Cytoplasmic male sterility and mitochondrial metabolism: evidence for low complex I contribution in male-sterile freshwater snail Physa acuta

Cytoplasmic male sterility (CMS) is a well-known example of mitonuclear genetic conflict over sex determination in hermaphrodite plants, where mitochondrial genes maternally inherited sterilize the male function while biparental inherited nuclear genes restore it. CMS has been recently discovered in animals, in the freshwater snail Physa acuta. In this species, CMS is associated with two extremely divergent mitogenomes D and K compared to the classical mitogenome N. D individuals are male-steriles, while male fertility is restored in K individuals. 

To better understand the physiological aspects of CMS and their potential link to overall organism fitness, we compared the following variables among the three mitogenomes: 

• phenotypic traits (male status, whole body mass and maximal shell length);

• mitochondrial aerobic metabolism (high resolution respirometry O2k, Oroboros);

• anaerobic cellular metabolism (enzymatic activities of the lactate dehydrogenase LDH, key enzyme of anaerobic metabolism; and the citrate synthase CS, key enzyme of aerobic metabolism). 

Description of the data and file structure

The data countains:

• Measurements of phenotypic traits:

  • Male status: male status of focal individual was assessed by pairing it to a virgin albino and by recording the pigmentation of the albino progeniture (unhatched offspring, about one week after the pairing using a binocular microscope) → pigmented offspring = focal individual was male-fertile, no or albino = focal individual was male-sterile. 

  Following analyses were conducted exclusively on N and K male-fertile and D male-sterile individuals.

  • Whole-body mass (shell+body): measured with a precision scale to the nearest 0.001mg.
  • Maximal shell length: photography analysed with the software ImageJ at the nearest 0.001mm. Digital photographs of the snails used for measuring maximum shell length with software ImageJ are available in the "shell_length_photos.zip" file.

• Measurements of mitochondrial aerobic metabolism: mitochondrial functionality using high resolution respirometry (O2k, Oroboros; DatLab software), SUIT=substrate-inhibitor-uncoupler titration protocol

• Measurements of enzymatic activities: Enzyme activities (lactate dehydrogenase LDH, and citrate synthase CS) were measured spectrophotometrically lactate dehydrogenase.

Files and variables

File: Rscript_phenotypic_analysis.R

Description: R script used for the analysis of phenotypic traits.

File: data_phenotype.csv

Description: Data related to the measurement of phenotypic traits (male status, whole-body mass and maximal shell length) in N, K and D individuals regarless of their male status.

Variables

• mitotype: N for normal hermaphrodite, K for restored hermaphrodite and D for male-sterile
• individu: individual's number
• poids: whole body mass (shell+body) in g
• taille: maximal shell length in mm 
• ponte albinos: presence (= 1) or absence (= 0) of eggs laid by the albino partner
• pigmentation ponte albinos: pigmentation of the offspring produced by the albino partner used to determine male status (1 = pigmented eggs; 0 = albinos)
• statut male: male status (sterile or fertile according to the pigmentation of the offsprings of the albino partner)

File: data_phenotype_sf.csv

Description: Data related to the measurement of phenotypic traits (male status, whole-body mass and maximal shell length) in N and K male-fertile individuals (normal hermaphrodite and restored hermaphrodite, respectively) and D male-sterile individuals.

Variables

• mitotype: N for normal hermaphrodite, K for restored hermaphrodite and D for male-sterile
• individu: individual's number
• poids:  whole body mass (shell+body) in g
• taille: maximal shell length in mm 
• ponte albinos: presence (= 1) or absence (= 0) of eggs laid by the albino partner
• pigmentation ponte albinos: pigmentation of the offspring produced by the albino partner used to determine male status (1 = pigmented eggs; 0 = albinos)
• statut male: male status (sterile or fertile according to the pigmentation of the offsprings of the albino partner)

File: Rscript_mitochondrial_metabolism.R

Description: R script used for the analysis of mitochondrial aerobic metabolism.

File: respi_correct.csv, ratio_correct.csv

Description: Data related to the assessment of mitochondrial functionality using high-resolution respirometry.

Variables in respi_correct.csv

• ID: individual's number 
• Mitotype: N = normal hermaphrodite, K = restored hermaphrodite and D = male-sterile
• Day_mito
• Run
• Oroboros
• Chamber
• State : refers to respiratory states which are AOX, CI OXPHOS, CI+II OXPHOS, CI+II LEAK, ETS, COX
• RespiO2 : values in pmol O2/s/mg

Variables in ratio_correct.csv

• ID: individual's number 
• Mitotype: N = normal hermaphrodite, K = restored hermaphrodite and D = male-sterile
• Day_mito
• Run 
• Oroboros
• Chamber
• mg_physe/mL
• endo
• sham
• pm
• adp
• S
• cytc
• S/cytc_max
• o
• FCCP
• aa
• sham
• asc
• tmpd
• azd
• AOX_endo
• CI_LEAK
• CI_OXPHOS
• CI+II_OXPHOS
• %_cytC
• CI+II_LEAK
• ETS
• COX
• OxCE
• OxCE_ETS
• ReserveCapacity
• CI_contrib
• CII_contrib

File: Rscript_enzymatic_activities_analysis.R

Description: R script used for the analysis of anaerobic metabolism.

File: data_enzymatic_activities.csv

Description: Data related to the assessment of enzymatic activities. 

Variables

• Mitotype: N = normal hermaphrodite, K = restored hermaphrodite and D = male-sterile
• ID: individual's number 
• Tissue_mass
• Tissue_concentration
• CS
• LDH
• LDH_CS

Code/software

Rscript_phenotypic_analysis.R associated with data files :

• data_phenotype.csv
• data_phenotype_sf.csv

Rscript_mitochondrial_metabolism.R associated with  data files :

• respi_correct.csv
• ratio_correct.csv

Rscript_enzymatic_activities_analysis.R associated with the data file :

• data_enzymatic_activities.csv