Studies On The Thermotolerance Of Artemia Cysts And Its Intrinsic Mechanism

The storage or heating experiment under certain conditions showed that there is different thermotolerance among four geographical Artemia cysts, and a series of comparative studies on biological aspects of Artemia cysts were carried out to find the reason of thermotolerance diversity occurred. The results are summarized as follows:1. Comparative study on cysts thermotoleranceThe experiment of cysts storage in different conditions, different temperatures, different cysts mass per unit volume (CMPV), different residual water, or different gas showed that the thermotolerance of Ga Hai (GH) and Aqqikkol Hu (AQK) cysts were better than the cysts of Jingyu Hu (JYH) and Xizang (XZ).2. Comparative observations on the cyst shellsThe quiescent Artemia cysts of seven geographical origins in China were examined with scanning and transmission electron microscopes. SEM observations on cysts of these Artemia strains showed that the surface topography of cyst shells could be categorized into 6 types: complete smooth surface; smooth surface with sparsely distributed glabrate humps; surface with densely arranged wart-like structures which are composed of packed minute tubercles; rugged surface; shallow-pocked surface; and surface with numerous and densely spaced pore-like fossulae. Some of the patterns were strain specific [e.g., cysts from GH are characterized by having a surface with wart-like ornaments that are composed of packed minute tubercles, rugged surface is only found in Chengkou (CK) cysts], and apparent intra-strain variation of cyst surface topography was found in XZ, JYH and Xie Chi (SIN) strains. The inner surface of embryonic membrane is characterized by a negative bas-relief of polygons, which are tightly arranged and possess ridgy edges. The polygons with five, six, seven, or eight edges were detected. TEM studies on the ultrastructure of cyst shells revealed an apparent divergence in the structure of outer cuticular membrane (OCM) and EC1/TOT among Artemia strains. In CK, AQK, SIN and GH strains, it is a normal, asymmetrical and multi-layered structure. In XZ, JYH and Lagkor Co (LGC) strains, however, the OCM is not obviously multi-layered. The EC1/TOT of seven Artemia strains ranks as GH>AQK=SIN>XZ=JYH=CK>LGC. The hypoplasia of OCM and thin EC1 in XZ and JYH strains may be the reason that cysts of these Artemia strains tend to suffer heavy mortality when stored at normal temperatures.3. Observations on embryonic stage and nuclear numberThe embryonic development stage of encysted embryos of four Artemia populations (GH, QAK, XZ, JYH) from Qinghai-Tibet Plateau was studied with paraffin section method, and the nuclei numbers of encysted embryos were estimated by counting nuclear on squashed preparations. The results showed that the encysted Artemia embryos of all four Artemia populations were at the stage of gastrula, and consisted of 3441±164, 3412±171, 3736±212, 3861±155 nuclei respectively, as well as no certain correlation with the embryo stage and nuclei number.4.Study on the biochemical adaption of Artemia cystsAnalysis of trehalose and glycerol in cysts of four geographical Artemia strains (GH, AQK, XZ, JYH) was performed by HPGC (High Performance Gel Chromatography). The contents of trehalose of four Artemia strains ranks as AQK>JYH>GH>XZ, and glycerol of four Artemia strains ranks as XZ>AQK>JYH>GH. This results suggested that the phenomenon of thermotolerance diversity in cysts storage was not correlated with trehalose and glycerol contents directly.The relative contents of stress protein hsp70 in nuclei or cytoplasm of four Artemia strain cysts (GH, AQK, XZ and JYH) were detected by western immunoblotting. Two conditions were examined: original cysts and cysts stored. The results showed that the relative contents of stress protein hsp70 in cytoplasm of four Artemia strain cysts were very similar, the hsp70 in original cysts and their treated counterparts were similar either, and no hsp70 in nuclei was detected. It means that hsp70 existed in dry cysts of four Artemia strains and inducing translocation of hsp70 to nuclei didn't occurred under the storage conditions used herein at least. The phenomenon of tolerance diversity in cysts storage was not correlated with hsp70 directly.5. Dehydration of intact and decapsulated cystsIntact and decapsulated cysts of four strains (GH, AQK, XZ, JYH) were dehydrated in a LGJ-10 Lyophilizer simultaneous. The water loss was quantified and recorded at intervals by weight loss. Residual water of intact and decapsulated cysts was estimated by interval recording and dried weight, which was weighed quickly after the heating of cysts at 103oC for 16-18h at the final of dehydration. The results showed that the residual water degradation of both intact and decapsulated cysts was very similar, A common and distinct phenomenon was that the dehydration of GH was obvious slower than that of AQK, which was dehydrated slower than XZ and JYH. Namely, the cysts of GH and AQK with multi-layered OCM and thick EC1 were better resistant to water-loss than the cysts of XZ and JYH with unmulti-layered OCM and thin EC1.In conclusion, based on the hypothesis of Clegg (2005), it suggests that multi-layered OCM and thick EC1can maintain the iner-environment of embryos and protect embryos from damage, nevertheless, our understanding of these organisms to survive environmental stress is still remarkably superficial, and the possibility of other aspects cannot be excluded.