Trolling for Nile perch (Lates niloticus) in Lake Kyoga

Nile perch were introduced into Lake Kyoga in the mid·1950s from Lake Albert. Murchison Falls on the River Nile, between the two lakes, prevented Nile Perch and other elements of the typical nilotic fish population from naturally reaching Lake Kyoga. The introduction has been successful and considerable stocks of Nile Perch now exist in Lake Kyoga. In 1967, 13,000 tons of Nile Perch were estimated to have been landed by the commercial fishermen, fish of 200 lb. being now caught and specimens of 100 lb. being fairly common. Large Nile perch are caught commercially on long lines baited with live Protopterus' spp. or Clarias spp. Large mesh gillnets uccasionally take Nile Perch of up to 30 lb., but the high cost of the nets does not, at the moment, appear to justify this method of fishing; a 10 in. net, stretched 100 yards long (unmounted). 15 meshes deep and 60-ply nylon. costs approximately U. Shs. 300. The long·lines used are extremely simple and cheap to make, but considerable labour is needed to catch bait. Small Protopterus are normally caught by turning over floating rafts of grasses and papyrus, and extracting the fish from the root mass; this is hard and dirty work. Other small fish, more readily available, do not, according to fishermen, work as well, possibly because they are not as durable as the Protopterus or Clarias. Dead bait is never used.

The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Identification of the p28 subunit of eukaryotic initiation factor 3(eIF3k) as a new interaction partner of cyclin D3.

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

How to estimate scavenger fish abundance using baited camera data

Baited cameras are often used for abundance estimation wherever alternative techniques are precluded, e.g. in abyssal systems and areas such as reefs. This method has thus far used models of the arrival process that are deterministic and, therefore, permit no estimate of precision.
Furthermore, errors due to multiple counting of fish and missing those not seen by the camera have restricted the technique to using only the time of first arrival, leaving a lot of data redundant. Here, we reformulate the arrival process using a stochastic model, which allows the precision of abundance
estimates to be quantified. Assuming a non-gregarious, cross-current-scavenging fish, we show that prediction of abundance from first arrival time is extremely uncertain. Using example data, we show
that simple regression-based prediction from the initial (rising) slope of numbers at the bait gives good precision, accepting certain assumptions. The most precise abundance estimates were obtained
by including the declining phase of the time series, using a simple model of departures, and taking account of scavengers beyond the camera’s view, using a hidden Markov model.

BRD7, a subunit of SWI/SNF complexes, binds directly to BRCA1 and regulates BRCA1-dependent transcription

We carried out a yeast two-hybrid screen using a BRCA1 bait composed of amino acids 1 to 1142 and identified BRD7 as a novel binding partner of BRCA1. This interaction was confirmed by coimmunoprecipitation of endogenous BRCA1 and BRD7 in T47D and HEK-293 cells. BRD7 is a bromodomain containing protein, which is a subunit of PBAF-specific Swi/Snf chromatin remodeling complexes. To determine the functional consequences of the BRCA1-BRD7 interaction, we investigated the role of BRD7 in BRCA1-dependent transcription using microarray-based expression profiling. We found that a variety of targets were coordinately regulated by BRCA1 and BRD7, such as estrogen receptor alpha (ERalpha). Depletion of BRD7 or BRCA1 in either T47D or MCF7 cells resulted in loss of expression of ERalpha at both the mRNA and protein level, and this loss of ERalpha was reflected in resistance to the antiestrogen drug fulvestrant. We show that BRD7 is present, along with BRCA1 and Oct-1, on the ESR1 promoter (the gene which encodes ERalpha). Depletion of BRD7 prevented the recruitment of BRCA1 and Oct-1 to the ESR1 promoter; however, it had no effect on the recruitment of the other Swi/Snf subunits BRG1, BAF155, and BAF57 or on RNA polymerase II recruitment. These results support a model whereby the regulation of ERalpha transcription by BRD7 is mediated by its recruitment of BRCA1 and Oct-1 to the ESR1 promoter.

The long-term impacts of fisheries on epifaunal assemblage function and structure, in a special area of conservation

