DOI: 10.5061/dryad.41ns1rnrg
title: Suppression of cytoplasmic incompatibility in the leaf-mining fly Liriomyza sativae with a nuclear Wolbachia insert

description: Cytoplasmic incompatibility (CI) drives maternally transmitted
 endosymbionts such as Wolbachia through insect populations by inducing
 embryonic mortality when infected males fertilize uninfected females. CI
 is controlled by Wolbachia cif operons that are categorized into multiple
 phylogenetic Types. CI strength is further shaped by poorly understood
 host factors, including development and genetic background. To study the
 strength of CI across different host species, we genotyped a Japanese
 field population of Liriomyza sativae. By uncovering paternal transmission
 of Wolbachia genic elements, we collected strong evidence of horizontal
 genome transfer, including Type I and Type V cif operons, from Wolbachia
 into the nuclear genome of L. sativae. We established a transinfection of
 wLtri in L. sativae, a Wolbachia variant that induces strong CI in
 Liriomyza trifolii. No CI was observed in both intraspecific and
 interspecific reciprocal crosses with L. trifolii, suggesting that both
 uninfected females and infected males of L. sativae completely suppress
 wLtri-mediated CI. Our results raise the appealing hypothesis that host
 suppression of Wolbachia-induced CI might evolve due to horizontal
 transfer of cif operons into the host nuclear genome.
description: crossing_experiment_results.csv: The dataset
 was collected through controlled crossing experiments involving four
 different strains of <em>Liriomyza trifolii</em> and
 <em>Liriomyza sativae</em> with varying <em>Wolbachia
 </em>infection statuses. Below is a breakdown of the data collection
 and processing methods: <strong>1. Experimental
 Design & Data Collection</strong><br>Strains
 Used:<br><em>L. trifolii</em>
 (<em>Wolbachia</em>-infected and antibiotic-treated uninfected
 strains)<br><em>L. sativae</em>
 (<em>Wolbachia</em>-transinfected and uninfected
 strains) Crossing Design:<br>A full factorial
 crossing experiment was performed, yielding 16 different
 combinations.<br>Individual Selection &
 Handling:<br>Only unmated individuals were used, ensuring
 standardization of reproductive conditions.<br><em>L.
 trifolii</em> uninfected individuals were used from the third
 generation after antibiotic treatment to avoid direct antibiotic
 effects.<br><em>L. sativae</em> infected individuals
 were used from the third generation after <em>Wolbachia
 </em>transinfection via microinjection.<br>Mating
 Conditions:<br>1 female + 2 males per test tube (7.5 cm × 1.0 cm;
 Thermo Fisher Scientific).<br>Individuals were placed in tubes for
 24 hours with 50% honey in water as food.<br>Egg Collection
 & Rearing:<br>Quail bean leaves were used as oviposition
 substrates.<br>Eggs were counted after 24 hours and then transferred
 to a conical flask with water.<br>After hatching, larvae were moved
 to square acrylic cases (9.5 cm × 6.5 cm × 6.5 cm) with mesh for further
 development.<br>Counts recorded: Number of eggs, hatched larvae,
 pupae, and emerging adults.<br>Adults were sexed and counted upon
 emergence.
 <strong>2. <em>Wolbachia</em> Infection
 Confirmation</strong><br>Diagnostic PCR using wsp primers was
 performed on:<br>Parental generation (P) individuals before
 crossing.<br>Offspring (F1) to confirm vertical transmission of
 <em>Wolbachia</em>. <strong>3.
 Environmental Conditions</strong><br>Controlled in a 16L:8D
 photoperiod at 25°C to standardize environmental effects.
 <strong>4. Data Processing & Statistical
 Analysis</strong><br>Measured Parameters:
 Number of eggs laid per female.<br>Hatching rate
 (larvae/eggs).<br>Emergence rate (adults/larvae).<br>Sex ratio
 (female/male proportion in F1).<br><em>Wolbachia
 </em>infection rate in offspring.
 Liriomyza_DNA_sequences.csv,
 Liriomyza_amino_acid_sequences.csv: This dataset
 contains nucleotide sequence data related
 to <em>Wolbachia</em> genes, including
 <em>fbpA</em><strong>,
 </strong><em>cifA</em><strong>,
 </strong><em>cifB</em><strong>,
 </strong><em>clpA</em><strong>,</strong> and
 <em>pyrD</em>, from <em>Liriomyza trifolii</em>,
 <em>Liriomyza sativae</em>. The dataset also includes
 predicted amino acid sequences. These data were generated as part of a
 study investigating <em>Wolbachia</em> horizontal gene
 transfer and cytoplasmic incompatibility mechanisms in leaf miner
 flies. The sequence data were obtained from PCR amplification of the
 target genes followed by Sanger sequencing. Methods:
 How was this dataset collected?<br>Primer Design & PCR
 Amplification Primers for fbpA, cifA, cifB, clpA, and
 pyrD were designed using Primer3 Plus, based on a previously published
 fragmented wLtri genome assembly.<br>PCR reaction mixtures (50 µL)
 contained:<br>KOD One® PCR Master Mix -Blue- (Takara) (25
 µL)<br>Distilled water (21 µL)<br>Primers (1.5 µL each,
 forward & reverse)<br>DNA extract (1.0 µL)<br>PCR
 conditions:<br>Initial denaturation: 98°C for 2 min<br>30
 cycles of:<br>98°C for 10 sec<br>53°C for 5 sec<br>68°C
 for 3 sec<br>Final extension: 68°C for 10 min<br>PCR products
 were verified via 1% agarose gel electrophoresis.<br>Sequencing
 & Processing PCR products were submitted to
 Fasmac, Inc. (Kanagawa, Japan) for Sanger sequencing.<br>Raw
 sequence data were quality-checked and trimmed. The ExPASy Translate Tool
 was used to predict amino acid sequences from nucleotide
 sequences.
