the other hand, the expression of some representative marker genes from different immune response pathways was only modulated in the midgut and not in the circulating hemocytes, suggesting that this feed supplementation can be used as an attractive strategy to enhance immunity in shrimp gut. Altogether, our results evidence the immunomodulatory properties of A. platensis supplemented feed in shrimp humoral and intestinal defenses and highlight the potential use of cyanobacteria-based immunostimulants in shrimp farming for protection against infectious diseases.

DNA Damage and Immunological Responses in the Whiteleg Shrimp (Litopenaeus vannamei) Exposed to Sublethal Levels of Mercury.

Litopenaeus vannamei juveniles were exposed to sublethal levels (2.33-18.03 µg/L) of inorganic mercury. Time of exposure (0, 24 and 168 h) was a source of DNA damage. Mean comet tail length not changed significantly with mercury concentrations and exposure time, and this parameter cannot be used to assess DNA damage in this shrimp. Total hemocyte count showed a trend to decrease according to the increase of mercury concentrations, although no significant difference between treatments with mercury was observed. The phenoloxidase (PO) activity was not influenced by the time of exposure. At the end of the experiment, the PO in organisms exposed to 18.03 µg/L was different from the control. The time of exposure has a more important influence in superoxide dismutase than the concentration of mercury. According to these results, a suitable criterion of water quality for long-term exposure of L. vannamei should be lower than 2 µg/L of mercury.