Fisheries can have profound effects on epifaunal community function and structure. We analysed the results from five dive surveys (1975–1976, 1980, 1983, 2003 and 2007), taken in a Special Area of Conservation, Strangford Lough, Northern Ireland before and after a ten year period of increased trawling activity between 1985 and 1995. There were no detectable differences in the species richness or taxonomic distinctiveness before (1975–1983) and after (2003–2007) this period. However, there was a shift in the epifaunal assemblage between the surveys in 1975–1983 and 2003–2007. In general, the slow-moving, or sessile, erect, filterfeeders were replaced by highly mobile, swimming, scavengers and predators. There were declines in the frequency of the fished bivalve Aequipecten opercularis and the non-fished bivalves Modiolus modiolus and Chlamys varia and some erect sessile invertebrates between the surveys in 1975–1983 and 2003–2007. In contrast, there were increases in the frequency of the fished and reseeded bivalves Pecten maximus and Ostrea edulis, the fished crabs Cancer pagurus and Necora puber and the non-fished sea stars Asterias rubens, Crossaster papposus and Henricia oculata between the surveys in 1975–1983 and 2003–2007. We suggest that these shifts could be directly and indirectly attributed to the long-termimpacts of trawl fishing gear, although increases in the supply of discarded bait and influxes of sediment may also have contributed to changes in the frequency of some taxa. These results suggest that despite their limitations, historical surveys and repeat sampling over long periods can help to elucidate the inferred patterns in the epifaunal community. The use of commercial fishing gear was banned from two areas in Strangford Lough in 2011, making it a model ecosystem for assessing the long-term recovery of the epifaunal community from the impacts of mobile and pot fishing gear.

User behaviour, best practice and the risks of non-target exposure associated with anticoagulant rodenticide use

Usage of anticoagulant rodenticides (ARs) is an integral component of modern agriculture and is essential for the control of commensal rodent populations. However, the extensive deployment of ARs has led to widespread exposure of a range of non-target predatory birds and mammals to some compounds, in particular the second-generation anticoagulant rodenticides (SCARS). As a result, there has been considerable effort placed into devising voluntary best practice guidelines that increase the efficacy of rodent control and reduce the risk of non-target exposure. Currently, there is limited published information on actual practice amongst users or implementation of best practice. We assessed the behaviour of a typical group of users using an on-farm questionnaire survey. Most baited for rodents every year using SGARs. Most respondents were apparently aware of the risks of non-target exposure and adhered to some of the best practice recommendations but total compliance was rare. Our questionnaire revealed that users of first generation anticoagulant rodenticides rarely protected or checked bait stations, and so took little effort to prevent primary exposure of non-targets. Users almost never searched for and removed poisoned carcasses and many baited for prolonged periods or permanently. These factors are all likely to enhance the likelihood of primary and secondary exposure of non-target species. (C) 2010 Published by Elsevier Ltd.

Habitat effects on the spatial ecology of the European badger (Meles meles)

This study investigates how habitat variation affects sett density, the number of animals per social group and group territory size in the badger (Meles meles). Identical methods were applied in three habitat types: lowland parkland with mixed woodland, pastoral farmland and upland rough pasture with moorland, representing areas of presumed good, medium and poor badger habitat, respectively. Contiguous main setts were identified and bait-marking was used to estimate territory size. Group size was estimated by direct enumeration. Variation in sett density, group size and territory size supported the hypothesis that badger group and territory size are influenced by habitat type. This was further supported by analyses of data from other studies in the British Isles. The implications for badger spatial ecology, badger survey techniques and the badger's role in the epidemiology of TB are discussed.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Protein phosphatases acting on the replication checkpoint

A génese de um cancro está dependente da acumulação de mutações genéticas que dão origem a instabilidade genómica, que por sua vez resulta na proliferação descontrolada. Para prevenir a acumulação destas mutações, as células têm mecanismos de controlo (checkpoints) que suspendem o ciclo celular e accionam as vias de reparação do ADN. Estes eventos são muitas vezes regulados por dinâmicas de (des)fosforilação de proteínas. As proteínas fosfatases (PPs), enzimas responsáveis pela remoção do grupo fosfato de resíduos fosforilados, desempenham funções cruciais na regulação de muitos mecanismos celulares. Enquanto que no início do projecto as cinases envolvidas no checkpoint da replicação estavam bem estabelecidas, as PPs envolvidas não eram conhecidas. A Chk1, um componente da maquinaria do checkpoint da replicação, é exemplo dessa regulação por (des)fosforilação, como sejam nos resíduos Ser317 e Ser345. Assim, como primeira abordagem para determinar quais os grupos de PPs envolvidos na regulação do checkpoint da replicação, decidimos investigar o seu papel na regulação da fosforilação da Chk1. A primeira conclusão é que a desfosforilação da Chk1 ao longo do tempo, tanto in vivo como in vitro, ocorre com uma dinâmica bi-fásica. Em segundo, a abordagem in vitro sugere que as famílias PP1, PP2A e PP2C estão envolvidas na desfosforilação da Chk1. Uma vez que a família PP2A foi a que mostrou a maior acção nesta reacção, decidimos investigar outros membros da família in vivo, primeiro com uma abordagem geral (tratando com OA ou sobreexpressando a PME-1), e depois com o knockdown específico da PP4 e PP6 (através de siRNA). Os resultados mostram que a inibição das PPs afectam tanto a desfosforilação como o estado de activação da Chk1 em resposta a tratamento com Hidroxiureia (HU). Todas as PPs testadas in vivo pareceram ser capazes de regular, a níveis diferentes, tanto a fosforilação como a desfosforilação da Chk1. A função das PPs foi também investigada ao nível: da regulação do disparo das origens de replicação, e da recuperação da suspensão da replicação, induzida pela HU. No último caso, os dados indicam que na situação simultânea de knockdown da PP4 com tratamento de HU, há um atraso do ciclo celular na resolução da transição de G2/M. No ensaio de replicação por pulse-chase, os resultamos mostram que tanto o tratamento com OA, como a sobre-expressão de I-2 ou PME-1, atrasam a cronologia do disparo programado das origens de replicação. No entanto, nenhum dos tratamentos efectuados parece desregular o início do checkpoint da replicação. Um rastreio de 2-híbrido de levedura com uma biblioteca de cDNA de testículo humano foi realizado, usando a Chk1 como isco, no sentido de descobrir novos interactores e definir novas possíveis funções para a Chk1 no contexto da meiose. Com base nos resultados do rastreio, duas novas funções são sugeridas: a interacção com a GAGE12 sugere uma função na recombinação genómica/vigilância do genoma durante a meiose, e as interacções com a EEF1α1 e a RPS5 sugerem uma função na regulação da síntese proteíca. Estas experiências fornecem um visão geral para a compreensão da diversidade de funções das proteínas fosfatases envolvidas no checkpoint da replicação, bem como, abre novos caminhos para o desenvolvimento de novas drogas para o tratamento do cancro.

