A suite of selective pressures supports the maintenance of alleles of a Drosophila immune peptide

Reviewer #1 (Public Review):

Summary:
In this manuscript, Unckless and colleagues address the issue of the maintenance of genetic diversity of the gene diptericin A, which encodes an antimicrobial peptide in the model organism Drosophila melanogaster.

Strengths:
The data indicate that flies homozygous for the dptA S69 allele are better protected against some bacteria. By contrast, male flies homozygous for the R69 allele better resist starvation than flies homozygous for the S69 allele.

Weaknesses:
-I am surprised by the inconsistency between the data presented in Fig. 1A and Fig. S2A for the survival of male flies after infection with P. rettgeri. I am not convinced that the data presented support the claim that females have lower survival rates than males when infected with P. rettgeri (lines 176-182).

-The data in Fig. 2 do not seem to support the claim that female flies with either the dptA S69 or the R69 alleles have a longer lifespan than males (lines 211-215). A comment on the [delta] dpt line, which is one of the CRISPR edited lines, would be welcome.

-The data in Fig. 2B show that male flies with the dptA S69 or R69 alleles have the same lifespan when poly-associated with L. plantarum and A. tropicalis, which contradicts the claim of the authors (lines 256-260).

Reviewer #2 (Public Review):

Summary: In this study, the authors delve into the mechanisms responsible for the maintenance of two diptericin alleles within Drosophila populations. Diptericin is a significant antimicrobial peptide that plays a dual role in fly defense against systemic bacterial infections and in shaping the gut bacterial community, contributing to gut homeostasis.

Strengths: The study unquestionably demonstrates the distinct functions of these two diptericin alleles in responding to systemic infections caused by specific bacteria and in regulating gut homeostasis and fly physiology. Notably, these effects vary between male and female flies.

Weaknesses: Although the findings are highly intriguing and shed light on crucial mechanisms contributing to the preservation of both diptericin alleles in fly populations, a more comprehensive investigation is warranted to dissect the selection mechanisms at play, particularly concerning diptericin's roles in systemic infection and gut homeostasis. Unfortunately, the results from the association study conducted on wild-caught flies lack conclusive evidence.

Major Concerns:

Lines 120-134: The second hypothesis is not adequately defined or articulated. Please revise it to provide more clarity. Additionally, it should be explicitly stated that the first part of the first hypothesis (pathogen specificity), i.e., the superior survival of the S allele in Providencia infections compared to the R allele, has been previously investigated and supported by the results in the Unkless et al. 2016 paper. The current study aims to additionally investigate the opposite scenario: whether the R allele exhibits better survival in a different infection. Please consider revising to emphasize this point.

Figures and statistical analyses: It is essential to present the results of significant differences from the statistical analyses within Figures 1B, 2B, and 3. Additionally, please include detailed descriptions of the statistical analysis methods in the figure legends. Specify whether the error bars represent standard error or standard deviation, particularly in Figure 3, where assays were conducted with as few as 3 flies.

Lines 317-318 (as well as 320-328): The data related to P. rettgeri appear somewhat incomplete, and the authors acknowledge that bacterial load varies significantly, and this bacterium establishes poorly in the gut. These data may introduce more noise than clarity to the study. Please consider revising these sections by either providing more data, refining the presentation, or possibly removing them altogether.

Lines 335-387 and Figure 4: Although these results are intriguing and suggest interactions between functional diptericin and fly physiology, some mediated by the gut microbiome, they remain descriptive and do not significantly contribute to our understanding of the mechanism that maintains the diptericin alleles.

Lines 399-400: The contrast between this result and statement and the highly reproducible data presented in Figures 2-4 should be discussed.

Lines 422-429 and Figure 5D: The conclusion regarding an association between diptericin alleles and Morganellaceae bacteria is not clearly supported by Figure 5D and lacks statistical evidence.

Reviewer #3 (Public Review):

Summary:
This paper investigates the evolutionary aspects around a single amino acid polymorphism in an immune peptide (the antimicrobial peptide Diptericin A) of Drosophila melanogaster. This polymorphism was shown in an earlier population genetic study to be under long-term balancing selection. Using flies with different AA at this immune peptide it was found that one allelic form provides better survival of systemic infections by a bacterial pathogen, but that the alternative allele provides its carriers a longer lifespan under certain conditions (depending on the microbiota). It is suggested that these contrasting fitness effects of the two alleles contribute to balance their long-term evolutionary fate.

Strengths:
The approach taken and the results presented are interesting and show the way forward for studying such polymorphisms experimentally.

Weaknesses:
1. A clear demonstration (in one experiment) that the antagonistic effect of the two selection pressures isolated is not provided.

The study is overwhelming with many experiments and countless statistical tests. The overall conclusion of the many experiments and tests suggests that "dptS69 flies survive systemic infection better, while dptS69R flies survive some opportunistic gut infections better." (line 444-446). Given the number of results, different experiments, and hundreds of tests conducted, how can we make sure that the result is not just one of many possible combinations? I suggest experimentally testing this conclusion in one experiment (one may call this the "killer-experiment") with the relevant treatments being conducted at the same time, side by side, and the appropriate statistical test being conducted by a statistical test for a treatment x genotype interaction effect.

2. The implication that the two forms of selection acting on the immune peptide are maintained by balancing selection is not supported.

The picture presented about how balancing selection is working is rather simplistic and not convincing. In particular, it is not distinguished between fluctuating selection (FL) and balancing selection (BL). BL is the result of negative frequency-dependent selection. It may act within populations (e.g. Red Queen type processes, mating types) or between populations (local adaptation). FL is a process that is sometimes suggested to produce BL, but this is only the case when selection is negative frequency dependent. In most cases, FL does not lead to BL.

The presented study is introduced with a framework of BL, but the aspects investigated are all better described as FL (as the title says: "A suite of selective pressures ..."). The two models presented in the introduction (lines 62 to 69; two pathogens, cost of resistance) are both examples for FL, not for BL.

Finally, no evidence is presented that the different selection pressures suggested to select on the different allelic forms of the immune peptide are acting to produce a pattern of negative frequency dependence.