The immunostimulatory property of glucan isolates from three marine yeasts (Debaryomyces hansenii S8, Debaryomyces hansenii S169 and Candida tropicalis S186) and one Baker's yeast (Saccharomyces cerevisiae S36) as examined for potential application as immunostimulants in Penaeus monodon postlarvae against White Spot Syndrome Virus (WSSV) infection. Structural characterization of the glucan component in the isolates by proton nuclear magnetic resonance (NMR) indicated similar structures containing (1-3)-linked anhydroglucose repeat units (AGRUs) in the backbone with (1-6)-linked AGRUs in side chains that are (1-6)-linked to the backbone AGRUs. Glucan from C. tropicalis (S186) with the highest molecular weight and the lowest level of branching supported maximum survival (69%) followed by the other two marine yeast (S169 and S8) glucans of 27% and 23% respectively while glucan from Baker's yeast, S. cerevisiae S36 with the lowest molecular weight and the highest level of branching exhibited poor survival (4%) in P. monodon post challenge WSSV. The present study showed that the glucan isolate from marine yeast with a higher molecular weight and a lower degree of branching acts as better immunostimulants in P. monodon postlarvae than did the glucan isolate from S. cerevisiae.

Le bruissement enzymatiques, appelé mélanose post mortem chez les crustacés est un phénomène enzymatique catalysé par des protéines à activités phénoloxydases (tyronase, catécholase, laccase et hémocyanine). L'utilisation de conservateurs de type sulfites (E220 à E228 et E539) reste à l'heure actuelle la solution la plus répandue pour éviter le développement de cette coloration peu attrayante pour le consommateur. Mais une partie de la population développe des réactions d'hypersensibilité en consommant des aliments sulfités. Dans l'onbjectif de rechercher une alternative à ces conservateurs, deux axes de recherche ont été développés durant ces travaux de thèse : la caractérisation biochimique des protéines responsables de la mélanose post mortem chez la crevette P. monodon, puis la recherche de molécules inhibitrices. Un fractionnement sur résine Phenyl Sepahrose™ CL-4B (HIC) suivie d'une séparation par électrophorèse SDS-PAGE ont montré la présence de trois protéines de 46, 82 et 89 kDa à activité principalement laccase. Une identification par RP-HPLC-Q/TOF a mis en évidence la présence d'hémocyanine uniquement. Un pH de 7,0 et une température comprise entre 37 et 50 °C ont mis en évidence les activités les plus importantes, en utilisant le dosage enzymatique dit "test au MBTH". Par ailleurs, un criblage à haut débit de 45 molécules potentiellement inhibitrices a été réalisé dans des conditions d'analyses standardisées grâce à l'outil de robotique de la plateforme Realcat. Une inhibition a été mise en évidence pour 23 composés, certains étant suffisamment efficaces pour être utilisés seuls. D'autres pourraient être introduits dans un cocktail de molécules inhibitrices aux fonctionnalités complémentaires. Les résultats des tests de trempage réalisés sur des crevettes entières ont montré qu'il était indispensable de compléter les études in vitro avec des essais à l'échelle de la matrice alimentaire dans son intégralité. / Enzymatic browning, called post mortem melanosis in crustaceans, is an enzymatic phenomenon catalyzed by proteins with phenoloxidase activities (tyronase, catecholase, laccase and hemocyanin). The use of sulfite preservatives (E220 to E228 and E539) remains at present the most widespread solution to avoid the development of this unattractive color towards consumers. But, a part of the population develops hypersensitivity reactions by consuming sulfited foods. With the objective to find an alternative to these conversators, two research axes have been planned : the biochemical characterization of the proteins responsible for post mortem melanosis in the P. monodon shrimp, then the search for inhibitory molecules. Fractionation on Phenyl Sepahrose™ CL-4B resin (HIC) followed by SDS-PAGE electrophoresis separation showed the presence of three proteins of 46, 82 and 89 kDa with mainly laccase activity. Identification by RP-HPLC-Q / TOF revealed the presence of hemocyanin only. A pH of 7.0 and a temperature between 37 and 50 °C showed the most important activities, using the enzymatic assay called "MBTH test". On the other hand, a high throughput screening of 45 potentially inhibitory molecules could be performed under standardized analysis conditions thanks to the robotic tools of the Realcat platform