description: # Suppression of cytoplasmic incompatibility in the leaf-mining fly
 *Liriomyza sativae* with a nuclear *Wolbachia* insert
 [https://doi.org/10.5061/dryad.41ns1rnrg](https://doi.org/10.5061/dryad.41ns1rnrg) ## Description of the data and file structure This dataset contains reproductive and *Wolbachia* transmission data from crossing experiments between infected and uninfected strains of *Liriomyza trifolii* and *Liriomyza sativae*. It includes egg counts, hatching rates, emergence rates, sex ratios, and *Wolbachia* infection rates. Additionally, the dataset comprises nucleotide sequences of *fbpA*, *cifA*, *cifB*, *clpA*, and *pyrD*, obtained through PCR amplification and Sanger sequencing. These data were collected to investigate the genetic composition and functional characteristics of *Wolbachia* genes, with a focus on horizontal gene transfer and cytoplasmic incompatibility mechanisms. This dataset provides insights into *Wolbachia* -host interactions and their evolutionary implications. ### Files and variables #### File: crossing\_experiment\_results.csv **Description:**  ##### Variables * Cross_ID: Unique identifier for each cross. Lt means *L. trifolii*, Ls means *L. sativae*. "+" and "-" indicate the *Wolbachia* infection status of Host ("+": Infected, "-": Uninfected). * Egg_Count: Number of eggs laid * Hatch_Count: Number of larvae hatched * Pupae_Count: Number of pupae formed * Adult_Count: Number of emerged adults * Female_Count: Number of female adults * Male_Count: Number of male adults * *Wolbachia*_Confirmed_F1: Number of *Wolbachia* infected F1 generation.  #### File: Liriomyza\_amino\_acid\_sequences.fasta **Description:** Translated amino acid sequences of the genes. #### File: Liriomyza\_DNA\_sequences.fasta **Description:** Nucleotide sequences of the *Wolbachia* genes from *Liriomyza sativae* (uninfected strain) and *Wolbachia* endosymbiont of *Liriomyza trifolii*. This dataset includes the following sequence files in FASTA format, each containing nucleotide sequences of Wolbachia genes from different host strains and experimental conditions. **Multi Fasta File Structure** Each sequence follows the standard FASTA format, where: The header line starts with "&gt;" and contains: * The species or strain name * The gene name * The GenBank accession number **Variables and Abbreviations** The dataset uses the following definitions: *Wolbachia* endosymbiont of *Liriomyza trifolii*: The *Wolbachia* strain from a *Liriomyza trifolii* Units of measurement: Nucleotide sequences are represented in 5' to 3' direction using standard IUPAC nucleotide codes. Amino acid sequences follow one-letter amino acid codes. ## Code/software #### File: crossing\_experiment\_results.csv Methods for Data Collection * Crossing experiments were conducted using unmated individuals of the *Liriomyza* species. - One female and two males were placed in test tubes for 24 hours, after which egg counts were recorded. * The eggs were monitored for hatching, and emerging adults were sexed and tested for *Wolbachia* infection using PCR. Data Processing * The dataset represents raw data collected from the experiments. * Statistical analyses were conducted using R (version 4.2.1). * *Wolbachia* infection was confirmed using the wsp gene PCR. #### File: Liriomyza\_amino\_acid\_sequences.fasta #### File: Liriomyza\_DNA\_sequences.fasta Software Required to View and Analyze Data: The dataset consists of FASTA files, which can be viewed and analyzed using widely available free or open-source software. **1. Viewing and Editing FASTA Files** FASTA format sequences can be opened and analyzed using: * BioEdit (Version 7.2.5 or later) – A free sequence alignment editor for Windows. * MEGA (Version 11 or later) – Used for sequence alignment and phylogenetic analysis. * AliView (Version 1.28 or later) – A lightweight alignment viewer. * Any standard text editor (e.g., Notepad++, VS Code, or nano). Sequence Analysis and Alignment * ClustalW (Version 2.1) – Used for multiple sequence alignment. * ExPASy Translate Tool (ExPASy Server) – Used for translating nucleotide sequences into amino acid sequences. Statistical and Bioinformatics Analysis * R (Version 4.2.1 or later) – Used for data analysis, visualization, and statistical tests. * Relevant R packages: tidyverse (data handling), ggplot2 (visualization) **2. Primer Design** * Primer3 Plus (Online tool) – Used to design primers for target genes. **3. PCR and Sequencing** * Sequences were obtained using Sanger sequencing at Fasmac, Inc. * Raw sequence data were checked and trimmed. * ExPASy Translate Tool was used for protein sequence prediction. ## Access information #### File: crossing\_experiment\_results.csv Other publicly accessible locations of the data: * This dataset is exclusively available on Dryad and has not been deposited in any other public repository. Data was derived from the following sources: * All data in this dataset were collected and generated as part of this study. Other publicly accessible locations of the data:  * GenBank: The accession number for accessing each sequence is listed at the end of the header line for each sequence. Data was derived from the following sources: * This dataset was generated as part of this study. However, the *Wolbachia* genome sequences used for primer design were obtained from: Pramono, A. K., Hidayanti, A. K., Tagami, Y., & Ando, H. (2024). Bacterial community and genome analysis of cytoplasmic incompatibility-inducing *Wolbachia* in American serpentine leafminer, *Liriomyza trifolii*. *Frontiers in Microbiology*, *15*, 1304401. DOI: [https://doi.org/10.3389/fmicb.2024.1304401](https://doi.org/10.3389/fmicb.2024.1304401)