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Sci Rep
2017 May 19;71:2173. doi: 10.1038/s41598-017-02171-8.
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Characterization of TRPA channels in the starfish Patiria pectinifera: involvement of thermally activated TRPA1 in thermotaxis in marine planktonic larvae.
Saito S
,
Hamanaka G
,
Kawai N
,
Furukawa R
,
Gojobori J
,
Tominaga M
,
Kaneko H
,
Satta Y
.
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The vast majority of marine invertebrates spend their larval period as pelagic plankton and are exposed to various environmental cues. Here we investigated the thermotaxis behaviors of the bipinnaria larvae of the starfish, Patiria pectinifera, in association with TRPA ion channels that serve as thermal receptors in various animal species. Using a newly developed thermotaxis assay system, we observed that P. pectinifera larvae displayed positive thermotaxis toward high temperatures, including toward temperatures high enough to cause death. In parallel, we identified two TRPA genes, termed PpTRPA1 and PpTRPA basal, from this species. We examined the phylogenetic position, spatial expression, and channel properties of each PpTRPA. Our results revealed the following: (1) The two genes diverged early in animal evolution; (2) PpTRPA1 and PpTRPA basal are expressed in the ciliary band and posterior digestive tract of the larval body, respectively; and (3) PpTRPA1 is activated by heat stimulation as well as by known TRPA1 agonists. Moreover, knockdown and rescue experiments demonstrated that PpTRPA1 is involved in positive thermotaxis in P. pectinifera larvae. This is the first report to reveal that TRPA1 channels regulate the behavioral response of a marine invertebrate to temperature changes during its planktonic larval period.
Figure 1. Thermotaxis assay system for starfish larva. (A) A 4-day-old-bipinnaria larvae of the starfish, P. pectinifera. Scale; 100 µm. (B) Thermal image of a representative 20–25 °C thermal gradient. The image was taken 15 min after placing the test chamber on the thermal stage. No obvious changes in the gradient were observed during the experiment. Scale; 1 cm. (C) Overview of the thermotaxis assay system. Five thermal sensors (white cables) were placed beneath the chamber and used to monitor temperature throughout the assay. Temperatures measured with the thermal sensors (shown in Fig. 2A) were essentially the same as those obtained by thermography as shown in Fig. 1B.
Figure 2. Thermotaxis assay of starfish larvae and the inhibitory effect on thermotaxis by a broad TRP channel blocker. (A) Changes in the number of larvae in each temperature range over time (n = 3). In each assay, approximately 50 larvae were added to the chamber. Larvae showed positive thermotaxis toward higher temperatures in each thermal gradient treatment. The table shows the upper and lower temperatures for each area (the chamber was equally divided into 4 areas) indicated by different colors according to the thermal gradient images in Fig. 1B. Proportions of larvae in each area are shown at 0, 30, and 60 min after the onset of the assay. (B) Morphology of a bipinnaria larva that stopped swimming at approximately 33 °C in the 31–38 °C thermal gradient. This photograph was taken at 10 min after the initiation of the assay. The arrow shows the area where the extracellular matrix (ECM) had separated from the epithelial cells. Scale; 100 µm. (C) High magnification image of the ectodermal epithelial cells in (B). Propidium iodide (PI) permeates into the nuclei of larval cells. White dotted lines represent the outline of the larval body. Scale; 20 µm. (D) Thermotaxis assay in the 20–25 °C thermal gradient with ruthenium red (RR, 20 µM)–treated larvae (n = 3). RR–treated larvae did not exhibit thermotactic behaviors as shown in A (middle).
Figure 3. Domain structures and phylogenetic positions of PpTRPA1 and PpTRPA basal. (A) Schematic drawing of PpTRPA1 and PpTRPA basal. The length of the amino acid sequences is indicated just above the lines. (B) The NJ-tree including known TRPA channels and PpTRPA1 and PpTRPA basal. Only the amino acid sequences of the transmembrane regions were used for phylogenetic analysis. Bootstrap values obtained from 1,000 replications are shown near the nodes. Because complete deletion reduces the number of amino acids, the pair-wise deletion option for calculation of genetic distances was used. The average number of amino acids used was 225.2.
Figure 4. The expression patterns of PpTRPA1 and PpTRPA basal genes at the bipinnaria stage. (A–F) Whole mount in situ hybridization (WISH) illustrating gene expression patterns of (A–C) PpTRPA1 and (D–F) PpTRPA basal at the bipinnaria stage as blue or fluorescent red signals, which were detected by NBT-BCIP or HNPP, respectively. Larvae are shown in the ventral view. (B, C) Images were created with the maximum-intensity method (B) using z-stack acquired by the confocal microscopy and (C) merged with blight field. (E, F) Coronal-sectioned images were acquired by the confocal microscopy and then (F) merged with blight field. (E) Open arrowhead, arrow, and filled arrowhead indicate the intestine, posterior end of the esophagus, and stomach of the bipinnaria larva, respectively. The scale bar is 50 µm. (G) Expression values of PpTRPA1 and PpTRPA basal determined by quantitative PCR analysis at the bipinnaria stage. The y-axis shows the number of copies per larva. Three independent batches were used to extract total RNA from the bipinnaria stage. 4dBp: 4-day-old bipinnaria.
Figure 7. Suppression of heat-evoked currents by PpTRPA1 MO in X. laevis oocytes injected with PpTRPA1 cRNA. The amplitudes for heat-evoked currents were compared among X. laevis oocytes injected with PpTRPA1 cRNA, PpTRPA1 cRNA with MO (1.5 or 15 pmol/µL) or PpTRPA1 cRNA with CMO (1.5 or 15 pmol/µL). Each dot indicates amplitude for the heat-evoked current obtained from each X. laevis oocyte. It should be noted that concomitant injection of PpTRPA1 cRNA and PpTRPA1 MO (15 pmol/µL) drastically suppressed heat-evoked activation. Average values are shown with bars.
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