Muscle color metric quantitatively predicts structural and functional remodeling in Gryllus lineaticeps
Data files
Sep 15, 2026 version files 2.53 MB
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cs_activity_dataset.ipynb
761.41 KB
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cs_activity_dlm_data.csv
5.59 KB
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cs_activity_dvm_data.csv
4.66 KB
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df_dlm_modeling.csv
15.26 KB
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df_dlm_variation.csv
10.13 KB
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df_dvm_modeling.csv
15.20 KB
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df_dvm_variation.csv
10.16 KB
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iron_protein_content_data.csv
6.84 KB
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iron_protein_content_dataset.ipynb
1.03 MB
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modeling_dataset.ipynb
654.07 KB
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README.md
11.58 KB
Abstract
Comparative and evolutionary approaches have advanced our understanding of muscle biology. However, methods for assessing muscle functional state are expensive, technically demanding, and difficult to scale. Here, we developed and validated a standardized framework for quantifying insect muscle color during flight muscle breakdown, using the wing-dimorphic cricket Gryllus lineaticeps. Our color metric robustly tracks variation in muscle color across remodeling stages and predicts muscle mass, protein content, and enzyme activity associated with mitochondrial abundance. By providing a standardized, accessible, and biologically informative proxy of muscle state, this framework not only advances the utility of G. lineaticeps as a model for muscle remodeling but will also facilitate comparative analyses in systems where direct measurement of muscle function is logistically, technically, or financially impractical. More broadly, quantitative muscle color analysis may facilitate comparative, ecological, and mechanistic studies of muscle function, dispersal, aging, seasonal plasticity, and environmental stress responses across diverse insect systems.
Dataset DOI: 10.5061/dryad.rv15dv4pr
Description of the data and file structure
Flight muscle color is commonly used as a qualitative proxy for muscle function (in insect flight muscles of the Gryllus genus), with red muscle indicating functional tissue and white muscle indicating non-functional tissue. However, this scoring is subjective, particularly when muscles are in intermediate stages of breakdown, and prone to variation among observers and laboratories. To reduce bias and improve reproducibility, we developed a digital method to quantitatively measure muscle color and track the progression of histolysis. We collected 65 long-winged and 31 short-winged females to capture a broad range of muscle colors. We dissected and imaged two muscle types: dorsal longitudinal muscles (DLMs) and dorsal ventral muscles (DVMs). Muscle color was categorized by the observer as red (R), red-pink (RP), pink (P), pink-white (PW), white (W), or transparent (T). Images of muscles were analyzed, and color was quantified with pixel intensity in RGB channels. We constructed predictive models that used RGB values and other engineered features to classify discrete color categories. We selected the best-performing model and then translated the model's coefficients and features into a continuous color metric. Using the color metric in two independent datasets, we explored the relationship between muscle color and structural (protein content, muscle mass) and functional characteristics (iron content, and citrate synthase activity as an index of mitochondrial abundance).
Files and variables
File: cs_activity_dvm_data.csv
Description: Muscle color and citrate synthase activity of dorsal ventral muscles (DVMs) dissected from long-winged female crickets (Gryllus lineaticeps). Data used to explore the relationship between muscle color and citrate synthase activity (a proxy of mitochondrial abundance).
Variables
- cricket_id: unique cricket ID
- muscle: muscle type: V = DVM
- age: age of adult crickets (days since adult molt)
- weight_mg: muscle mass in mg
- color: muscle color category (W = white, P = pink, R = red)
- abs_rate: absorption rate determined in citrate synthase assay
- raw_protein_conc_mg_per_ml: protein concentration mg/ml of sample
- protein_conc_starting_homogenate_mg_per_ml: protein concentration of homogenate mg/ml
- total_protein_in_sample_mg: total protein in sample (in mg)
- protein_per_tissue_mass: protein per mg of tissue
- cs_activity_microMcit_0ver_mgPro_min: cs activity
- cs_act_tissue: total cs activity of tissue sample
- mean_b: average blue pixel intensity
- mean_g: average green pixel intensity
- mean_r: average red pixel intensity
- color_index: color index: 9.414*(R /B) + 0.030*(R - B) - 0.019*G -14.173
File: cs_activity_dlm_data.csv
Description: Muscle color and citrate synthase activity of dorsal longitudinal muscles (DLMs) dissected from long-winged female crickets (Gryllus lineaticeps). Data used to explore the relationship between muscle color and citrate synthase activity (a proxy of mitochondrial abundance).
