Electroneutral Artificial Hosts for Oxoanions Active in Strong Donor Solvents

Electroneutral Artificial Hosts for Oxoanions Active in Strong Donor Solvent

The effect of ethanol on odorant attraction.

The odorant attraction of Canton S or w1118 third instar larvae was determined on 2.5% agarose containing different concentrations of ethanol. (A) Canton S larvae showed significantly reduced attraction to amyl acetate (AM) and 2-heptanone (2-Hep) in the presence of 8% ethanol, whereas their attraction to BA differed significantly when larvae were placed on 8% ethanol-containing or 5% ethanol-containing agarose (AIAM 0% = 0.51, ci = 0.41–0.59; AIAM 5% = 0.7, ci = 0.58–0.82; AIAM 8% = 0.19, ci = 0.11–0.25; AIBA 0% = 0.37, ci = 0.32–0.43; AIBA 5% = 0.48, ci = 0.41–0.56; AIBA 8% = 0.12, ci = 0.1–0.23; AI2-Hep 0% = 0.69, ci = 0.42–0.72; AI2-Hep 8% = 0.3, ci = 0.23–0.43). (B) The w1118 larvae did not change their attraction in the presence of ethanol (AIAM 0% = 0.25, ci = 0.2–0.38; AIAM 5% = 0.35, ci = 0.04–0.49; AIAM 8% = 0.31, ci = 0.18–0.49; AIBA 0% = 0.3, ci = 0.09–0.51; AIBA 5% = 0.38, ci = 0.16–0.58; AIBA 8% = 0.33, ci = 0.24–0.35; AI2-Hep 0% = 0.29, ci = 0.12–0.37; AI2-Hep 8% = 0.29, ci = 0–0.61). N = 8–15 groups of 20 larvae. Two groups were compared using Student’s t test, and more than two were compared with one-way ANOVA and a post hoc Bonferroni Holm correction. Significant differences from random choice were determined using the one-sample sign test and are labeled with the letter “a”. * P P < 0.01.</p

Influence of order of reinforcement during training on outcome of test.

Odorant attraction after three training cycles starting on ethanol-supplemented agarose. (A) When starting on ethanol-containing agarose with AM exposure, the Canton S larvae were not significantly attracted to AM or BA during the test. Extending the training-test interval by 5 min caused a significant attraction to AM (AIAM 3-cycle odor = 0.23, ci = 0.1–0.4; AIAM 3-cycle = 0.19, ci = 0.02–0.27; AIAM 3-cycle+ 5 min = 0.24, ci = 0.05–0.35). (B) When the training started with BA and ethanol, the Canton S larvae were indifferent to both odorants in the test. Extending the training-test interval by 5 min did not change the outcome (AIAM odor = -0.16 ci = -0.2–0.04; AIAM 3-cycle = -0.09, ci = -0.18–0.01; AIAM 3-cycle+5 min = -0.12, ci = -0.19–0.18). (C) The w1118 larvae were significantly attracted to AM in all tested conditions when training started with an AM exposure reinforced with ethanol (AIAM odor = 0.42, ci = 0.33–0.62; AIAM 3-cycle = 0.33, ci = 0.29–0.37; AIAM 3-cycle+ 5 min = 0.45, ci = 0.39–0.58). (D) Similar results were obtained when the training started with BA exposure paired with ethanol (AIAM odor = 0.5, ci = 0.26–0.63; AIAM 3-cycle = 0.33, ci = -0.22–0.53; AIAM 3-cycle+ 5 min = 0.42, ci = 0.11–0.59). (A and C) For comparison, the first boxes show the data from Fig 3B and 3E. N = 9–12 groups of 20 larvae. The red dots reflect the odorant container with AM, and the blue dots reflect the odorant container with BA. The one-sample sign test was used to determine significant differences from random choice, and the letter “a” indicates significant differences. One-way ANOVA with a post hoc Bonferroni Holm correction was used for differences between groups.</p

Learning index without reinforcer during test.

