Gene expression during early ascidian metamorphosis requires signaling by Hemps, an EGF-like protein

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

The effect of larval age on morphology and gene expression during ascidian metamorphosis

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

海鞘（Ciona savignyi）性腺组织细胞、血细胞和生殖干样细胞的体外培养

随着对海洋无脊椎动物免疫、发育以及细胞生物学等方面研究的需要，海洋无脊椎动物细胞培养日益受到关注。然而，由于海洋无脊椎动物的细胞代谢途径以及生长特性与陆生哺乳动物有很大差异，细胞培养难度较大,至今尚未有连续的细胞系建立。海鞘（Ciona savignyi）属于尾索动物亚门(Urochordata)，是典型的被囊类动物。作为无脊椎动物中进化地位较高等的一类动物，海鞘在免疫、胚胎发育、细胞学等各个领域对研究脊椎动物的系统发生都有着非常重要的作用。 本文分别以海鞘的性腺组织细胞和血细胞为材料，建立和优化海鞘细胞体外培养方法和条件；另外，还识别了成体海鞘性腺内的生殖样干细胞，并对其进行了体外培养和鉴定，为进一步开展海鞘细胞体外培养最终建立细胞系积累了资料。 首先比较了4种基础培养基L-15、M199、DMEM和RPMIl640 与各种培养添加物组合、不同温度和PH值对海鞘性腺组织细胞和血细胞的体外生长的影响。结果表明，20℃，pH6.8，M199基础培养基添加10%胎牛血清最适合海鞘性腺组织细胞生长。同时对海鞘性腺的分离方法即机械解离细胞法和酶学解离细胞法进行了比较，发现机械解离细胞法最适合海鞘性腺组织细胞的分离，分离得到的细胞贴壁率和成活率高。对于海鞘血细胞的培养，20℃，pH6.8条件下，L-15基础培养基并添加10%胎牛血清对血细胞的体外存活和生长效果较好。另外，还成功的利用原代培养的血细胞检测了海鞘肿瘤坏死因子配体家族成员（CsTL）基因的表达变化。 论文还研究了海鞘细胞培养中细菌污染的鉴定和控制方法。对于培养过程中的细菌污染，通过细菌分离、培养和纯培养发现两类菌株检出率较高，均为革兰氏阴性菌。经PCR 扩增16S rDNA 基因序列片断，结果显示这两类菌株分别属于弧菌属和施万氏菌属。药敏试验结果表明，亚胺培南和氯霉素等对受检施万氏菌的敏感度较高；而受检弧菌对氯霉素和环丙沙星的敏感度高。为控制培养中的微生物污染，比较了几种抗生素组合的使用效果，其中氯霉素和亚胺培南与双抗的抗生素组合有较好的抑菌效果并对培养细胞的贴壁和生长没有影响。然而，海鞘性腺组织细胞和血细胞在体外的传代培养并未取得成功，本论文对来源于成体海鞘性腺的生殖样干细胞进行了体外培养和鉴定。结果表明，成体海鞘性腺内存在生殖样干细胞，且在体外可以生长，繁殖并且可能具有分化潜能。体外培养的过程中，生长的细胞克隆明显具有类似胚胎干细胞的形态和基因表达特点。 本研究克隆了海鞘肿瘤坏死因子配体家族成员（CsTL）基因。CsTL全长995个核苷酸编码281个氨基酸。组织表达结果显示，CsTL在性腺组织的表达水平相对比较高，提示CsTL可能对海鞘性腺的发育或分化等起着一定的作用。利用昆虫杆状病毒表达系统表达纯化了CsTL蛋白。结果显示，重组CsTL对L929细胞显示了明显的细胞毒性作用，说明CsTL是具有生物学活性的重组蛋白。但是尝试用获得的重组CsTL蛋白作为培养添加物培养海鞘性腺组织细胞，但并未检测到CsTL对海鞘性腺组织细胞的生长或凋亡有任何影响。 总之，本文筛选到了适合海鞘性腺组织细胞和血细胞生长的培养基，并成功的将这两类细胞在体外进行了原代培养，并且虽然细胞传代未获成功，但为今后继续深入开展海鞘细胞培养研究奠定了基础，另外，海鞘生殖样干细胞的识别和培养也将为海洋无脊椎动物的细胞培养提供一条新途径。

Physiological-Responses Of Mytilus-Edulis L To Parasitic Infestation By Mytilicola-Intestinalis

Various levels of infestation by Mytilicola had no effect on the rates of oxygen consumption by Mytilus edulis in laboratory experiments. However, high levels of infestation (> 10 parasites per mussel) caused a depression in the feeding rate of the host at high temperatures (22° or 23° C) and low ration (maintenance or sub-maintenance). This depression of feeding resulted in a decline in the scope for growth, which would result in time in a decline in the “condition” of the host. It is concluded that similar effects may occur in the field when large numbers of small parasites are present at a time of high metabolic demand and low food availability.

Population-Dynamics Of Mytilicola-Intestinalis In Mytilus-Edulis In South West England

The population dynamics of Mytilicola intestinalis Steuer in mussels (Mytilus edulis L.) from the River Lynher, Cornwall, England, have been studied over 3 years. By transplanting uninfested mussels from the River Erme, South Devon, into the Lynher mussel bed, the study was extended to the growth and development of new infestations under natural conditions. Female Mytilicola intestinalis are shown to breed twice, and two generations of parasites coexist for most of the year, with recruitment taking place in summer and autumn. One generation contributes its first brood to the autumn recruits before overwintering and contributing its second brood to the following summer's recruits. The other generation overwinters as juvenile and immature stages to contribute its two broods successively to the summer and autumn recruits. Environmental temperatures are believed to control the rates of development at all stages rather than acting as triggers in the onset or cessation of breeding at specific times. There is no evidence to support the contention that heavily infested mussels are killed, and parasite mortality is shown to be density-independent.

Relationship Between Infestation By Mytilicola-Intestinalis Steuer (Copepoda Cyclopoidea) And Condition Index Of Mytilus-Edulis In Southwest England

Seasonal cycles in the condition index of Mytilus edulis from three sites in southwest England are described. These are analysed in relation to host length, stage of gonad development and parasite burden by linear regression analysis. An effect on the condition index due to the presence of Mytilicola intestinalis can be detected only in the sublittoral mussels in those few winter months when the mean intensity of infestation is over about 25 parasites per host. In all cases studied, the magnitude of the effect due to variation in host length, stage of gonad development, seasonal cycles and environmental factors is greater than that due to parasitism.

Occurrence Of Mytilicola-Intestinalis Steuer An Intestinal Copepod Parasite Of Mytilus In Southwest Of England

The occurrence of Mytilicola intestinalis in populations of mussels in south-west England is recorded and compared with previous data. Since 1955 there have been two main changes in the distribution of Mytilicola: (a) it has invaded all the major estuarine mussel populations on the Bristol Channel coast, and (b) many previously uninfested open-coast populations all round the peninsula are now lightly infested. It is suggested that differences in infestation levels between estuarine and open-coast populations of mussels are due primarily to differences in the degree of exposure to wave action although factors such as size, population density and location of the hosts also influence infestation. The chance of the establishment of breeding pairs of Mytilicola depends on the parasite population size and its distribution through the host population.