Result 1 Chemotaxsis assay
Preparation of two-choice preference assay plates:
- Take a 1L conical bottle and wash it
- We obtained a specific concentration of algal suspension, filtrated a portion of it, and took the culture medium as a control for two-choice preference assay. t
- We designed a T-type experimental area on the plates and pleaced 100µl of each inoculum diagonally opposite onto assay plates.
The testing procedure of the two-choice preference assay:
- We recorded the movement of C. elegans on the two-choice preference assay plates by video recording.
- We first explored the choice bias of C. elegans when the concentration of algal suspension is 10^7 cells /L.
- We observed that C. elegans moved back and forward between the algal suspension and the control culture medium at the beginning of the experiment.
A few minutes later, we observed a significant difference in the number of C. elegans on the algal suspension and on the control culture medium.
Later in the experiment, most of the C. elegans on the control culture medium leaving and approaching the algal suspension were observed. But most of the C. elegans on the algal suspension remained in this region. The result showed that the number of C. elegans on the algal suspension was much greater than that on the control culture medium. After two-choice preference assay using different concentrations of algal suspension, We found that the minimum/boundary concentration of algal suspension for the formation of C. elegans choice bias was 2*10^6 Cell/L.
2.Data analysis of two-choice preference assays
- The chemotaxis index (i) is an important index for the analysis of the results of the two selection experiments.
- i=[(No.of worms on Liquid of algae)-(No.of worms on culture medium)]/Total number of worms on plates
- Generally, when the chemotaxis index is greater than 0, it indicates that
prefers algal suspension to culture medium. The closer the chemotactic index is to 1, the more attractive the algal fluid is to nematodes. - In the actual experiment, if the average chemotactic index of the same concentration is greater than 0.5, it can be shown that the algal suspension is more attractive to C. elegans than the culture medium.
Figure 1. Responses of Wild Type to Different Concentrations of Microcystis aeruginosa Unoperated wild-type animals weretested for their responses to Microcystis aeruginosa. Chemotaxis index =(animals at the Microcystis aeruginosa -animals at the control)/total animals on the plate. A chemotaxis index of 1.0 indicates complete attraction; a chemotaxis index of -1.0 indicates complete repulsion. Each column represents the average of three independent chemotaxis assays, using ~100 animals in each assay. Error bars equal the SEM. This result has been repeated ten times and each repeat shows similar tendency.
- Based on the results of two-choice preference assay(Figure 1), which is C. elegans preferred algal suspension, indicating that Microcystis aeruginosa can attract C.elegans and influence its behavior.
- C. elegans have the ability to detect whether the water contains Microcysts aeruginosa.
- When the concentration of algal suspension was 10^7 cells /L, the chemotactic index was about 0.6,which was the highest value under all experimental conditions.
- The chemotaxis index decrease slowly while the concentration decrease.
- The results showed that the index is still around 0.5 when the concentration of algal suspension is 2*10^6 cell/L, until the concentration is 10^6 cell/L, there is a sudden drop of index, finally at the 10^5 cell/L, C.elegans can hardly sense the existence of Microcystis aeruginosa(Figure 1).
- According to the standard [1], it defines the "no significant water blooming" concentration is 2*10^6 to 10^7cell/L, and the sudden drop of the index overlap with this standard concentration. So our device has the expected function to detect the water blooming before it really happens and leads to environment damage.
- As the same time, our experimental data showed that the locomotor behavior ofi C. elegans can be observed without relying on the microscope, which indicated that our device have the potential to shrink the size and perform the real-time examine at the sampling places without the microscope.
Result Ⅱ Color Reaction Assay
Figure 2. Fluorescence Images of the Phsp-6:GFP Reporter Strain after MC-LR Treatment (A) Brightfield and fluorescence images of the Phsp-6:GFP reporter strain after conventional culture and MC-LR treatment. Animals were moved to control or testing plate at L4 stage, the photos were taken after 24h treatment. (B) Quantification of Phsp-6:GFP fluorescence intensity (n ≥ 15; n is the number of biological replicates; Mean is shown by bar; ***p is smaller than 0.0001 by two-tailed t-test).
However, various organisms are contained in natural water bodies, and we need to further confirm that Microcystis aeruginosa plays a leading role in worm attracting. Microcystis aeruginosa secrets microcystin LR (MC-LR). It has been reported that MC-LR increases ROS levels in C.elegans, while ROS induces mitochondrial unfolded protein response (UPRmt). Accordingly, we designed our second step assay, using Phsp-6, a promoter specifically activated by UPRmt, to confirm whether MC-LR induces UPRmt and increases expression of reporter genes downstream of Phsp-6. We treated Phsp-6:GFP transgenic strain with 100 μg/L MC-LR for 24h and found that, compared to the control, the fluorescence intensity significantly increased (Figure 2), indicating that MC-LR treatment activated UPRmt and induced high expression of GFP.In summary, as our two-step experiments have shown, worms are attracted by algal suspension, while MC-LR activates UPRmt of nematodes. These results strongly suggest that there may be high concentrations of Microcystis aeruginosa in the water, which may lead to water blooms.
In order to detect the UPRmt level of worms in a convenient assay, independent on expensive and unportable fluorescence microscopes, we replaced GFP with lacZ to construct the plasmid Phsp-6:SV40 NLS:lacZ:SL2:mcherry (Figure 3). lacZ is a bacteria-derived gene, encoding β-Galactosidase, which transforms X-gal substrate to blue insoluble precipitate. This obvious discoloration can be easily observed by naked eyes. We provides a method for easier detection of UPRmt activation level through color reactions (data not shown).
Figure 3. A Schematic of Plasmid.