A new three-phase culture method for Manila clam, Ruditapes philippinarum, farming in northern China

Studies on reproduction, hatchery management, and culture of Manila clams Ruditapes philippinarum were carried out in an attempt to optimize their culture conditions and techniques. Results from these studies led to the development of a three-phase culture method for Manila clam farming in northern China. The key components of the new method were: 1) early spawning and over-wintering indoors (greenhouse); 2) optimized larval culture conditions and techniques; 3) juvenile rearing in shallow, fertilized nursery ponds; 4) optimized stocking size and density and substrate for mudflat grow out. Broodstock were maturated indoors for a month from early April to early May. Primarily because of higher water temperatures in the greenhouse the clams spawned more than one month earlier than in the natural environment. From May to July, juveniles were reared for 1-2 months indoors to a size of 2.0-3.0 mm in shell length before being moved to outdoor, pre-disinfected, nursery ponds. Juveniles were then reared in the nursery ponds for one month to about 1.0 cm before being transferred to the mudflat for grow out. Juvenile clams in nursery ponds grew considerably faster than in the natural environment probably because of higher temperatures and more abundant natural food. During grow out, the clams were reared for 4-7 months until they reached a market size (3.0-3.3 cm). Juveniles produced after August were over-wintered in the greenhouse in which the water temperature was about 3 degrees C higher than that of the outdoor environment. Juveniles grew at an average rate of > 20 mu m day(-1), while in the natural environment no growth was observed during winter because of low temperatures. Juveniles in the greenhouse grew to 2-3 mm by the following March before being moved into outdoor nursery ponds. The three-phase culture method not only shortened the production period from spawn to market size from 24-36 months to about 10-14 months, but also prolonged the spawning season from 2 to 7 months, resulting in increased production of seed and market-size clams. Compared with the traditional method, the new method could increase the yield of market-size clams by 10-11 times, and increase the profit per ha mudflat by as much as 124 times and the profit per kg market-size clams produced by 13 times. (c) 2006 Elsevier B.V. All rights reserved.

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

The analysis of Bcr-Abl—Nox signalling in chronic myeloid leukaemia

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterised by increased proliferation of haematopoietic stem cells. CML results following generation of the chimeric protein Bcr-Abl, a constitutively active tyrosine kinase which induces oncogenesis in part by promoting increased cell survival and proliferation. Since the development of Bcr-Abl-specific tyrosine kinase inhibitors (TKIs) there has been a substantial improvement in the clinical treatment of CML. Unfortunately, residual disease and the development of TKI resistance has become an ever growing concern, resulting in the need for a greater understanding of the disease in order to develop new treatment strategies. Interestingly, constitutive expression of the Bcr-Abl in CML is known to produce elevated levels of Reactive Oxygen Species (ROS) which are known to influence a variety of cellular processes. Previous studies have demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, enhancing survival signalling. The objective of this study was to elucidate how Nox protein activity was influenced downstream of Bcr-Abl while examining how Nox-derived ROS influenced CML disease phenotype to identify the potential in targeting these proteins to improve CML treatment. These studies demonstrated that inhibition of Bcr-Abl signalling, led to a significant reduction in ROS levels which was concurrent with the GSK-3dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. siRNA knockdown of p22phox identified it to have a significant role in cellular proliferation and cell viability, demonstrating the importance of Nox protein activity in CML disease phenotype. Furthermore, removal of p22phox was demonstrated to make cells significantly more susceptible to Bcr-Abl-specific TKI treatment, while pharmacological silencing of Nox activity in combination with TKIs was demonstrated to produce substantial, synergistic increases in cell death through augmentation of apoptosis, demonstrating the therapeutic potential of targeting Nox proteins in combination with Bcr-Abl inhibition.

