No description available.

Microbial communities associated with gastrointestinal tract of animals play a critical role in gut development, digestion and resistance to disease, thus the prospect of altering these communities beneficially by using probiotics is attractive. In terrestrial animals, the gut provides a stable, moist habitat in an otherwise moisture-limited environment, thus microbial communities tend to be very stable. In contrast, farmed aquatic animals reside within an environment that can support microbes in high densities, and as many marine animals drink continuously for osmoregulation, they are subjected to potential re-inoculation. Consequently, little is known of the stability of gut microbial communities in marine shrimp or whether it is possible to establish beneficial bacteria in the gut. The aims of this thesis were therefore to examine the midgut microbial community associated with farmed black tiger shrimp, Penaeus monodon, and to investigate whether the introduction of potentially probiotic Bacillus could alter the species diversity or abundance of the present microbes. Using culture methods it was found that B. pumilus was able to transfer between animals via the water column and persisted in the midgut for at least 7 days, while B. subtilis was only recovered from animals directly fed the bacteria and persisted for less than 24 h in the midgut. V. parahaemolyticus, a known shrimp pathogen,remained in the tanks it was originally found in, and did not transfer via the water column to other tanks and is therefore tightly associated with its host. A bacterium with apparent probiotic qualities was isolated from control animals in the above study and identified as a strain of B. pumilus. Its safety for food animal use was confirmed due to the absence of B. cereus toxin genes, and the isolate’s pH and salt tolerances were investigated. Moreover, the isolate was highly inhibitory to crustacean pathogens in the family Vibrionaceae. Methods to investigate the gut microbiota using the full cycle 16S rRNA methodology were optimized. Fluorescence in situ hybridization (FISH) probes designed specifically targeting B. pumilus, B. subtilis and B. licheniformis, commercially available probiotics, were validated for specificity and optimal hybridization conditions. For FISH analysis of bacteria in situ in histological sections of shrimp midgut trunks, fixation times in 4 % paraformaldehyde wereoptimizedfor bacterial RNA retention whilst maintaining tissue integrity. Due to the broad range of autofluorescence in the shrimp tissue, spectral imaging is required to adequately differentiate between host tissue and multiple bacterial probes. The richness and diversity of the midgut microbiota of animals treated with the novel strain of B. pumiluswere analyzed using 16S rRNA gene clone libraries and FISH analysis of histological sections. It was confirmed that B. pumilus can enter the midgut via top-coated feed and through water inoculation. In the tanks that were treated with B. pumilus the proportion of Vibrio sp. in the microbial community decreased, however, only in the systems in which B. pumilus was recovered from the shrimp midgut did the proportion of pathogenic Vibrio species decrease. The application of the B. pumilus caused a shift in the shrimp midgut microbiota, but the community returned to its initial diversity over time. The midgut microbiota of P. monodon is relatively stable but can be adjusted using probiotics. The transience or residence of the probiotics is strain-specific and should be tested for any new strains before determining optimum application protocols. The methods designed in this study are applicable to future research in this field.

