DOI: 10.5061/dryad.66t1g1kcp
title: Defensive fungal symbiosis on insect hindlegs

description: Tympanal organs, as “insect ears,” have evolved repeatedly. Dinidorid
 stinkbugs were reported to possess a conspicuous tympanal organ on
 females’ hind legs. Here we report an unexpected discovery that the
 stinkbug’s “tympanal organ” is actually an external symbiotic organ. The
 organ’s surface is not membranous but a porous cuticle, where each pore
 connects to glandular secretory cells. In reproductive females, the hind
 leg organ is covered with fungal hyphae growing out of the pores. Upon
 oviposition, the females skillfully transfer the fungi from the organ to
 the eggs. The eggs are covered with fungal hyphae and physically protected
 against wasp parasitism. The fungi are mostly benign Cordycipitaceae fungi
 and show considerable diversity among insect individuals and populations,
 indicating environmental acquisition of the specific fungal associates.
description: # Defensive fungal symbiosis on insect hindlegs Dataset DOI:
 [10.5061/dryad.66t1g1kcp](10.5061/dryad.66t1g1kcp) ## Description of the
 data and file structure The results of SEM observations of the insect
 materials, amplicon sequence analyses of fungal microbiota of the stink
 bugs, RNAseq analyses of the insect hindlegs, the insect morphometries,
 phylogenetic analyses of the insect mitochondria. ### Files and variables
 #### File: Data_For_Defensive_fungal_symbiosis_on_insect_hindlegs.zip
 **Description:**  The ZIP-compressed file contains folders corresponding
 to the figures in the manuscript. Each folder includes the digital images
 and/or source data files containing the raw data used to prepare the
 figures. **Folder: Fig1** SEM images and their metadata for Fig. 1. *
 **Mg_Ad_Mal_Hind_R20200311.000004.tif** – SEM image of the hind tibia of a
 mature adult male *M. gracilicorne*. *
 **Mg_Ad_Mal_Hind_R20200311.000004.txt** – Metadata of equipment settings
 when capturing the above image. * **Mg_Ad_Mal_Hind_R20200311.000007.tif**
 – Magnified SEM image of the hind tibia of a mature adult male *M.
 gracilicorne*. * **Mg_Ad_Mal_Hind_R20200311.000007.txt** – Metadata of
 equipment settings when capturing the above image. *
 **Mg_Te_Fem_Hind_R20151026.000185.tif** – SEM image of the hind tibia of
 an immature adult female *M. gracilicorne*. *
 **Mg_Te_Fem_Hind_R20151026.000185.txt** – Metadata of equipment settings
 when capturing the above image. * **Mg_Te_Fem_Hind_R20151026.000187.tif**
 – Magnified SEM image of the hind tibia of an immature adult female *M.
 gracilicorne*. * **Mg_Te_Fem_Hind_R20151026.000187.txt** – Metadata of
 equipment settings when capturing the above image. *
 **Mg_Ad_Fem_Hind_R20200311.000001.tif** – SEM image of the hind tibia of a
 mature adult female *M. gracilicorne*. *
 **Mg_Ad_Fem_Hind_R20200311.000001.txt** – Metadata of equipment settings
 when capturing the above image. * **Mg_Ad_Fem_Hind_R20200311.000004.tif**
 – Magnified SEM image of the hind tibia of a mature adult female *M.