The reproductive biology of the exploited razor clam, Ensis siliqua, in the Irish Sea

Knowledge of the reproductive cycle of a species is a prerequisite for sustainable management of a fishery. The infaunal marine bivalve, Ensis siliqua, is a commercially important species in Europe, and is exploited in many countries, including Ireland, where it is sold by wet weight. Seasonal variations in the reproductive cycle of subtidal razor clams from the Skerries region of the Irish Sea, an important fisheries area, were examined between June 2010 and September 2011 while monitoring weight. Histological examination revealed that the E. siliqua sex-ratio was not different from parity, and no hermaphrodites were observed in the samples collected. In the summer months of 2010 all female clams were either spent or in early development, with just a small percentage of males still spawning. The gonads of both sexes developed over the autumn and winter months of 2010, with the first spawning individuals recorded in January 2011. Spawning peaked in March 2011, but unlike in 2010, spawning continued through June and July with all animals spent in August 2011. The earlier and longer spawning period found in this species in 2011 compared to 2010 may have been due to the colder than normal temperature observed during the winter of 2010 plus the relatively warmer temperatures of Spring 2011, which could have affected the gametogenic development of E. siliqua in the Irish Sea. It was noted that wet weight dropped in the summer months of both years, immediately after the spawning period which may impact on the practicality of fishing for this species during this period. Timing of development and spawning is compared with other sites in the Irish Sea and elsewhere in Europe, including the Iberian Peninsula.

The reproductive biology of the soft shell clam Mya arenaria in Ireland and the possible impacts of climate variability

Little is known about the biology of the softshell clam in Europe, despite it being identified as a potential species to culture for food in the future. Monthly samples of the softshell clam, Mya arenaria, were collected intertidally from Co. Wexford, Ireland, over a period of sixteen months. The mean weight of sampled individuals was 7 4 ± 4 . 9  g and mean length was 8 . 2 ± 0 . 2  cm. Histological examination revealed a female-to-male ratio of 1 : 1.15. In 2010, M. arenaria at this site matured over the summer months, with both sexes either ripe or spawning by August. A single spawning event was recorded in 2010, completed by November. Two unusually cold winters, followed by a warmer-than-average spring, appear to have affected M. arenaria gametogenesis in this area, potentially affecting the time of spawning, fertilisation success, and recruitment of this species. No hermaphrodites were observed in the samples collected, nor were any pathogens observed. Timing of development and spawning is compared with the coasts of eastern North America and with other European coasts.

Intersex in the clam Scrobicularia plana: A sign of endocrine disruption in estuaries?

The phenomenon of endocrine disruption is currently a source of growing concern. Feminisation of male fish in UK rivers has been shown to occur extensively and has been linked with exposure to endocrine-disrupting compounds present in the environment. Much less is known of the extent and scale of endocrine disruption in estuarine and marine ecosystems, particularly in invertebrates. We present evidence that intersex, in the form of ovotestis, is occurring in the common estuarine bivalve Scrobicularia plana, which is considered to be inherently gonochoristic. We report varying degrees in the severity of ovotestis in male S. plana, and have adopted and developed a grading method to assess the extent of this intersex condition. These findings indicate that S. plana offers potential for widespread screening and investigation of endocrine disruption, helping to focus remediatory strategy.

Intersex in the clam Scrobicularia plana (Da Costa): Widespread occurrence in English Channel estuaries and surrounding areas

Estuarine clams Scrobicularia plana were sampled from 108 intertidal locations around the English Channel and adjacent areas. Although S. plana is believed to be a strict gonochorist, 58% of the populations sampled included intersexed individuals (described as male clams exhibiting ovotestis). Over the entire region, on average, 8.6% of male clams exhibited intersex, although proportions of affected males ranged from 0% to 53% depending on location. The severity of intersex was assessed using a simple classification scale, with the majority of individuals showing low levels of impact. Sex ratios were significantly skewed at some sites. There were no significant relationships between the incidence and severity of intersex; or of associations with size or parasitism of individual clams. Intersex in S. plana is a useful tool to assess endocrine disruptive effects in estuaries, although mechanisms of impact and causative agents remain uncertain.