A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus

1) Please strengthen the analysis of the role of the amphid sheath cell. Either ablate amphid sheath in Ppa or express Ppa obi-1 (or C06G1.1) in C. elegans under an amphid sheath cell promoter to see if C. elegans now becomes sensitive to ZTDO.

We constructed C. elegans transgenic strains that expressed Ppa-obi-1 cDNA under the amphid sheath cell-specific promoter T02B11.3 and F16F9.3. The translational fusion protein Ppa-OBI-1:GFP is expressed faithfully in amphid sheath cells and improved the ability of F16F9.3p::obi-1:gfp worms to resist ZTDO-induced paralysis in the L4 larvae (Figure 5–figure supplement 2). However, misexpression of Ppa-OBI-1 in the C. elegans amphid sheath cells did not result in changes in chemotaxis response to ZTDO. We attempted but were not able to train a researcher to use a cell ablation setup in a facility nearby to definitively answer if amphid cells mediate ZTDO sensitivity in P. pacificus. We have thus tempered our speculation that Ppa-OBI-1is required only in the amphid sheath cells in P. pacificus. However, given the growing evidence by the Shaham lab at the Rockefeller University that glial cells are important for amphid neuron ontogeny and function in C. elegans, and that secreted proteins in the sheath cell have not been actively studied, we feel obliged to discuss several models for how Ppa-OBI-1 proteins may function in the amphid sheath cells.

2) RNAi of C06G1.1 to resolve its role in C. elegans.

We have performed RNAi against C06G1.1 in C. elegans using two fragments of the coding sequence and measured their body dimensions (Figure 4–figure supplement 4). RNAi resulted in longer and slenderer body, consistent with the reported RNAi phenotype in www.wormbase.org based on results published by Simmer et al. 2003.

3) Please include all data that support the claim that tu404 and tu405 are allelic.

We have made a typographical error and meant to indicate that Ppa-obi-1(tu404) and Ppa-obi-3(tu405) are not allelic because they do complement each other in the F1 cross. This passage was corrected in the main text.

4) Strengthen or remove the feeding rescue experiment, which appears weak.

We have removed the feeding rescue experiment due to the weak effects.

5) Strengthen or more explicitly discuss the caveat of the difference in gene expression of Cel-obi-1.

We acknowledge that small changes in amino acid sequence can have profound effects on protein function, so we have removed the statement that suggests changes in gene expression pattern is more likely than changes in protein function to contribute to the differences in ZTDO sensitivity between C. elegans and P. pacificus. The statement now reads “While small changes in sequence can lead to large changes in protein function, the absence of expression of the C. elegans obi-1 ortholog in amphid sheath cells suggests changes in gene expression pattern may also play a role in determining ZTDO sensitivity.” We also mentioned other possible tissues types that can mediate ZTDO sensing.

Reviewer #1:

1) Can the authors specify what percentage of arrested Dauer Larva are paralyzed rather than some and majority? Is the developmental block specific for dauer exit or do they not exit the dauer stage because of paralysis?

Very few wild-type DL became paralyzed or died on plates with 0-0.001% ZTDO. Approximately 40% wild-type DL remained active after two days on food with 0.01% ZTDO (Figure. 2G). However for the Ppa-obi-1 mutant, the dauer-exit defect is exacerbated by the hypersensitivity to ZTDO, in which 79±17% of all two-day old dauers became paralyzed and died on plates without ZTDO, and 94.6±5% DL became paralyzed on 0.001% ZTDO. Therefore, 0.01% ZTDO can block active wild-type P. pacificus DL from progressing to the reproductive stage without causing paralysis, but 0.001-0.01% ZTDO significantly reduced dauer exit in Ppa-obi-1 DL primary by inducing paralysis and death.

We have explained this finding more explicitly in the main text.

2) I did not completely follow the rationale for the egl-4 experiment is not entirely clear. Is the role of egl-4 in chemosensation and body size important? Also reference Etoille 2002 should that be Daniels 2000?

We have added the reference to Daniels 2000 regarding egl-4’s role in dauer development. Daniels 2000 showed that C. elegans egl-4 loss-of-function allele n479 is daf-c (dauer constitutive), whereas we detected a weak but statistically significant dauer exit phenotype in the P. pacificus egl-4(tu374) allele (Figure 2G).

3) No egg laying data are presented so show the data or add “(data not shown)”

We have added the egg-laying data for Ppa-obi-1(tu404) as Table 1.

4) How do you know they are in equal abundance? RT-PCR? qPCR? “(data not shown)”.

We subcloned Ppa-obi-1 cDNA from multiple RT-PCR products using primers from the first and last exons and sequenced at least 6 clones from both strains. We found 3 inserts of each short and long splice variants.

5) Since splice form variation does not contribute to the difference in insect pheromone attraction, not sure why this is “Interestingly”.

We have reworded this passage to “In the two Ppa-obi-1 splice forms, the longer transcript contains a VKDDDPR peptide that may be the result of an alternative splice donor site. Both splice variants were isolated from the California and Washington strains in equal abundance by subcloning RT-PCR products (data not shown) and no differences in nucleotide sequence were found in the coding region of these two strains.”

6) Why are the data in Figure 4C different from those in Figure 1?

The results for Ppa-obi-1(tu404)’s neutral response to ZTDO in Figure 1 were obtained in Germany 6 years ago, while Ppa-obi-1’s avoidance response in Figure 4C was obtained in the PI’s new lab at California State University Northridge. This more recent result is highly repeatable and was also confirmed by testing animals from a frozen Ppa-obi-1 strain. The ZTDO chemical supplier remained the same. Thus, it is possible that unlike wild-type PS312 (whose response to ZTDO remained unchanged), Ppa-obi-1 is also responding to differences in other chemical compositions (e.g. water or media) in the current lab environment. All chemoattraction assays were performed with concurrent controls and indicated successful transgenic rescue of Ppa-obi-1(tu404).

Reviewer #2:

Two alleles of obi-1 – tu404 and tu405 – were isolated by screens for mutants defective in beetle pheromone sensing. The authors refer to data from complementation testing but do not show these data. Please include all data that support the claim the tu404 and tu405 are allelic. Also, the authors only report the molecular characterization of tu404. Is there no coding mutation in the tu405 strain? Does the transgene that rescues the tu404 mutant phenotype also rescue the tu405 mutant phenotype? In its current form the data in the manuscript leave open the possibility that tu404 and tu405 are not allelic but display some interesting interactions.

The typographical error was corrected as mentioned previously. Ppa-obi-1(tu404) and Ppa-obi-3(tu405) are not allelic because they do complement each other in ZTDO attraction. Ppa-obi-3(tu405) also does not share the body proportion and ZTDO hypersensitivity phenotypes of Ppa-obi-1(tu404).

Reviewer #3:

1) One important point made by the authors is the difference in expression between Ppa and Cel. However, the expression pattern in C. elegans is based solely on a transgenic reporter assay. Since this is a key point, I think smFISH of Cel-C06G1.1 (Cel-obi-1) is necessary to establish a convincing negative expression result.

We agree that transcriptional reporters based on a single promoter fragment may not comprehensively represent endogenous mRNA expression patterns. Due to the technical expertise needed to perform the suggested smFISH experiments for C06G1.1, we hope to address this caveat in our future research.

2) I am confused and concerned by the feeding rescue experiment. It just seems weird to me. I suppose it could be acting in the intestine. This result needs shoring up with something like localized expression of obi-1 in gut, nervous system, etc.

We have removed the feeding rescue experiment.