BackgroundEven subtle changes in environmental factors can exert behavioral effects on creatures, which may alter interspecific interactions and eventually affect the ecosystem. However, how changes in environmental factors impact complex behaviors regulated by neural processes is largely unknown. The freshwater planarian Dugesia japonica, a free-living flatworm, displays distinct behavioral traits mediated by sensitive perception of environmental cues. Planarians are thus useful organisms for examining interactions between environmental changes and specific behaviors of animals.ResultsHere we found that feeding behavior was suppressed when the concentration of ions in the breeding water was low, while other behaviors were unaffected, resulting in differences in population size. Notably, the decline in feeding behavior was reversed in an ion-concentration-dependent manner soon after the planarians were moved to ion-containing water, which suggests that ions in environmental water rapidly promote feeding behavior in planarians. Moreover, the concentration of ions in the environmental water affected the feeding behavior by modulating the sensitivity of the response to foods. Finally, we found that calcium ions in the aquatic environment were required for the feeding behavior, and exposure to higher levels of calcium ions enhanced the feeding behavior, showing that there was a good correlation between the concentration of calcium ions and the responsiveness of planarians to foods.ConclusionsEnvironmental calcium ions are indispensable for and potentiate the activity level of the feeding behavior of planarians. Our findings suggest that the ions in the aquatic environment profoundly impact the growth and survival of aquatic animals via modulating their neural activities and behaviors.
The in vivo imaging of mice makes it possible to analyze disease
progress non-invasively through reporter gene expression. As the removal of hair improves
the accuracy of in vivo imaging, gene-modified mice with a reporter gene
are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous
Hrhr mutation that exhibit a hair loss phenotype. However,
it is time consuming to produce mice carrying both the reporter gene and mutant
Hrhr gene by mating. In addition, there is a risk that
genetic background of the gene-modified mice would be altered by mating. To resolve these
issues, we established a simple method to generate hairless mice maintaining the original
genetic background by CRISPR technology. First, we constructed the pX330
vector, which targets exon 3 of Hr. This DNA vector (5
ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced
Hr gene mutations were found in many founders (76.1%) and these
mutations were heritable. Next, we performed in vivo imaging using these
gene-modified hairless mice. As expected, luminescent objects in their body were detected
by in vivo imaging. This study clearly showed that hairless mice could be
simply generated by the CRISPR/Cas9 system, and this method may be useful for in
vivo imaging studies with various gene-modified mice.
Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.
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