Distinct protocerebral neuropils associated with attractive and aversive female-produced odorants in the male moth brain

The pheromone system of heliothine moths is an optimal model for studying principles underlying higher-order olfactory processing. In Helicoverpa armigera, three male-specific glomeruli receive input about three female-produced signals, the primary pheromone component, serving as an attractant, and two minor constituents, serving a dual function, that is, attraction versus inhibition of attraction. From the antennal-lobe glomeruli, the information is conveyed to higher olfactory centers, including the lateral protocerebrum, via three main paths – of which the medial tract is the most prominent. In this study, we traced physiologically identified medial-tract projection neurons from each of the three male-specific glomeruli with the aim of mapping their terminal branches in the lateral protocerebrum. Our data suggest that the neurons’ widespread projections are organized according to behavioral significance, including a spatial separation of signals representing attraction versus inhibition – however, with a unique capacity of switching behavioral consequence based on the amount of the minor components.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: In this study, no power analysis nor specific sample sizes justification was performed. With regards to the calcium imaging data, the sample size of eight was designed, as similar sample sizes (n: 6 to 9) has previously proven sufficient to demonstrate general response tendencies across the glomerular units (Sachse & Galizia, 2002;Wu, Xu, Hou, Huang, Dong, & Wang, 2015). As for sharp intracellular recordings, sample sizes are completely dependent on time and success-rate restrictions. We gathered as much data as possible within a three-year window.
eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014 2 -We collected data whenever the insect materials were available.
-With respect to the intracellular recordings, each stimulus was presented in multiple trials, and in several distinct animals. As such, both technical and biological replication was considered, respectively. Details see Method and material. Comparisons across trials for the low-concentration protocol is available in the supplementary materials, while between subject variation is addressed in the main results. For the calcium imaging experiments, an example of across-trial variation is presented in Fig. 1C, while between-subjects variability was considered in Fig. 1D.
-Examples of all reported data is presented in the main article, while more detailed summaries and individual neuron data can be found in the supplementary materials.
-None of the odor responses were considered to be outliers. For the calcium imaging data, the staining procedure was previously described (e.g. Ian, Kirkerud, Galizia and Berg,2017; Chu, Heinze, Ian, & Berg, 2020), and the post scanning confirmed that the responses was sampled from the target neuron population. Intracellularly recorded responses would not be appropriate to exclude, as the electrophysiological data was evidently linked with specific labelled morphologies.
-For the intracellular data, we solely included medial-and mediolateral-tract projection neurons arborizing exclusively/primarily in the MGC. All labelled neurons that did not display such characteristics were excluded, irrespective of physiological responses to odor stimulation.
-No sequence data were utilized in this study. 3

Statistical reporting
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Group allocation
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Not relevant.
eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014