 gracilicorne*. * **Mg_Ad_Fem_Hind_R20200311.000004.txt** – Metadata of
 equipment settings when capturing the above image. **Folder: Fig2** ITS
 amplicon seq data for Fig. 2 and Fig. S3. * **Fig2abcd_S3abcdef.tsv** –
 Number of reads for each OTU in the *M. gracilicorne* ITS amplicon
 experiment. * **First column (id):** OTU IDs. * **Second to 76th
 columns:** Sample names (headers); rows contain the number of reads for
 each OTU ID. * **Last column (taxon):** Taxon assignments for each OTU ID
 **Folder: Fig4** Data of suppression of wasp parasitism by egg-covering
 fungal hyphae for Fig. 4. * **Fig4e_parasitized rate in wild.csv** -
 Parasitism rates of eggs in wild. * **egg no:** Egg ID. * **egg mass
 group:** Egg mass ID. * **parasitized:** Egg parasitized (1) or not (0). *
 **fertilized:** Egg fertilized (1) or not (0). * **disappeared:** Egg
 disappeared (1) or not (0). * **unfertilized:** Egg unfertilized (1) or
 not (0). - **Fig4f_parasitism success rate.csv** - Parasitism success
 rates of fungus-covered or fungus-removed eggs. * **egg no:** Egg ID. *
 **egg mass group:** Egg mass ID. * **parasitized:** Egg parasitized (1) or
 not (0). * **fertilized:** Egg fertilized (1) or not (0). *
 **unfertilized**: Egg unfertilized (1) or not (0). * **treatment:**
 "Fungus-covered eggs" or \ "Fungus-removed eggs". -
 **Fig4g_oviposition trials.csv** - Oviposition trials by parasitic wasps
 for fungus-covered or fungus-supressed eggs. * **oviposition**: Yes (1) or
 no (0). * **abandon**: Yes (1) or no (0). * **treatment**:
 "Fungus-covered eggs" or "Fungus-supressed eggs". -
 **Fig4h_parasitism success rate.csv** - Parasitism success rate
 of fungus-covered or fungus-supressed eggs. * **parasitized eggs**: Egg
 parasitized (1) or not (0). * **hatched eggs**: Egg hatched (1) or not
 (0). * **unhatched eggs**: Egg unhatched (1) or not (0). * **treatment**: 
 "Fungus-covered eggs" or "Fungus-supressed eggs". -
 **Fig4i_wasp survival.csv** - Survival of T. brevinotaulus experimentally
 forced to contact with conidia of the hindleg-derived fungi. *
 **survival**: Wasp survival days * **treatment**: "Untreated
 wasps" or "Fungus-treated wasps". **Folder: FigS03** Number
 of isolates for each OTU in the *M. gracinicorne* fungal isolate
 experiment for Fig. S3. * **FigS3abcdef.tsv** - Number of isolates for
 each OTU in the *M. gracinicorne* fungal isolate experiment. * **First
 column (OTU)**: OTU IDs. * **Second column (id)**: Corresponding IDs of
 Amplicon-seq OTUs. * **Third to last columns:** Sample names
 (headers). rows contain the number of isolates. **Folder: FigS04** Read
 distribution assigned to each taxonomic class in the fungal isolate and
 Amplicon-seq experiment for Fig. S4 * **FigS4.tsv** - Read distribution
 assigned to each taxonomic class in the fungal isolate and Amplicon-seq
 experiment. * **Phylum and Class columns:** Taxonomic names of each rank.
 * **TotalIsolate and %:** Number and percentage of isolates in each
 taxonomic group. * **TotalRead and %**: Number and percentage of reads in
 each taxonomic group. **Folder: FigS05** Multiple alignment of ITS
 sequences for Fig. S5. * **FigS5.fasta** - aligned sequences used for the
 phylogenetic analysis in Fig S5. **Folder: FigS06** TPM expression levels
 from the RNAseq experments for Fig. S6. * **FigS6ab.tsv** - TPM expression
 levels from the RNAseq experments. **Folder: FigS07** SEM images and their
 metadata for Fig. S7. * **Mg_egg_0day20160309.000231.tif** – SEM image of
 egg surface at Day 0 (immediately post-oviposition). *
 **Mg_egg_0day20160309.000231.txt** – Metadata of equipment settings when
 capturing the above image. * **Mg_egg_0day20160309.000236.tif** –
 Magnified SEM image of egg surface at Day 0. *
 **Mg_egg_0day20160309.000236.txt** – Metadata of equipment settings when
 capturing the above image. * **Mg_egg_7day20180607.000007.tif** – SEM
 image of egg surface at Day 7 (7 days post-oviposition). *
 **Mg_egg_7day20180607.000007.txt** – Metadata of equipment settings when
 capturing the above image. * **Mg_egg_7day20180607.000011.tif** –
 Magnified SEM image of egg surface at Day 7. *
 **Mg_egg_7day20180607.000011.txt** – Metadata of equipment settings when
 capturing the above image. **Folder: FigS08** SEM images and their
 metadata for Fig. S8. * **Mg_egg_ddw20180122.000001.tif** – SEM image of
 egg surface after extraction with distilled water. *
 **Mg_egg_ddw20180122.000001.txt** – Metadata of equipment settings when
 capturing the above image. * **Mg_egg_MerUre20180406.000008.tif** – SEM
 image of egg surface after extraction with 8 M urea + 1%
 2-mercaptoethanol. * **Mg_egg_MerUre20180406.000008.txt** – Metadata of
 equipment settings when capturing the above image. *
 **Mg_egg_SamBuf20180420.000002.tif** – SEM image of egg surface after
 extraction with 10% SDS + 1% 2-mercaptoethanol. *
 **Mg_egg_SamBuf20180420.000002.txt** – Metadata of equipment settings when
 capturing the above image. **Folder: FigS09** Survaival data of *M.
 gracilicorne* and *P. stali* populations after inoculation for Fig S9. *
 **survival_rate.csv**: - Counts of surviving *M. gracilicorne* and *P.
 stali* populations at each days post-inoculation, used for constructing
 survival curves. **Folder: FigS10** Fungal growth level on eggs for Fig.