A series of studies were conducted on an emerging disease in farmed penaeid shrimp. This disease was first named as Early Mortality Syndrome (EMS) or more descriptively as Acute Hepatopancreatic Necrosis Syndrome (AHPNS). As part of the outcome of this research, the etiology of EMS/AHPNS was demonstrated. EMS was first classified as an idiopathic disease because no causative agent had been identified. Preliminary studies conducted in Vietnam in 2012 by the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) indicated that EMS is infectious (Tran et al., 2013). The agent was identified as a unique strain of Vibrio parahaemolyticus. Hence, EMS has a bacterial etiology confirmed by satisfying Koch's Postulates. Further studies focusing on the bacterial isolate causing EMS revealed that the agent could produce toxin(s), which is responsible for the primary pathology in affected shrimp. Since the causative agent has been identified, we propose a new name for EMS as Acute Hepatopancreatic Necrosis Disease (AHPND). Characterizations of the AHPND-causing Vibrio parahaemolyticus: Biochemical methods and molecular methods were used. Based on these results, various diagnostic methods were developed including polymerase chain reaction (PCR) test and biochemical tests. Other aspects of the AHPND causing V. parahaemolyticus were also run to determine such as antibiogram and the development of resistance mechanism of the bacteria exposed to farm conditions with antibiotic medications, pathogenicity, and infection dose of the bacteria, mode of infection, mechanisms governing the toxin production, and effects of environmental parameters on the invasion of the agent. Some proposed control measures for AHPND: Several antibiotic-free approaches were tested to determine viable control methods for AHPND. The principle proposed control method is to increase biosecurity. With the PCR method that has been developed, potential sources of the pathogen such as post-larvae and broodstock can be tested. As more and more insights of the pathogen were explored, the behavior of the pathogen was further elucidated. Based on this, control methods such as using polyculture with tilapia, probiotics, and bioflocs system were also tested. Several improvements in shrimp farming practices that may reduce the outbreak of the disease were also proposed.

Klimatförändringarna har en stor påverkan på de arktiska valarna grönlandsval (Balaena mysticetus), vitval (Delphinapterus leucas) och narval (Monodon monoceros), mer än vad som tros vid en första tanke. I dagsläget får dessa valar utstå stora utmaningar som troligenkan komma att förvärras i framtiden om inte isens smältande kan bromsas. En del av utmaningarna innebär att valarna måste genomgå stora förändringar för att överleva, vilket innebär att deras förmåga att anpassa sig spelar en stor roll. Människans jakt på valen har alltid varit ett stort problem för de arktiska valarna, tack vare restriktioner om fångstkvoter och vem som får jaga val ser framtiden ljusare ut i alla fall för grönlandsvalen och vitvalen. För narvalen ser det dock inte lika ljust ut eftersom det är en art som är känsligare än många andra arktiska arter för effekterna som den globala uppvärmningen har på den arktiska miljön. I dagsläget har en del effekter på valarna blivit synliga såsom ändrade migrationsvanor och ökad predation. På grund av bristande data från perioden innan klimatförändringarna är detsvårt att dra konkreta slutsatser, därför fokuserar mycket forskning på att förutse vad som kommer att ske i framtiden. Fokus på framtiden är viktigt eftersom det som sker idag redan är försent att göra någonting åt, det vi kan göra är att se till att det inte blir ännu värre. Den smältande isens effekter är svåra att skilja på då de överlappar en del, till exempel leder tillgången på föda till förändringar i habitat. Man har i dagsläget sett små skillnader i tillgång på föda, beståndet av istorsken har minskat, eftersom det är en viktig föda för de arktiska valarna kan det ha en effekt. En minskning av en viss typ av plankton har också observerats och eftersom ingen ersättande art har setts kommer detta få effekter på näringsväven i de arktiska haven och därmed alla arter som lever där inklusive de arktiska valarna. I framtiden tror man att primärproduktionen kommer att öka på grund av den höjda vattentemperaturen och den ökande ytan med öppet vatten, detta kommer eventuellt ha en positiv effekt på de arktiska valarna.

Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

De part son profil équilibré en acides aminés essentiels (AAE), la farine de poisson (FP) est la source protéique principale utilisée dans l’alimentation des crevettes d’élevage. Cependant, compte tenu des enjeux du développement durable de la production aquacole, son utilisation doit être réduite, et remplacée par d’autres sources protéiques comme les protéines végétales (PV), qui sont souvent carencées en lysine et méthionine mais riches en cystine. Les conséquences d’un tel changement alimentaire sont peu connues chez la crevette tigrée Penaeus monodon. Pour les évaluer, nous avons utilisédes aliments semi-purifiés reflétant les carences/excès en AA des PV pour estimer les besoins en protéine, lysine et méthionine pour l’entretien et la croissance. Tout en confirmant les données antérieures sur les besoins pour la croissance des stades postlarves, nous avons pu préciser la contribution de l’apport en ces deux acides aminés pour l’entretien. Au niveau métabolique, la variation de l’apport protéique (10, 30, 50% protéine brute) et la carence en méthionine (-30% par rapport au besoin) entraînent une modification de l’activité des enzymes du catabolisme des AA, mais pas celle des voies de reméthylation et transsulfuration. En revanche, et pour la première fois chez la crevette, nos résultats démontrent une épargne de la méthionine par la cystine (et la choline), soulignant l’importance de l’apport en AA soufrés totaux (methionine + cystine). Nos résultats illustrent aussi l’importance qu’il convient d’accorder à la disponibilité des AAE dans les études de remplacement de la FP par un mélange de PV pour améliorer l’utilisation azotée chez la crevette P. monodon. / Due to its well balanced essential amino acid (EAA) profile, fishmeal (FM) is the major protein source used in the formulation of aquafeed for cultured shrimp. To sustain farming systems, its incorporation, however, must be reduced and substituted by other protein sources less well nutritionally balanced, such as plant protein ( PP) which are often low in lysine and methionine but rich in cystine. The metabolic consequences of such a shift in dietary profile are not well known for the black tiger shrimp, Penaeus monodon. To describe these consequences, we used semi-purified diets limiting in lysine and methionine (to reflect PP profile) to determine juvenile requirements of protein, lysine and methionine for both maintenance and growth, applying a factorial approach. Our results confirm the previous data on growth requirement for post-larvalstages of P. monodon while also providing new data on maintenance requirements. At the metabolic level, a variation in the dietary protein level (10, 30, 50 % crude protein) and methionine (adequate or 30% lower) resulted in a significant change in the activity of transdeaminating enzyme, but not those of remethylation and transsulfuration. Nevertheless, we found for the first time that methionine utilisation for body protein accretion can be spared by cystine and choline (up to 50%) in this species, illustrating the importance to consider total sulphur AA supply. Our data also show that full consideration should be given to AA availability in order to develop practical diets with low FM levels for P. monodon.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Comparative analyses of genetic diversity, population structure and evolutionary relationships among co–distributed species can provide useful insights into fisheries management. In this study, mitochondrial DNA control region (mtCR) sequences were used to investigate genetic population structure and recruitment patterns of three co–occurring shallow water penaeid prawn species; Penaeus monodon, Fenneropenaeus indicus and Metapenaeus monoceros. These taxa dominate artisanal and commercial prawn catches in the Malindi–Ungwana Bay in Kenya, where juvenile prawns inhabit estuarine habitats, and adults occur further offshore, on mudbanks in the bay. A total of 296 [i.e. (P. monodon; n = 129), (F. indicus; n = 96), (M. monoceros; n = 71)] specimens were sampled from five sites; two estuarine nursery areas (juveniles), a nearshore mid–station (adults), and two offshore areas (adults). The sites were chosen to represent the bulk of the Kenyan fishery activities, and to include juvenile and adult cohorts that are presumably connected to each other through larval dispersal processes and migrations. Juveniles were obtained during 2010 from