Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Background:Bitter taste receptors(Tas2rs)are generally considered to sense various bitter compounds to escape the intake of toxic substances.Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.Methods:To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues,multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique.A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes.Then,T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice.Quantitative real-time polymerase chain reaction(qRT-PCR)and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds.Perception to taste substance was also studied using twobottle preference tests.Results:We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique.Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice.But qRT-PCR results revealed the changed expression profile of m Tas2rs gene in taste buds of these mutant mice.With two-bottle preference tests,these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105.In addition,these mutant mice showed a loss of taste perception to quinine dihydrochloride,denatonium benzoate,and cucurbitacin B(CuB).Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.Conclusions:These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

A Music Preference Test System for Rats

In music preference experiments, housing conditions and the control of parameters for animals can affect experimental results. However, the needs of animals are indeed insufficiently considered in many reports of animal experiments. In order to evaluate which music rats prefer, we developed a new music preference test system. Dwelling time and visiting frequency can be recorded automatically when rats moving among different compartments of the system. We can also observe the behaviors of rats captured by a video camera. By this system, the music preference can be found, and then the related music can be used in following various studies. In this paper, we described the design of this music preference test system of rat, and some primary results were reported.

Quality assessment of local Trichogramma species (Hymenoptera, Tri- chogrammatidae) to control the olive moth Prays oleae Bern. (Lepidop- tera, Yponomeutidae)

Assessment of quality of biological control agents is an important step for the success of any biocontrol program.Experiments were carried out to select candidate Trichogramma species for use in biological control of the olive moth Prays oleae Bern(Lepidoptera,Yponomeutidae).Thirteen local strains were preselected on the basis of the geographical origin and the availability from mass production of the rearing host Ephestia kuehniella ZELL.(Lepidoptera,Pyralidae) under laboratory conditions.Selection tests were focusing on acceptance and preference for P.oleae eggs.In the acceptance test,a single parasitoid female was offered P.oleae eggs on "tricho-card" for 6 hours at 25°C,70% HR and L16:D9 and assessed for parasitism after 6 days.The preference was determined by offering the choice and observing contacts with eggs of the target pest P.oleae and eggs of the host E.kuehniella.Parasitism was therefore assessed 6 days after the experiment under similar laboratory conditions.In host acceptance test,most eggs of P.oleae were parasited by strains belonging to the species T.oleae,T.bourarachae and T.cacoeciae.These strains also showed a high number of contacts with olive moth eggs in preference tests.However,a small number of T.bourarachae observed ovipositions resulted in successful parasitism.Therefore,T.oleae and T.cacoeciae were selected as candidates for further experiments.

Assessment of commonly used tests in experimental depression studies according to behavioral patterns of rodents

Considering the main factor that causes or triggers depression in humans is stress.Several stress factors are applied to form depression-like symptoms in rodents.Depression tests are used to analyze the nature and patterns of depression.Well-founded modeling and versatile evaluation of tests are necessary to investigate a hypothesis that is related to depression.It is impossible to model or test all aspects of depression in humans by using experimental animals.As a result,the aims of the study should be determined specifically in depression models.The correct interpretation of the tests that are suitable for these aims is indispensable for the reliability of the data.To achieve this goal,the biological basis of the depression-related behaviors of animals should be well known.In this review,model and test concepts related to depression are discussed and behavioral patterns of rodents are explained with several examples.

A novel quantified bitterness evaluation model for traditional Chinese herbs based on an animal ethology principle

Traditional Chinese herbs(TCH) are currently gaining attention in disease prevention and health care plans. However, their general bitter taste hinders their use. Despite the development of a variety of taste evaluation methods, it is still a major challenge to establish a quantitative detection technique that is objective, authentic and sensitive. Based on the two-bottle preference test(TBP), we proposed a novel quantitative strategy using a standardized animal test and a unified quantitative benchmark. To reduce the difference of results, the methodology of TBP was optimized. The relationship between the concentration of quinine and animal preference index(PI) was obtained. Then the PI of TCH was measured through TBP, and bitterness results were converted into a unified numerical system using the relationship of concentration and PI. To verify the authenticity and sensitivity of quantified results,human sensory testing and electronic tongue testing were applied. The quantified results showed a good discrimination ability. For example, the bitterness of Coptidis Rhizoma was equal to 0.0579 mg/mL quinine, and Nelumbinis Folium was equal to 0.0001 mg/mL. The validation results proved that the newassessment method for TCH was objective and reliable. In conclusion, this study provides an option for the quantification of bitterness and the evaluation of taste masking effects.

Cumulative effects of volcanic ash on the food preferences of two Orthopteran species

Inert dusts are an early form of insecticide which is still in use. One of the most common inert dusts is volcanic ash. In order to study the reaction ofrangeland grasshoppers, Dichroplus vittigerum （Acrididae） and a katydid, t]wgilis mendosensis （Phaneropteridae）, to the presence of volcanic ash in their food sources and how this reaction changed as a function of time, we conducted paired preference tests between clean leaves of their pre- ferred host plant and leaves exposed to volcanic ash of different grain size. The behavioral response was measured as the rating on the Thurstonian preference scale of leaves with ash in relation to clean leaves. The results showed that the avoidance of volcanic ash increased as a function of time in both species. Both species studied are occasionally exposed to vol- canic activity, and come from an area in which a volcanic eruption had recently occurred. As their populations did not decrease after the ash fall, we propose that some behavioral responses such as avoidance of places with ash, works as tolerance mechanism to inert dusts exposure.