Deschamps, M.-H. | Search bioRxiv

bioRxiv2025

Reference Gene Selection for Accurate RT-qPCR Normalization in Four Tissues and Whole-Body Samples of Acheta domesticus

House crickets (Acheta domesticus) are increasingly recognized as a sustainable protein source for food and feed systems. However, despite their growing relevance, molecular research on this species remains extremely limited, particularly concerning robust normalization strategies for gene expression analysis. This study is the first to identify and validate suitable reference genes for RT-qPCR analysis in A. domesticus across different tissues, an essential step for accurate quantification of host and pathogen target gene expression. Six candidate reference genes commonly used in insects (AdoNEOPT, EF2, 18S rRNA, EF1, Histone H3, and GAPDH) were evaluated for expression stability in five tissue types (abdomen, legs, wings, head, and whole body). Gene stability was assessed using five computational tools: BestKeeper, geNorm, NormFinder, Delta Ct, and the integrated platform RefFinder. Additional validation was performed using the R statistical software. The results identified EF1, AdoNEOPT, EF2, and 18S rRNA as the most stable reference genes across all the selected tissues, while GAPDH and His H3 showed high variability and were generally unsuitable except in the head, where GAPDH demonstrated stable expression. This study provides the first validated set of reference genes for A. domesticus, laying a foundation for accurate and reproducible gene expression studies. Moreover, our study will enable the development of new diagnostic tests based on qPCR and molecular signatures. These tests will be essential tools for health monitoring in insect farms, which remain exposed to emerging diseases.

molecular biology↗

bioRxiv2025

Temperature outweighs diet in shaping developmental performance in two cricket species via growth delays and physiological limits

Understanding how chronic environmental stressors shape animal development is essential for predicting ecological responses and optimizing rearing systems. This perspective complements the use of short-term tolerance assays, which overlook the cumulative effects of sustained stress. Temperature and nutrition affect key life-history traits such as growth, development rate, and survival, which are closely tied to reproductive success and fitness. While both factors have been widely studied, their relative impacts arent clearly defined. We investigated how constant temperature (26- 41{degrees}C) and dietary protein-to-carbohydrate (P:C) ratio (0.15-2.18) influence development in two cricket species, Acheta domesticus and Gryllodes sigillatus. Growth trajectories were modelled using a unified-logistic equation to estimate asymptotic mass and relative growth rate. This approach captures the growth trajectory in a simplified and interpretable way, enabling comparisons across treatments. Asymptotic mass was combined with developmental rate and survival to calculate a composite metric of developmental performance. Developmental performance peaked at 35{degrees}C but fell at thermal extremes due to delayed development (in cold) or reduced mass and survival (in heat). Diet had more modest effects. Performance was stable across most P:C ratios, declining only at extreme imbalances. Notably, the performance cost of the most unbalanced diets was comparable to a 4-5{degrees}C shift from thermal optimum. Our results demonstrate that temperature, more than diet, drives variation in developmental performance during ad libitum feeding. This integrative framework provides a robust approach to quantify environmental sensitivity, define performance limits, and guide us toward the mechanisms underlying those limits and/or performance trade-offs. Summary statementTemperature more strongly influences growth trajectories and developmental performance than diet, due to delayed development and reduced survival at thermal extremes.

physiology↗