Smz 10

In the first stage, the non-disaggregated material from the beige and brown strips was observed with a binocular magnifying glass (Nikon SMZ-10) with variable magnification ranging from 10× to 40× to identify the structural elements.

Every sample was loaded parallel to the adhesive interface that formed between the dentin and test material. The measurement of bond strength was conducted with a universal testing machine (UltratesterTM, Ultradent, South Jordan, UT, USA), which subjected specimens to a shear load at a crosshead speed of 0.5 mm/min using recommended parameters for testing [10 (link)]. The required force to break the bond between the sealer and dentin was recorded in kg. The measurement was conducted using a special device Sentan kb3 (Sentran, LLC 4355NLoweell street, Ontario, CA, USA) connected to the testing machine. The megapascal (MPa) was the measurement unit for the shear strength of the bond.
After the shear bond testing, all samples were inspected with a stereomicroscope (Nikon SMZ10, Tokyo, Japan) at 20× magnification to determine the failure mode. The failure mode was recorded as adhesive when bonding failure was observed at the substrate–adhesive interface; cohesive when debonding was observed within the adhesive material not involving the interface; or mixed (cohesive and adhesive), which involved debonding within the adhesive material and at the interface [10 (link)].

We bought T. sazae from fishermen in Japan who dive in the vicinity of Jyogashima, Kanagawa prefecture, Najima in Hayama, Kanagawa prefecture and Okinoshima in Tateyama, Chiba prefecture, respectively. Hayama and Tateyama are relatively sheltered shores in the Sagami Bay and the Tokyo Bay, respectively, and suffering from a major coastal desertification in recent years [37 ]. On the other hand, Jyogashima is more exposed and has been recovering from a major coastal desertification since 2014 [38 ] and food sources for T. sazae are more abundant compared to Hayama and Tateyama.
Specimens were brought to the laboratory within the day when they are caught, and dissected to collect the shells. Photographs of the shells were taken using a digital camera (Canon EOS Kiss x7) with a micro lens (Canon EF100mm f/2.8L Micro IS USM) and magnified photographs of the spines were taken using dissection microscope (Nikon SMZ10) equipped with a digital camera (Canon EOS Kiss x7). We collected and photographed for analysis 12 specimens from Hayama, 16 from Jyogashima, and nine specimens from Tatayama.

The root exudates were collected and processed as reported in a previous study [40 (link)]. After collection, the root exudates were filtered through a 0.22-μm membrane (Millipore, Merck KGaA, Germany), freeze-dried, and then stored at – 80 °C for the chemotaxis assay.
A modified method based on the Petri-plate assay [55 (link)] was used for the chemotaxis assay. Semisolid 1/10 CTT medium (0.1% casitone, 10 mM Tris-HCl [pH 7.6], 8 mM MgSO₄, 1 mM KH₂PO₄, and 0.5% agar) was poured into 6 cm sterile plastic plates, and 3 μl of strain EGB suspension prepared as above was spotted onto each plate 2–4 mm away from the root exudates. Then, the plates were incubated in the dark at 30 °C for 24 h, and the swarming of strain EGB was observed and recorded with a stereoscopic microscope (Nikon SMZ-10) every 6 h. The chemotactic responses of strain EGB towards 13 substances detected in cucumber root exudates [29 (link), 30 (link)] were further analyzed. The substances were as follows: butanedioic acid, citric acid, fumaric acid, malic acid, maltitol, sorbitol, arabitol, tryptophan, tyrosine, alanine, aspartic acid, valine, maltose, and glucose.

After 1 day in the tanks, three mussels per day for 30 days were checked for R. pseudosericeus larvae. The presence of larvae on the four gills (left or right, outer or inner) of U. d. sinuolatus mussels was checked using a mussel‐opening device that enabled mussels to be opened to approximately 10 mm. The adductor muscle of mussels was cut to examine the position, number, and developmental stage of bitterling eggs/embryos/larvae. Mussels without bitterling eggs/embryos/larvae were housed in different tanks.
To evaluate the changes in larval position in the mussels, the gills were divided into nine parts (Figure 1); from the gill demibranch to its point of contact with the suprabranchial cavity, the gill was divided into lower part (L), middle part (M), and upper part (U); it was also divided into three parts in the other direction, 3 being the farthest from the outlet, followed by 2 and 1. Moreover, the larvae’s position was accurately recorded and photographed (Canon, Mark II, Tokyo, Japan) by measuring the transverse length of the siphon of the mussel and the longitudinal length from the suprabranchial cavity to the gill demibranch. The developmental stages of R. pseudosericeus larvae were determined under a stereoscopic microscope (Nikon, SMZ‐10, Tokyo, Japan) using AxioVision LE program (version 4.5, Carl Zeiss, Germany), as described by Kim, Kang, and Kim (2006).

After treatment with or without Al, whole roots were stained with hematoxylin (0.2% hematoxylin in 0.02% sodium iodide, w/w, pH 4.8) for 15 min as described by Khan et al. (2009) (link), and Al accumulation in the root tip was observed under a stereoscope (SMZ-10, Nikon, Tokyo, Japan).