Variables
- cricket_id: unique cricket ID
- muscle: muscle type: L = DLM
- age: age of adult cricket (days since adult molt)
- weight_mg: muscle mass in mg
- color: muscle color category (W = white, P = pink, R = red)
- abs_rate: absorption rate (from citrate synthase activity assay)
- raw_protein_conc_mg_per_ml: protein concentration mg/ml of sample
- protein_conc_starting_homogenate_mg_per_ml: protein concentration mg/ml of homogenate
- total_protein_in_sample_mg: total protein in sample (in mg)
- protein_per_tissue_mass: protein per mg of tissue
- cs_activity_microMcit_0ver_mgPro_min: cs activity
- cs_act_tissue: total cs activity of tissue sample
- mean_b: average blue pixel intensity
- mean_g: average green pixel intensity
- mean_r: average red pixel intensity
- color_index: color metric = 76.228*(R / G) - 0.244*(R-G) + 0.053R - 94.166
File: iron_protein_content_data.csv
Description: Muscle color and iron and protein content of dorsal longitudinal and dorsal ventral muscles dissected from 5-day-old long-winged female crickets (Gryllus lineaticeps). Data used to explore the relationship between muscle color and iron and protein content of the muscle.
Variables
- cricket_id: unique cricket ID
- muscle: muscle type (0 = DVM, 1 = DLM)
- mmass: muscle mass (mg)
- mean_b: average blue pixel intensity
- mean_g: average green pixel intensity
- mean_r: average red pixel intensity
- iron: iron content (mM)
- raw_protein_conc_ug_per_ul: protein concentration of sample (ug/ul)
- protein_conc_starting_homogenate_ug_per_ul: protein concentration of homogenate (ug/ul)
- total_protein_in_sample_mg: total amount of protein in sample (mg)
- color: muscle color category (W = white, P = pink, R = red)
File: df_dvm_variation.csv
Description: The replicate RGB values and color categories of dorsal ventral muscles (DVMs) dissected from long- and short-winged female crickets (Gryllus lineaticeps). Data used to explore within-muscle color variation.
Variables
- cricket_id: unique cricket ID
- replicate: replicate number: 1, 2, or 3 (each muscle color was quantified with 3 ROIs)
- B: blue pixel intensity of replicate
- G: green pixel intensity pixel intensity of replicate
- R: red pixel intensity pixel intensity of replicate
- dvm_color: muscle color category (red, red/pink, pink, pink/white, white, transparent)
File: df_dlm_variation.csv
Description: The replicate RGB values and color categories of dorsal longitudinal muscles (DLMs) dissected from long- and short-winged female crickets (Gryllus lineaticeps). Data used to explore within-muscle color variation.
Variables
- cricket_id: unique cricket ID
- replicate: replicate number: 1, 2, or 3 (each muscle color was quantified with 3 ROIs)
- B: blue pixel intensity of replicate
- G: green pixel intensity of replicate
- R: red pixel intensity of replicate
- dlm_color: muscle color category (red, red/pink, pink, pink/white, white, transparent)
File: df_dlm_modeling.csv
Description: The RGB values and color categories of dorsal longitudinal muscles (DLMs) dissected from long- and short-winged female crickets (Gryllus lineaticeps). Data used to develop a color metric.
