Abstract
The basal media M199 or MEM was utilized in the classical method of vitro culture of glochidia where 1–5% CO2 was required to maintain stable physiological pH for completion of non-parasitic metamorphosis. The classical method encounters a great challenge to those glochidia which undergo development of visceral tissue but significantly increase in size during metamorphosis. The improved in vitro culture techniques and classical methods were firstly compared for non-parasitic metamorphosis and development of glochidia in pink heelsplitter. Based on the improved method, the optimal vitro culture media was further selected from 14 plasmas or sera, realizing the non-parasitic metamorphosis of axe-head glochidia for the first time. The results showed that addition of different plasma (serum) had significant effect on glochidial metamorphosis in pink heelsplitter. Only glochidia in the skewband grunt and red drum groups could complete metamorphosis, the metamorphosis rate in skewband grunt was 93.3±3.1% at 24±0.5°C, significantly higher than in marine and desalinated red drum. Heat-inactivated treatment on the plasma of yellow catfish and Barbus capito had significant effect on glochidia survival and shell growth. The metamorphosis rate also varied among different gravid period, and generally decreased with gravid time. Further comparison of free amino acid and fatty acid indicated that the taurine of high concentration was the only amino acid that might promote the rapid growth of glochidial shell, and the lack of adequate DPA and DHA might be an important reason leading to the abnormal foot and visceral development. Combined with our results of artificial selection of host fish, we tentatively established the mechanism of its host specialists in pink heelsplitter for the first time. This is the first report on non-parasite metamorphosis of axe-head glochidia based on our improved vitro culture method, which should provide important reference to fundamental theory research of glochidia metamorphosis and also benefit for better understand of mechanism of host specialists and generalists of Unionidae species.
Highlights
Most Unionidae species are obligated to undergo short period parasite on special fish or amphibian to complete the metamorphosis and maintain nature reproduction [1,2,3]
There was about 50%glochidial shell opened with different angle but not died in L15 group, while all glochidial shell remained closed with significant growth of the new shell in M199 group (Fig 1F), but the mantle tissue of some glochidia abnormally proliferated and inflated
To 12th day, there were still about 50%glochidial shell opened with short growing foot in L15 group (Fig 1G), while 80% glochidial mantle tissue abnormally expanded without foot and visceral mass developing in M199 group (Fig 1H)
Summary
Most Unionidae species are obligated to undergo short period parasite on special fish or amphibian to complete the metamorphosis and maintain nature reproduction [1,2,3]. 41 Unionidae species had successful report on non-parasitic metamorphosis through the classical virto culture method [9]. All these species tested possess large valves as glochidia belonging to hooked or hookless type, and only undergo development of visceral tissue with little growth in size. The genus Leptodea possess glochidia less than 60 μm in shell length that must develop visceral tissue, but amass an increase in shell length four-fold before they become fully developed juveniles [7] It is a hotspot and difficulty in the study of glochidia vitro culture. Based on the results of Owen [7], apart from quality of glochidia and control of bacteria and fuga contamination, the selection of plasma or critical nutrition should become an improtant sally port to the metamorphosis of those glochidia
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