Ecotoxicological evaluation of pesticide mixtures

Neste trabalho o risco da aplicação de moluscicidas (metaldeído e metiocarbe) para o isópode terrestre Porcellionides pruinosus foi avaliado usando como parâmetros a mortalidade e biomarcadores de exposição. O tempo até à morte dos isópodes (após contacto com os moluscicidas) foi muito curto, especialmente no caso da exposição ao metiocarbe. Os vários biomarcadores revelaram-se úteis para a compreensão do modo de acção dos dois moluscicidas neste isópode, particularmente o efeito do carbamato metiocarbe na inibição da enzima acetilcolinesterase (AChE). Os efeitos de combinações binárias de três produtos de protecção das plantas (PPP) dimetoato, glifosato e espirodiclofeno foram avaliados testando o comportamento de evitamento de P. pruinosus, o sucesso reprodutivo do colêmbolo Folsomia candida e o crescimento das plantas Brassica rapa e Triticum aestivum, usando os dois modelos de referência de concentração de adição (CA) e acção independente (IA). O modelo MIXTOX foi usado para avaliar possíveis desvios (devido a interacções entre os pesticidas) dos dois modelos de referência. Os resultados obtidos permitem constatar que estes PPP quando aplicados segundo a dose recomendada não acarretam efeitos perniciosos para os organismos testados. Foi detectado sinergismo na mistura feita com glifosato e espirodiclofeno no isópode P. pruinosus e na mistura com glifosato e dimetoato na planta T. aestivum. Um ecossistema terrestre em pequena escala (“STEM”) foi desenvolvido, contendo um solo agrícola mediterrânico. Nestes STEM, minhocas (Eisenia andrei), P. pruinosus, B. rapa e “bait-lamina” foram incorporados no sentido de avaliar os efeitos da aplicação de dimetoato com espirodiclofeno e glifosato com dimetoato. A dose recomendada de aplicação dos PPP, quer na exposição individual quer nas misturas binárias não teve quaisquer efeitos nas espécies testadas. As minhocas foram sensíveis à aplicação conjunta de dimetoato com espirodiclofeno (10 vezes a dose recomendada) na sua distribuição vertical ao longo da coluna do STEM, e foi detectado sinergismo (i.e. mais minhocas escaparam do que a predição feita pelo modelo IA). Em todas as misturas binárias feitas com glifosato e dimetoato registou-se um decréscimo no consumo de “bait-lamina”, indicando sinergismo (menos “bait-lamina” consumidos que o esperado). Dos quatro biomarcadores (Catalase, AChE, GST e LPO) avaliados nos isópodes, verificaram-se diferenças significativas na actividade da enzima AChE (quando dimetoato foi aplicado no solo) e LPO (aumento da actividade devido à aplicação de glifosato e dimetoato).