The AI values of the learning indices were calculated as follows: [(number of larvae on reinforced odorant side)—(number of larvae on nonreinforced odorant side)]/[(number of larvae on both sides + neutral zone)], unlike the distributional visualization in the previous figures. Each plot is the average of the two reciprocals’ PI values as indicated above the box blot. The data used are from Figs 3 and 4. (A) The learning index of Canton S larvae showed that ethanol functioned as a positive reinforcer when the reinforcer was presented at the first odorant exposure, but this effect disappeared when the test was delayed by 5 min (LI1 = -0.01 ci = -0.07–0.08; LI2 = 0.15, ci = 0.07–0.19; LI3 = -0.03, ci = -0.17–0.05: LI4 = 0.11, ci = 0.04–.0.25). (B) Exchanging the order of reinforcement during training resulted in no significant positive or negative association with 5% ethanol (LI1 = 0.06, ci = 0.03–0.2: LI2 = 0.05 ci = -0.05–.0.12; LI3 = 0.08 ci = -0–0.19; LI4 = 0.01, ci = -0.1–0.2). (C) The learning index of w1118 larvae showed that ethanol functioned as a negative reinforcer when the reinforcer was presented at the second odorant exposure. This effect did not disappear when the test was delayed by 5 min (LI1 = -0.11, ci = -0.17 –-0.07; LI2 = 0.025, ci = -0.1–0.11; LI3 = -0.18, ci = -0.28 –-0.04: LI4 = 0, ci = -0.11–.0.24). (D) Exchanging the order of reinforcement during training resulted in no significant positive or negative association with 5% ethanol (LI1 = -0.11, ci = -0.12–0: LI2 = -0.05, ci = -0.1–.0.05; LI3 = -0.08, ci = -0.09 –-0.04; LI4 = -0.04, ci = -0.2–0.1). Significant differences from random choice were analyzed with a one-sample sign test and labeled with the letter “a”. (TIF)</p

Statistics.

Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae.</div

Data for figures.

Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae.</div

Movement of <i>w</i>^<i>1118</i> larvae on 2.5% agarose with scarring for 5 min.

Movement of w1118 larvae on 2.5% agarose with scarring for 5 min.</p

Influence of ethanol in the substrate on the behavior of the larvae.

(A) Placed on agarose, Canton S larvae explored the edges of the ethanol-containing area, but they primarily stayed in the agarose area. When starting in an ethanol-containing area, the larvae stayed in the ethanol-containing area (AI agar5% = -0.1, ci = -0.22 –-0.05, AIagar10% = -0.21, ci -0.38 –-0.04; AIEtOH 5% = 0.2, ci = 0.18–0.38; AIEtOH 10% = 0.45, ci = 0.27–0.59). (B) The w1118 larvae moved to the boundary between the ethanol- and nonethanol-containing areas when they were placed on agarose. When they started on the ethanol-containing area, the larvae stayed in the 5% ethanol-containing area but moved out of the 10% ethanol-containing area (AI agar5% = -0.45, ci = -0.6 –-0.23; AIagar10% = -0.13, ci = -0.34 –-0.01; AIEtOH 5% = 0.23, ci = 0.15–0.41; AIEtOH 10% = -0.38, ci = -0.54 - -0.06). N = 8–14 groups of 20 larvae. Significant differences from random choice were determined using the one-sample sign test and are labeled with the letter “a”. To determine significant differences between groups, Student’s t test was used. *** P < 0.001.</p

Genotype-specific change in behavior after odorant presentation.

