SummaryBiarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium. In order to establish this tagging system in Dictyostelium, the filamin gene (FLN) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT 2 or ReAsH-EDT 2 , the fluorescence intensity of cells increased in a time-dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH-EDT 2 reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN-mRFP fusion protein, but the fluorescence signal from the ReAsH-labelled protein was stronger. Our findings suggest that the ReAsHtetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium.
Unlike high‐altitude Rhacophorus moltrechti breeding in spring and summer and middle‐altitude populations breeding throughout the year, one possible mechanism causing lowland populations to breed in winter may be that high summer temperatures at low altitudes are stressful for tadpoles and lowland populations so they breed in winter to avoid this stress. However, breeding in the winter, which is the dry season in Taiwan, causes high densities as the water bodies they breed in are smaller and more isolated. We tested whether high summer water temperatures impose a cost and high tadpole densities lead to a benefit in growth, development and survival of lowland tadpoles by rearing tadpoles at three temperatures (17 and 22°C are two typical winter water temperatures and 27°C is a representative summer water temperature) and four different densities (5, 10, 20 and 30 tadpoles per box). We found that tadpoles metamorphosed earlier and at smaller sizes at 22°C (the higher winter water temperature) than tadpoles raised at either 17 or 27°C. Tadpoles raised at 27°C exhibited a longer larval period and a smaller metamorphic size than those raised at 22°C. Likewise, under the two winter water temperatures, but not the summer water temperature, increased tadpole density enhanced larval growth, translating into greater metamorphic mass without changing time to metamorphosis or decreasing survival rates. Nevertheless, tadpoles survived to metamorphosis at 27°C and at rates equal to those at 17 and 22°C. Our study suggests that lowland tadpoles are better adapted to maturing at cooler, winter water temperatures and that the summer water temperatures may be stressful to their growth and development. This leads to winter breeding for lowland populations. It also suggests that lowland populations breed at high tadpole densities because high densities benefit the larval growth and development.
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