Made available in DSpace on 2016-06-02T20:21:27Z (GMT). No. of bitstreams: 1 3871.pdf: 3349931 bytes, checksum: 699bd6092d3da7218a1550f52bb10cdb (MD5) Previous issue date: 2011-05-27 / Financiadora de Estudos e Projetos / Litopenaeus vannamei is known as Pacific white shrimp and is the main species marketed worldwide. Its geographical distribution includes the Pacific coast, ranging from Mexico to Peru. Due to its outstanding economic importance, this species has been farmed and showing great adapting levels to captivity, having been introduced in several countries, including Brazil. However, despite all the concern to properly manage the farming populations, many countries have not had a renewal of their breeding herds because of the risk of introduction of exogenous pathogens, which has increased the degree of inbreeding of these stocks and decreased levels of genetic diversity. To monitor this loss, microsatellite markers or SSRs (Simple Sequence Repeats), from genome arbitrary and expressed regions have been used. In the specific case of SSRs present in expressed regions of the genome (ESTs, Expressed Sequence Tags) or SSRs-ESTs, they allow the access to the genetic variability of populations, and the ESTs markers devoid of SSRs may be related to genes of interest, subsidizing the development of breeding programs based on Marker Assisted Selection (MAS). Moreover, EST-SSRs loci may show an excellent rate of transferability in taxonomically related species, since they are in more conserved regions of the genome. Within this context, this study aimed (i) the validation population of EST-SSR loci isolated through dataming from L. vannamei ESTs database (www.shrimp.ufscar.br), (ii) the genomic annotation of ESTs and EST-SSRs markers (iii) the determination of EC numbers (Enzyme Codes), aiming respectively, (i) the characterization of polymorphic markers (ii) the description of genes and their protein products and (iii) the establishment of information to describe the possible pathways for the penaeid group. Therefore, we tested 32 EST-SSRs loci in PCR reactions. After establishing the best pattern of reaction and subsequent loci genotyping, nine SSRs-ESTs were polymorphic, with allele number ranging from two to 20, levels of observed heterozygosity from 0.32 to 0.86 and average PIC (Polymorphism Information Content) of 0.78. No pair of loci presented in linkage disequilibrium. However, after Bonferroni correction, we found that one of these showed a significant deficit of heterozygotes. The nine polymorphic loci from L.vannamei showed satisfactory amplification in at least one of the seven native species tested: the marine ones Xiphopenaeus kroyeri, Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Rimapenaeus constrictus and Litopenaeus schmitti and the freshwater ones Macrobrachium amazonicum and Macrobrachium jeskii, being useful also for genetic studies of these species. The genome annotation performed by access in ShEST website (Litopenaeus vannamei EST Genome Project) showed that only three of nine loci 4 have the gene and its protein product described. The other loci showed no matches with any other genomic database available for research. In this work, gene product could be elucidated for 99% of ESTs, being possible to establish 209 EC numbers, highlighting enzymes responsible for xenobiotics metabolism, immunity, energy production, reproduction, oxidative stress, among others. These codes were used for construction of some metabolic pathways present in the penaeid group, contributing for building a wide database of the genome of this important animal group. These data may be applied in genetic improvement programs as well as gene expression studies, such as realtime PCR and microarrays. / Litopenaeus vannamei é conhecido como camarão branco do Pacífico e é a principal espécie de peneídeo comercializada no mundo. Sua distribuição geográfica compreende a costa do Oceano Pacífico, indo desde o México até o Peru. Devido a sua relevante importância econômica, esta espécie passou a ser cultivada, adaptando-se bem às condições de cativeiro, tendo sido introduzida em diversos países, incluindo o Brasil. Entretanto, apesar de toda preocupação em manejar adequadamente as populações de cultivo, muitos países não têm tido renovação de seus estoques reprodutores ou plantéis, devido ao risco de introdução de patógenos exógenos, o que tem aumentado o grau de endogamia desses estoques e diminuição dos níveis de diversidade genética. Para monitorar esta perda de variabilidade genética, marcadores microssatélites ou SSRs (Simple Sequence Repeats), oriundos de regiões arbitrárias e expressas do genoma desta espécie, vêm sendo utilizados. No caso específico de SSRs presentes em regiões expressas do genoma (ESTs, Expressed Sequence Tags) ou SSRs-ESTs, além destes permitirem acessar a variabilidade genética das populações, as ESTs desprovidas de SSRs podem estar relacionadas a genes de interesse, podendo auxiliar o desenvolvimento de programas de melhoramento genético baseados na Seleção Assistida por Marcadores (MAS). Além disso, locos SSRs-ESTs podem apresentar uma excelente taxa de transferabilidade em espécies taxonomicamente relacionadas, uma vez que se encontram em regiões mais conservadas do genoma. Dentro deste contexto, este trabalho teve como objetivos (i) a validação populacional de locos SSRs-ESTs, isolados via dataming no banco de dados de ESTs de L. vannamei (www.shrimp.ufscar.br); (ii) a anotação genômica de marcas ESTs e locos SSRs-ESTs (iii) e a determinação dos EC numbers (códigos enzimáticos), visando, respectivamente, (i) a caracterização de marcadores polimórficos, (ii) a descrição de genes e seus respectivos produtos protéicos e (iii) o estabelecimento de informações para descrever as possíveis vias metabólicas para o grupo de peneídeos. Para tanto foram testados 32 locos SSRs-ESTs em reações de PCR. Após estabelecimento do melhor perfil de reação e posterior genotipagem dos locos, nove SSRs-ESTs mostraram-se polimórficos, com número de alelos variando de 4 a 20, níveis de heterozigozidade observada de 0,32 a 0,86 e valores médios de PIC (Polymorphism Information Content) de 0,78. Nenhum loco apresentou-se em desequilíbrio de ligação. Entretanto, após correção de Bonferroni, constatou-se que um deles apresentou déficit significativo de heterozigotos. Os nove locos polimórficos para L. vannamei apresentaram amplificação satisfatória em pelo menos uma das sete espécies nativas testadas: as marinhas Xiphopenaeus kroyeri, 2 Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Rimapenaeus constrictus e Litopenaeus schmitti e as de água doce Macrobrachium amazonicum e Macrobrachium jeskii podendo se constituir em marcas úteis para os estudos genéticos também dessas espécies. A anotação genômica realizada via acesso à página de anotação do Projeto ShEST (Projeto Genoma EST de Litopenaeus vannamei) demonstrou que apenas três dos nove locos polimórficos têm o seu gene e produto protéico já descritos. Os demais locos não apresentaram blasts positivos com nenhuma outra base de dados genômicos disponíveis para pesquisa. No caso das sequências anotadas, o produto gênico pôde ser elucidado para 99% destas, sendo possível estabelecer 209 EC numbers, destacando-se enzimas responsáveis pela degradação de xenobióticos, imunidade, produção de energia, reprodução, estresse oxidativo, dentre outras. Estes códigos enzimáticos foram utilizados para determinação de algumas vias metabólicas presentes no grupo dos peneídeos, contribuindo assim para a construção de uma ampla base de dados do genoma deste importante grupo animal, podendo ser utilizada em estudos de conservação, programas de melhoramento genético e em análises de expressão gênica, como PCR em tempo real e microarrays.

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.