(A) Canton S larvae were equally attracted to both odorants when placed for 5 min on agarose with a source of AM (red dot) and BA (blue dot) (AIbalance0% = -0.1, ci = -0.22–0.11; AIbalance5% = -0.15, ci = -0.22–0.03). (B) When larvae were initially exposed to AM followed by BA with the reinforcer, the attraction shifted significantly toward AM after 3 cycles of odorant exposure in Canton S larvae. Pairing BA with 5% ethanol did not change the initial balance between AM and BA. The delay of 5 min after training did not change the results (AIAM 1-cycle odor = 0.15, ci = -0.02–0.23; AIAM 3-cycle odor = 0.23, ci = 0.10–0.4; AIAM 1-cycle = 0.05, ci = -0.12–0.29; AIAM 3-cycle = -0.11, ci = -0.19–0.13; AIAM 3-cycle+ 5 min = 0.1, ci = -0.08–0.25). (C) When starting with BA exposure, the larvae were not attracted to AM or BA after one or three cycles of training. Delaying the test for 5 min did not change the balance, and neither did the pairing of AM with ethanol (AIAM 1-cycle odor = -0.17, ci = -0.28–0.04; AIAM 3-cycle odor = -0.16, ci = -0.2–0.04; AIAM 1-cycle = 0.19, ci = -0.07–0.26; AIAM 3-cycle = -0.03, ci = -0.11–0.09; AIAM 3-cycle+ 5 min = -0.06, ci = -0.11–0.14). (D) After 5 min on agarose or 5% ethanol-containing agarose, the w1118 larvae were significantly more attracted to the odorant (AM 1:100) than to BA (AIbalance0% = 0.29, ci = 0.15–0.47; AIbalance5% = 0.28, ci = 0.17–0.41). (E) When starting the training on agarose in the presence of AM, the attraction of the w1118 larvae shifted significantly toward AM within 3 cycles of repetition. Pairing BA exposure with different concentrations of ethanol resulted in a significant attraction toward AM (AIAM 1-cycle odor = 0.64, ci = 0.54–0.7; AIAM 3-cycle odor = 0.42, ci = 0.33–0.62; AIAM 1-cycle = 0.63, ci = 0.49–0.80; AIAM 3-cycle = 0.54, ci = 0.44–0.56; AIAM 3-cycle+ 5 min = 0.60, ci = 0.44–0.75). (F) When the training started with BA exposure, the w1118 larvae were significantly attracted to AM after one or three cycles of training. Three cycles of training with ethanol resulted in significant attraction to AM. A delay of 5 min before the test did not abolish the attraction (AIAM 1-cycle odor = 0.53, ci = 0.29–0.66; AIAM 3-cycle odor = 0.5, ci = 0.26–0.63; AIAM 1-cycle = 0.17, ci = -0.07–0.37; AIAM 3-cycle = 0.31, ci = 0.09–0.44; AIAM 3-cycle+ 5 min = -0.25, ci = -0.19–0.38). N = 8–17 groups of 20 larvae. Significant differences from random choice were determined using a one-sample sign test and marked with the letter “a”, and significant differences between groups were analyzed using one-way ANOVA with a post hoc Bonferroni Holm correction.</p

A Simple Biosensor-Based Assay for Quantitative Autoinducer‑2 Analysis

Bacteria produce and react to interspecies signaling molecules in order to control the expression of genes that are particularly beneficial when they are expressed by a bacterial community. In addition to intraspecies communication, the signaling molecule autoinducer-2 (AI-2) can also serve for interspecies communication between Gram-positive and Gram-negative bacteria and is therefore of particular interest. The analysis and quantification of AI-2 are essential for understanding population density-dependent changes in bacterial behavior and pathogenicity. However, currently available bioassays for AI-2 quantification are rather complex, have narrow detection ranges, and are very sensitive to trace components of, for example, growth media. To facilitate and improve the detection of AI-2, we have developed an Escherichia coli biosensor-based assay that is sensitive, cheap, fast, robust, and reliable in the quantification of biologically active AI-2. The bioassay is based on an lsr promoter-fluorescent reporter gene fusion cassette that we chromosomally integrated in a biosensor strain, but the cassette can also be used in a low-copy number plasmid for the application in other Gram-negative bacterial species. We show here that AI-2 quantification was possible in a concentration range from 400 nM to 100 μM and that a critical interpretation of the kinetics of the measurements can reveal sugar interference. With the help of our biosensor strain, coculture experiments were done to test the capability and kinetics of AI-2 secretion by various Gram-negative bacteria in real time. Finally, calibration curves were used to calculate the absolute AI-2 concentration in cell-free bacterial samples