Development of techniques to culture shrimp haemocytes and purify white spot syndrome virus (WSSV) in order to study WSSV-haemocyte interactions

The knowledge on immunity of farmed crustaceans is still at an early developmental stage when compared to that of terrestrial livestock. There are many fundamental questions that still need to be answered and others are currently under academic debate. In contrast, the shrimp farming industry has experienced a huge expansion in the last four decades. The conjugation of these two factors originated the emergence of many infectious diseases that threaten the sustainable development of the shrimp farming industry. Shrimp viruses are the biggest threat. They are associated with high mortalities, which provoke huge economic losses every year. The solution for these problems requires a deeper knowledge on shrimp immunity

Experimenten op het effect van temperatuur op white spot syndrome virus infectie in Litopenaeus vannamei garnalen

Dissertação de mest., Aquacultura e Pescas, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2006White Spot Disease (WSD) is an aggressive and devastating viral disease caused by the White Spot Syndrome Virus (WSSV). This highly pathogenic and widespread disease, present throughout Asia and the Americas, can cause up to 100% mortality within 3-7 days after infection. It is annually responsible for huge ecological and economical losses in the main producing countries and forms as such one of the greatest threats for the further sustainable development of shrimp aquaculture. Previous research showed that manipulation of physical factors gave promising results: manipulation of the environmental factors such as temperature produced the most interesting and promising results. For this thesis three experiments were performed, all in which pacific white shrimp (Litopenaeus vannamei) were intramuscularly inoculated with a well-defined viral dose (30 and/or 10000 SID50) and exposed to high water temperature via standardised protocols. The first experiment looked at the efficacy of elevated temperature for protecting shrimp against WSSV. Practically, four temperature treatments in which an elevated temperature (33 °C) was either applied before virus inoculation, after the inoculation, both before and after inoculation, and in the fourth treatment a low temperature (27ºC) was used throughout the test. In the second series of experiments the protective value of high temperature after an initial period of viral replication was evaluated. Water temperature was raised from 27ºC to 33ºC at 0, 12 or 24 hours post WSSV inoculation. Maintaining and controlling such high water temperatures for longer periods of time is of course very unpractical in field conditions and probably economically unfeasible, so the third experiment evaluated the effectiveness of shorter cyclic exposure periods to high water temperature. Hence, the shrimp were exposed to daily temperature cycles (33ºC/27ºC) with 6, 12 and 18 hours of high water temperature, during five consecutive days. Experiment 1 demonstrated a total blocking of disease progression when hyperthermia was applied immediately post inoculation. The protection was very effective even with a high viral dose (10000 SID50). The second experiment, at a low viral dose (30 SID50), showed that high temperature to some extent also worked therapeutic in that previously 24 hours of virus replication could be allowed. At a high infection dose (10000 SID50) the level of protection was however not so effective. In Experiment 3, only a minimum of 18 hours at 33°C resulted in a significant lower I mortality with the infected shrimp. The results from all the experiments clearly show the potential of high water temperature for preventing mortality in WSSV infected shrimp

Susceptibility of juvenile Macrobrachium rosenbergii to different doses of high and low virulence strains of white spot syndrome virus (WSSV)

As some literature on the susceptibility of different life stages of Macrobrachium rosenbergii to white spot syndrome virus (WSSV) is conflicting, the pathogenesis, infectivity and pathogenicity of 2 WSSV strains (Thai-1 and Viet) were investigated here in juveniles using conditions standardized for Penaeus vannamei. As with P. vannamei, juvenile M. rosenbergii (2 to 5 g) injected with a low dose of WSSV-Thai-1 or a high dose of WSSV-Viet developed comparable clinical pathology and numbers of infected cells within 1 to 2 d post-infection. In contrast, a low dose of WSSV-Viet capable of causing mortality in P. vannamei resulted in no detectable infection in M. rosenbergii. Mean prawn infectious dose 50% endpoints (PID50 ml(-1)) determined in M. rosenbergii were in the order of 100-fold higher for WSSV-Thai-1 (10(5.3 +/- 0.4) PID50 ml(-1)) than for WSSV-Viet (10(3.2 +/- 0.2) PID50 ml(-1)), with each of these being about 20-fold and 400-fold lower, respectively, than found previously in P. vannamei. The median lethal dose (LD50 ml(-1)) determined in M. rosenbergii was also far higher (similar to 1000-fold) for WSSV-Thai-1 (10(5.4 +/- 0.4) LD50 ml(-1)) than for WSSV-Viet (10(2.3 +/- 0.3) LD50 ml(-1)). Based on these data, it is clear that juvenile M. rosenbergii are susceptible to WSSV infection, disease and mortality. In comparison to P. vannamei, however, juvenile M. rosenbergii appear more capable of resisting infection and disease, particularly in the case of a WSSV strain with lower apparent virulence

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60 000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80 000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1 000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures

Moult cycle of laboratory-raised Penaeus (Litopenaeus) vannamei and P. monodon

This study was carried out to gather quantitative data on the moult cycle and stages in laboratory-raised shrimp, kept at a constant temperature of 27A degrees C. The stages of the moult cycle were differentiated and characterised by microscopic analysis of cuticle, epidermis and moulting processes in the uropods of P. vannamei and P. monodon. Five major moult stages were defined: early- and late post-moult (A and B), inter-moult (C) and early- and late pre-moult (D1 and D2). Total moult cycle duration was around 5 and 6.5 days for 2-g P. vannamei and P. monodon and 11 and 12 days for 15-g P. vannamei and P. monodon, respectively. Overall, the relative duration of the moult stages within the cycle was 5-10% for A, 9-16% for B, 12-20% for C, 28-36% for D1 and 30-38% for D2 stage. It was concluded from this study that the pre-moult stages comprised the dominant phase of the cycle and that P. monodon moulted at a significantly slower rate than P. vannamei, under the given conditions. Without the use of invasive techniques, the moult process was charted in laboratory-raised shrimp in Europe, providing a tool for taking into account this important physiological factor in further experiments