Variables
- cricket_id: unique cricket ID
- morph: wing morph (SW = short-winged, LW= long-winged
- age_days: age in days of adult cricket (days since adult molt)
- femur_length_um: length of femur (um)
- ovary_mass_g: ovary mass (g)
- category: dlm muscle color category: red, red/pink, pink, pink/white, white, transparent
- dvm_color: dvm muscle color category: red, red/pink, pink, pink/white, white, transparent
- dlm_blue_1: blue pixel intensity of replicate 1
- dlm_blue_2: blue pixel intensity of replicate 2
- dlm_blue_3: blue pixel intensity of replicate 3
- dlm_green_1: green pixel intensity of replicate 1
- dlm_green_2: green pixel intensity of replicate 2
- dlm_green_3: green pixel intensity of replicate 3
- dlm_red_1: red pixel intensity of replicate 1
- dlm_red_2: red pixel intensity of replicate 2
- dlm_red_3: red pixel intensity of replicate 3
- B: mean blue pixel intensity
- G: mean green pixel intensity
- R: mean red pixel intensity
- y: color category ordinal rank (red = 1, red-pink = 2, pink = 3, pink-white = 4, white = 5, transparent = 6)
File: df_dvm_modeling.csv
Description: The RGB values and color categories of dorsal ventral muscles (DVMs) dissected from long- and short-winged female crickets (Gryllus lineaticeps). Data used to develop a color metric.
Variables
- cricket_id: unique cricket ID
- morph: wing morph (SW = short-winged, LW= long-winged
- age_days: age in days of adult cricket (days since adult molt)
- femur_length_um: femur length (um)
- ovary_mass_g: ovary mass (g)
- dlm_color: dlm muscle color category: red, red/pink, pink, pink/white, white, transparent
- category: dvm muscle color category: red, red/pink, pink, pink/white, white, transparent
- dvm_blue_1: blue pixel intensity of replicate 1
- dvm_blue_2: blue pixel intensity of replicate 2
- dvm_blue_3: blue pixel intensity of replicate 3
- dvm_green_1: green pixel intensity of replicate 1
- dvm_green_2: green pixel intensity of replicate 2
- dvm_green_3: green pixel intensity of replicate 3
- dvm_red_1: red pixel intensity of replicate 1
- dvm_red_2: red pixel intensity of replicate 2
- dvm_red_3: red pixel intensity of replicate 3
- B: mean blue pixel intensity
- G: mean green pixel intensity
- R: mean red pixel intensity
- y: olor category ordinal rank (red = 1, red-pink = 2, pink = 3, pink-white = 4, white = 5, transparent = 6)
File: iron_protein_content_dataset.ipynb
Description: Python code used to visualize and analyze the relationship between muscle color and iron and protein content.
File: modeling_dataset.ipynb
Description: Python code used to quantify flight muscle color and build color metrics via predictive modeling.
File: cs_activity_dataset.ipynb
Description: Python code used to visualize and analyze the relationship between muscle color and citrate synthase activity (a proxy of mitochondrial abundance).
Code/software
We used Python (v3.11.5) with the following packages: numpy, pandas, matplotlib. pyplot, seaborn, sklearn, skimage, scipy, and statsmodels.
RGB values from images of two muscle types (DLM and DVM) were analyzed in Python using a JupyterLab Notebook with the pandas, numpy, skimage, scipy, and statsmodels libraries and were visualized with the matplotlib and seaborn libraries. Muscle color variability within individuals was quantified using replicate color measurements obtained from each muscle type. Overall variation of the samples was described, and within-muscle color variation was compared across muscle color categories.
We then developed a portable, continuous color metric capable of capturing the full extent and progressive nature of muscle histolysis. To achieve this, we constructed predictive models that used RGB values to classify discrete color categories and then translated the model coefficients and features into a continuous color metric. Importantly, model performance was evaluated using a rank-based framework to reflect the ordered progression of pigmentation loss. Model creation, model evaluation, and data visualization were carried out in Python with numpy, pandas, matplotlib, scipy, math, statsmodels, scikit-image, scikit-learn, matplotlib, and seaborn libraries. Once selected, the final model was evaluated on the held-out test set (20% of samples), and the coefficients were extracted.
Using the color metric, we explored the relationship between muscle color and structural (protein content, muscle mass) and functional characteristics (iron content, and citrate synthase activity as an index of mitochondrial abundance) in two independent datasets. To characterize the relationships between the color metric and three functional metrics (protein content, iron content, and citrate synthase activity) as well as muscle mass, we evaluated three candidate models: linear, quadratic, and exponential. Analysis and data visualization were performed in Python using the numpy, pandas, matplotlib, seaborn, statsmodels, and scipy libraries.