Diversity and biology of polychaeta of Diopatra Genus

Os anelídeos poliquetas são elementos importantes em ambientes estuarinos e costeiros, pela sua elevada biodiversidade e abundância e pelo papel que têm nas cadeias tróficas. Algumas espécies são intensivamente exploradas para serem utilizadas como isco na pesca desportiva e profissional, como é o caso de Diopatra neapolitana. Apesar da importância económica, existem poucos estudos sobre a sua biologia e ecologia. No decorrer deste estudo foram identificadas duas outras espécies do género Diopatra em Portugal: D. marocensis, inicialmente descrita para a costa de Marrocos e cuja distribuição actual se sabe estender-se a toda a costa Portuguesa e Norte de Espanha e, D. micrura, espécie nova para a ciência. O presente estudo tem como objectivos principais estudar a diversidade e reprodução do género Diopatra, bem como a capacidade de regeneração da espécie D. neapolitana. Este trabalho aborda a distribuição espacial de D. marocensis ao longo da costa Portuguesa e descreve a espécie D. micrura, uma nova espécie do género Diopatra Audouin and Milne Edwards, 1833. As três espécies coabitam em águas transicionais, onde as espécies D. micrura e D. marocensis facilmente se confundem com juvenis de D. neapolitana. Foi realizada uma comparação morfológica e genética entre as três espécies. A espécie D. neapolitana coexiste em algumas áreas da Ria de Aveiro com a D. marocensis. Apesar destas duas espécies apresentarem padrões reprodutivos muito diferentes, Maio a Agosto é o período principal para a reprodução de ambas as espécies. D. neapolitana apresenta um desenvolvimento larvar planctónico, e os óocitos presentes na cavidade celómica são esverdeados e apresentam um diâmetro de 40-240 μm (média = 164.39±40.79 μm) e as fêmeas contêm no celoma milhares de óocitos. Contrariamente, a espécie D. marocensis reproduz-se por desenvolvimento directo no interior do tubo parental. Os óocitos observados no celoma são amarelos com um diâmetro entre 180 e 740 μm (média = 497.65 ± 31.38 μm) e o seu número varia entre 44 e 624 (276.85 ± 161.54). Por seu turno, o número de ovos observados no interior dos tubos varia entre 75 e 298, com um diâmetro entre 600 e 660 μm, e o número de larvas entre 60 e 194. A proporção machos: fêmeas foi de 1:1 para a população de D. neapolitana e entre 1:2 e 1:4 para a população de D. marocensis, em que as fêmeas dominam a população durante todo o ano. O estudo da capacidade de regeneração da espécie D. neapolitana, avaliada a partir de experiências de laboratório, revelou que esta espécie é capaz de sobreviver à perda de alguns setígeros. Durante a captura de D. neapolitana para vender como isco são normalmente cortados mais de 20 setígeros e de acordo com os nossos resultados a extremidade posterior que fica no tubo não é capaz de regenerar a extremidade anterior; a espécie consegue no entanto recuperar de ataques por predadores.

Population dynamics and fisheries assessment of the bigeye thresher (Alopias superciliosus) in the Atlantic: a comparison between North Atlantic and South Atlantic stocks

Tese de doutoramento, Ciências do Mar, da Terra e do Ambiente (Biologia Pesqueira), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Efectos del tamaño de anzuelo sobre capturas y tallas del perico Coryphaena hippurus

Se evidenció alta selección interespecífica del espinel superficial experimental, al capturar 130 ejemplares de perico Coryphaena hippurus (87,8%) del total de 148 especímenes que incluyeron 8 especies entre peces, tortugas y cefalópodos; como pesca incidental se registró 1 enganche por lance de tortugas (verde Chelonia mydas y amarilla Caretta caretta). El anzuelo más eficiente para la captura de esta especie, fue el de menor calibre (Nº 6) que capturó 24,6%, el menos eficiente fue el Nº 5 (16,9%); sin embargo, mediante el ANOVA de dos vías se estableció que no existía efecto del tamaño de anzuelo sobre el número de ejemplares capturados (p= 0,963). Los anzuelos no evidenciaron una selección de tallas, al no establecerse diferencias significativas entre distribución de tallas (p= 0,538), excepto para los anzuelos Nº 2 y 6 (p= 0,084), al identificarse diferencia por efecto del tamaño de anzuelo. Las longitudes medias estimadas fueron 119,7 cm, 116,8 cm, 118,3 cm, 115,0 cm y 113,7 cm, para los anzuelos Nº 2, 3, 4, 5 y 6, respectivamente. El tamaño de anzuelo mostró un fuerte efecto sobre el tipo de enganche (p= 0,002), presentándose con mayor frecuencia los enganches en la agalla (53,1%) y boca (44,6%). Generalmente los enganches en la agalla son ocasionados por los anzuelos grandes (Nº 2 y 3), mientras que los de boca son realizados por los anzuelos más pequeños (Nº 5 y 6). La captura por unidad de esfuerzo (CPUE) global del longline experimental fue 1,2 ejemplares de perico para un esfuerzo de 1000 anzuelos por hora, lo que representa la tercera parte de la CPUE estimada para la flota comercial.