local fishermen, and adult prawns during 2011 using a commercial prawn trawler. Analysis of the mtCR sequences indicated high haplotype diversity (P. monodon; h = 0.9996 ± 0.0010; F. indicus; h = 0.9998 ± 0.0015; M. monoceros; h = 0.9815 ± 0.0110) for all three species. Genetic differentiation results for each species using AMOVA indicated no significant population differentiation (P. monodon; ΦST = 0.000, = p > 0.05; F. indicus; ΦST = 0.000, = p > 0.05; M. monoceros; ΦST = 0.0164, = p > 0.05) and pairwise ΦST statistics among sampling sites indicated the complete absence of spatial differentiation of female genes for all three species. In addition, the mtDNA data of P. monodon (i.e. n = 103) was augmented by using six polymorphic nuclear microsatellite loci. The pattern of panmixia was supported by the microsatellite analyses of P. monodon where AMOVA (i.e. RST = 0.00113, = p > 0.05), pairwise RST statistics (i.e. RST = 0.0000–0.0223, = p > 0.05) and STRUCTURE all confirmed the complete absence of genetic differentiation, among all sampled localities. Based on the absence of genetic population structure, each of the three species can be regarded as a single management unit throughout the Malindi–Ungwana Bay area. Spatial management strategies for prawn fisheries in the bay should therefore rely on factors other than genetic metapopulations, such as seasonal prawn recruitment and distribution patterns, ecosystem functioning and socio–economic implications to fishing communities and commercial trawl fishing companies. / AFRIKAANSE OPSOMMING: Vergelykende analise van genetiese diversiteit, bevolkings stuktuur en evolutionêre verwantskappe tussen spesies wat 'n verspreidingsgebied deel kan nuttige insigte lewer oor vissery bestuur. In hierdie studie was die mitokondriale DNS kontrole area (mtCR) volgordebepalings gebruik om die bevolkings genetiese stuktuur en werwingspatrone van drie mede-verspreide vlak water penaeid garnaal spesies; Penaeus monodon, Fenneropenaeus indicus and Metapenaeus monoceros te ondersoek. Hierdie taksa domineer die ambagtelike en kommersiële vangste in die Malindi-Ungwanabaai in Kenya waar, onvolwasse garnale in riviermondings voorkom en volwassenes in dieper waters op modderbanke in die baai voorkom. 'n Totaal van 296 [(P. monodon; n = 129), (F. indicus; n = 96), (M. monoceros; n = 71)] monsters was geneem vanaf vyf lokaliteite; twee in riviermondings (onvolwassenes), 'n nabykus mid stasie (volwasse) en twee diep water (volwasse) areas. Hierdie lokaliteite was gekies om die oorgrote meerderheid van Kenya se vissery aktiwiteite, asook die onvolwasses en volwassene kohorte te verteenwoordig wat vermoedelik geneties verbind is aan mekaar deur larwale verspreidingsprosesse en migrasies. Onvolwasse diere was verkry in 2010 vanaf plaaslike vissermanne en volwasse diere was in 2011 gekollekteer deur gebruik te maak van 'n kommersiële garnaal vissersboot. Analise van die mtCR volgorde bepaling het gewys dat daar 'n hoë haplotipiese diversiteit (P. monodon; h = 0.9996 ± 0.0010; F. indicus; h = 0.9998 ± 0.0015; M. monoceros; h = 0.9815 ± 0.0110) vir al drie spesies bestaan. Genetiese differensiasie resultate vir elke spesie, bepaal deur 'n AMOVA toets, dui op geen beduidende bevolking differensiasie nie (P. monodon; ΦST = 0.000, = p > 0.05; F. indicus; ΦST = 0.000, = p > 0.05; M. monoceros; ΦST = 0.0164, = p > 0.05) en paarsgewyse ΦST statistiek tussen die lokaliteite waar monsters geneem was, dui op geen ruimtelike differensiasie van die vroulike gene in al drie spesies nie. Hierbenewens is die mtDNS datastel van P. monodon (i.e. n = 103) uitgebrei deur ses polimorfiese kern mikrosatelliete in te sluit. Die patroon van mtCR panmixia was ondersteun deur die mikro-satelliet analise van P. monodon waar die AMOVA (i.e. RST = 0.00113, = p > 0.05), paarsgewyse RST statistiek (i.e. RST = 0.0000-0.0223, = p > 0.05) en STRUCTURE bevestig het dat daar totale afwesigheid is van genetiese differensiasie tussen alle vergelyk-te lokaliteite. Gebaseer op die afwesigheid van genetiese bevolking-struktuur kan elk van die drie spesies beskou word as 'n enkele bestuurseenheid deur die Malindi-Ungwanabaai area. Die bestuurstrategieë vir garnaal vissery aktiwiteite in die baai moet dus steun op ander faktore as genetiese meta-bevolking. Belangrike faktore om in ag te neem is seisoenale garnaal werwing en verspreidings patrone, ekosisteem funksionering en sosio-ekonomiese implikasies van vissers gemeenskappe en kommersiële visserymaatskappye.