 S10. * **FigS10h_fungal growth on eggs.csv** - Fungal growth level on eggs
 under each treatment. * **level**: Fungal growth level; 3 (+++), 2 (++), 1
 (+). * **treatment**: Female treatments; "No treatment",
 "Midlegs cut", or "Hindlegs cut". **Folder:
 FigS11-12** Field experiment data on the effects of fungal cover on
 parasitization susceptibility for Figs S11 and S12. *
 **FigS11j_S12abcdef.tsv** - Original data from field experiments that
 examined the effects of fungal cover on parasitization susceptibility. *
 **Oviposition**: Date of oviposition. * **Set**: Date of field settlement.
 * **Retrieve**: Date of retrieval of the sample from the field * **Site**:
 Field site IDs * **Sheet**: Sheet IDs * **Position:** Position IDs on the
 sheet * **Treat**: N indicates intact, while R indicates fungal removal *
 **Retrieved**: Retrieved (1) or lost (0) * **Egg_N**: Total number of eggs
 in a mass * **Predated**: Number of predated eggs * **Hatch**: Number of
 hatched eggs * **Embryonic**: Number of dead eggs with a developing embryo
 * **Non-embryonic**: Number of dead eggs without a visible embryo *
 **Ooencyrtus**: Number of eggs parasitized by Ooencyrtus wasp *
 **Trissolcus**: Number of eggs parasitized by Trissolcus wasp *
 **Unknown**: Number of eggs parasiteized by wasps that could not be
 identified due to developmental failure **Folder: FigS13** Morphometric
 data of diverse dinidorid stinkbugs. * **FigS13ef.tsv** - prothorax
 widths, hind tibia widths and associated metadata. * **Sampler**: Name of
 samplers * **ID1 and ID2**: insect identifiers * **Species**: Cc
 = *Coridius chinensis,* Cp = *Cyclopelta parva,* Mb =
 *Megymenumbrevicorne,*  Mg = *M. gracilicorne,* MgTW = *M.
 gracilicorne* (Taiwan), Ep = *Eumenotes pacao* * **Sex**: Sex of
 individuals * **TW**: Prothorax widths (mm) * **HLW**: Hindleg tibia
 widths (mm) **Folder: FigS14** Multiple alignment and partition
 information for Fig. S14. *  **figS14_alignment.phy** - PHYLIP-formatted
 amino acid sequence alignment used for phylogenetic inference. The file
 was generated by concatenating alignments of all 13 mitochondrial
 protein-coding genes. Sequence names are 8 or 9 characters, each of which
 corresponds to the accession number shown in the brackets next to the OTU
 name of Fig. S14. - **figS14_part.nex** - NEXUS-formatted partition file
 for IQ-TREE analysis. The file shows gene alignment regions, partition
 assignments, and evolutionary models. **Folder: FigS15** ITS-amplicon data
 of various Dinidorinid stinkbugs for Fig. S15. * **FigS15abcd.tsv** -
 Number of reads for each OTU in the ITS-Amplicon experiment. * **First
 column (id)**: OTU IDs * **Second to 58th columns**: Sample names
 (headers); rows contains number of reads for each OTU ID * **The last
 column (taxon):**  taxon assignments  ## Code/software * For text files,
 conventional text editors, spreadsheet softwares. - For SEM images,
 conventional imaging softwares. ## Access information Other publicly
 accessible locations of the data: * Nucleotide sequence data obtained in
 this study were deposited in the DNA Data Bank of Japan (DDBJ) with the
 following accession numbers: fungal ITS sequence data, LC792825 –LC793396;
 Fungal ITS amplicon sequencing data, DRX509842 – DRX509916 and
 DRX637795-DRX637852; RNA sequencing data, DRX509810 – DRX509841; insect
 mitochondrial genome data, LC670767 and PV423521-PV423524.