Suppression of cytoplasmic incompatibility in the leaf-mining fly Liriomyza sativae with a nuclear Wolbachia insert

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.

Methods
crossing_experiment_results.csv:
The dataset was collected through controlled crossing experiments involving four different strains of Liriomyza trifolii and Liriomyza sativae with varying Wolbachia infection statuses. Below is a breakdown of the data collection and processing methods:

1. Experimental Design & Data Collection
Strains Used:
L. trifolii (Wolbachia-infected and antibiotic-treated uninfected strains)
L. sativae (Wolbachia-transinfected and uninfected strains)

Crossing Design:
A full factorial crossing experiment was performed, yielding 16 different combinations.
Individual Selection & Handling:
Only unmated individuals were used, ensuring standardization of reproductive conditions.
L. trifolii uninfected individuals were used from the third generation after antibiotic treatment to avoid direct antibiotic effects.
L. sativae infected individuals were used from the third generation after Wolbachia transinfection via microinjection.
Mating Conditions:
1 female + 2 males per test tube (7.5 cm × 1.0 cm; Thermo Fisher Scientific).
Individuals were placed in tubes for 24 hours with 50% honey in water as food.
Egg Collection & Rearing:
Quail bean leaves were used as oviposition substrates.
Eggs were counted after 24 hours and then transferred to a conical flask with water.
After hatching, larvae were moved to square acrylic cases (9.5 cm × 6.5 cm × 6.5 cm) with mesh for further development.
Counts recorded: Number of eggs, hatched larvae, pupae, and emerging adults.
Adults were sexed and counted upon emergence.

2. Wolbachia Infection Confirmation
Diagnostic PCR using wsp primers was performed on:
Parental generation (P) individuals before crossing.
Offspring (F1) to confirm vertical transmission of Wolbachia.

3. Environmental Conditions
Controlled in a 16L:8D photoperiod at 25°C to standardize environmental effects.

4. Data Processing & Statistical Analysis
Measured Parameters:

Number of eggs laid per female.
Hatching rate (larvae/eggs).
Emergence rate (adults/larvae).
Sex ratio (female/male proportion in F1).
Wolbachia infection rate in offspring.

Liriomyza_DNA_sequences.csv, Liriomyza_amino_acid_sequences.csv:
This dataset contains nucleotide sequence data related to Wolbachia genes, including fbpA, cifA, cifB, clpA, and pyrD, from Liriomyza trifolii, Liriomyza sativae. The dataset also includes predicted amino acid sequences. These data were generated as part of a study investigating Wolbachia 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?
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.
PCR reaction mixtures (50 µL) contained:
KOD One® PCR Master Mix -Blue- (Takara) (25 µL)
Distilled water (21 µL)
Primers (1.5 µL each, forward & reverse)
DNA extract (1.0 µL)
PCR conditions:
Initial denaturation: 98°C for 2 min
30 cycles of:
98°C for 10 sec
53°C for 5 sec
68°C for 3 sec
Final extension: 68°C for 10 min
PCR products were verified via 1% agarose gel electrophoresis.
Sequencing & Processing

PCR products were submitted to Fasmac, Inc. (Kanagawa, Japan) for Sanger sequencing.
Raw sequence data were quality-checked and trimmed. The ExPASy Translate Tool was used to predict amino acid sequences from nucleotide sequences.