The growth of the Penaeus monodon prawn aquaculture industry in Australia is hampered by a reliance on wild-caught broodstock. This species has proven difficult to breed from if broodstock are reared in captivity. Studies were therefore carried out to investigate factors controlling reproduction and influencing egg quality. Results of the studies revealed that patterns of nutrient accumulation during early ovary development are altered by captive conditions, possibly contributing to reduce larval quality. The sinus gland hormones were shown, together with the environment, to regulate two stages of ovary development. In a separate study it was further revealed that the hormone methyl farnesoate (MF) could negatively regulate the final stages of ovary development. Lastly it was shown that broodstock reared in captivity are less likely to mate and that this is due to inherent problems in both the male and the female prawns.

Three series of studies were conducted to quantify the effectiveness of dietary incorporation of b-1,3-glucan (BG) from Schizophyllum commune in enhancing the immunity and resistance of grass prawn Penaeus monodon to vibriosis and white spot syndrome virus (WSSV) infections. In the first series of studies, three experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on shrimp growth and resistance to vibriosis. Weight gain, survival and feed efficiency of juvenile shrimp (0.5 ¡Ó 0.1 g) were not significantly different (P>0.05) after being fed the diets containing BG 0, 0.2, 2, 10 g/kg diet for 18 weeks. Subadult shrimp (20.4 ¡Ó 2.1 g) fed the diet containing of BG 2 g/kg diet for 10 days showed a significantly (P<0.001) enhanced resistance against vibriosis. Postlarvae fed with the BG diet (2 g/kg diet) were more resistant (P<0.001) against starvation and V. harveyi challenges than the postlarvae fed non-BG diet. Additive disease resistance was observed when polyphosphorylated L-ascorbic acid (PAA) was used together with BG. In challenge tests with V. damsela, shrimp fed with PAA (0.2 g/kg diet) + BG (2 g/kg diet) diet for 20 days had a survival rate up to 60%. In the second series of studies, two experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on wound healing and immunity in spawners. Dietary supplement of BG reduced the chance of infections, but did not help wound healing as did the supplement of PAA. And regardless of indoor or outdoor rearing, the survival rate of brooder (135 ¡Ó 25 g) fed the BG (2 g/kg diet) diet was higher (P<0.001) than that of the non-BG group. Fed the BG brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production then the control group. Third series of studies evaluated the effectiveness against white spot syndrome virus (WSSV). Six days after being challenged with WSSV, 12.2 % of the BG-treated (2 g/kg diet for 15 days) postlarvae (PL15) and 20 % BG-treated (2 g/kg diet for 20 days) juveniles (5.5 ¡Ó 0.5 g) were still alive; while all non-BG-treated shrimp died. In order to quantify the effectiveness of BG to WSSV, juveniles (6.5 ¡Ó 0.4 g) were fed diets containing graded levels of BG. The results showed that shrimp fed the diet containing BG 10 g/kg for 20 days had the highest (P<0.001) survival rate (42 %) among all groups. Shrimp that received diets supplemented with BG at a dosage >2 g/kg recuperated 9 ~ 12 days after WSSV challenge; while the group fed diets with no or 1 g/kg BG suffered from rapid decrease in total haemocyte count, phagocytic activity, phenoloxidase, O2-, superoxide dismutase (SOD) production and subsequent high mortality. The results in this study showed that b-1,3-glucan is effective in enhancing the phagocytic activity, phenoloxidase, O2- and SOD productions and consequently the resistance of postlarval, juvenile, subadult and brooder P. monodon against vibriosis and viral infections. Since prolonged use of BG, even at optimal dietary levels, decreased the immunity of the shrimp, care therefore must be taken to maximize its effectiveness. A cycle of dietary BG supplement of 2 ~ 10 g/kg diet for 20 days with an intermission of 10 days may serve the purposes.