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Video S2 from Successful escape of bombardier beetles from predator digestive systems

Predation and emetic behaviour of a juvenile <i>Bufo japonicus</i>. The toad easily swallowed an adult Pheropsophus jessoensis. However, the toad vomited the beetle up 88 min after swallowing it; that is, the beetle escaped from the toad's mouth 88 min after being swallowed

Electronic supplementary material from Successful escape of bombardier beetles from predator digestive systems

Electronic supplementary materia

Morphology-based optical separation of subpopulations from a heterogeneous murine breast cancer cell line

<div><p>Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated <i>in vitro</i> by using a spheroid assay and <i>in vivo</i> by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics.</p></div

An overview of the optical cell separation procedure.

<p>(A) Cell encapsulation in the PD-gelatin hydrogel. 4T1E cells were encapsulated in photodegradable hydrogels prepared by mixing azide-gelatin solution and DBCO-PC-4armPEG solution. (B) Optical cell separation from the PD-gelatin hydrogel. The target cells are irradiated with UV light and subsequently retrieved after hydrogel degradation. (C) Microscopic images of 4T1E cells, and colony- and granular-type cells to be separated by use of the optical cell separation process.</p

Tumor growth in mice transplanted with 4T1E cells, and colony- (C1 and C2) and granular-type (G1 and G2) cells separated from 4T1E cells.

<p>(A) Tumor growth. (B) Body weight of mice. The table below the figure shows the statistical profile as analyzed by using Scheffe’s test for tumor growth. Asterisks in the table indicate statistical significance. One asterisk indicates <i>p</i>-value less than 0.05 and double asterisks indicate <i>p</i>-value less than 0.01. Error bars indicate the standard deviation (n = 3–4).</p

Frequency of metastasis in tissues obtained 38 days post-transplantation of 4T1E cells, and colony- (C1 and C2) and granular-type (G1 and G2) cells separated from 4T1E cells.

<p>Arrows indicate metastatic tumors on the surface of the tissues. The table shows the frequency of metastases confirmed by observation of the dissected tissues. The number of tumors detected in lung, liver, colon, kidney, and intestine is indicated as none (-), one (+), one to five (++), and more than five (+++). The upward-pointing arrows in the table indicate spleen enlargement.</p

Microscopic images of 4T1E cells, and colony- and granular-type cells separated from 4T1E cells.

<p>The separated cells were established as colony- (C1 and C2) and granular-type (G1 and G2) cells by optical separation based on morphology in PD-gelatin hydrogels. 2D, cell culture on a culture dish; 3D, cell culture in the PD-gelatin hydrogels. The images were taken after 3 days of culture under 2D and 3D conditions. (A) Raw images. The scale bar indicates 500 μm. (B) Digitally magnified images. The scale bar indicates 125 μm.</p

Genetic Structure and Potential Environmental Determinants of Local Genetic Diversity in Japanese Honeybees (<i>Apis cerana japonica</i>)

<div><p>Declines in honeybee populations have been a recent concern. Although causes of the declines remain unclear, environmental factors may be responsible. We focused on the potential environmental determinants of local populations of wild honeybees, <i>Apis cerana japonica</i>, in Japan. This subspecies has little genetic variation in terms of its mitochondrial DNA sequences, and genetic variations at nuclear loci are as yet unknown. We estimated the genetic structure and environmental determinants of local genetic diversity in nuclear microsatellite genotypes of fathers and mothers, inferred from workers collected at 139 sites. The genotypes of fathers and mothers showed weak isolation by distance and negligible genetic structure. The local genetic diversity was high in central Japan, decreasing toward the peripheries, and depended on the climate and land use characteristics of the sites. The local genetic diversity decreased as the annual precipitation increased, and increased as the proportion of urban and paddy field areas increased. Positive effects of natural forest area, which have also been observed in terms of forager abundance in farms, were not detected with respect to the local genetic diversity. The findings suggest that <i>A</i>. <i>cerana japonica</i> forms a single population connected by gene flow in its main distributional range, and that climate and landscape properties potentially affect its local genetic diversity.</p></div

Locations of sites (<i>numbers</i>) where <i>Apis cerana japonica</i> workers were collected.

<p>Borders (<i>broken lines</i>) between prefectures are shown.</p