A team of researchers led by Carlos Eduardo Aguilar-Castillo and Obdulia Lourdes Segura-León at Colegio de Postgraduados in Texcoco, Mexico, compared the head transcriptomes of two mass-reared A. ludens strains, the Bisexual strain and the Tapachula-7 genetic sexing strain, with wild males collected from infested sweet oranges in Chiapas. Using RNA sequencing on the BGISEQ-500 platform, the researchers generated an average of 46.64 million raw reads per library across nine biological replicates, with quality values exceeding 89 percent of bases at Q30 or higher. After filtering and alignment to the A. ludens reference genome, mapping rates averaged 93.99 percent, with wild individuals showing the highest rates at 96.97 to 97.82 percent and the Tap-7 strain showing the lowest at 87.21 to 88.16 percent.

Principal component analysis revealed a striking separation between the mass-reared strains and wild host-associated flies. The first principal component accounted for 92 percent of total transcriptomic variance, while the second explained an additional 5 percent, together capturing 97 percent of the variation across all samples. This clear separation suggests that mass-reared flies occupy a fundamentally different head transcriptomic state compared with their wild counterparts, reflecting the combined effects of colony history, larval diet based on corn protein rather than natural host substrates, and rearing background.

Differential expression analysis identified more than 1,300 differentially expressed genes per strain. The Bisexual strain showed 1,329 differentially expressed genes, with 745 downregulated and 584 upregulated, while Tap-7 exhibited 1,289, with 723 downregulated and 566 upregulated. Approximately 680 genes were shared between both comparisons, representing roughly 51 to 52 percent of the differentially expressed genes in each contrast. The predominance of gene repression in both mass-reared strains suggests that transcriptional downregulation of specialized functions is a shared response to the reduced environmental complexity of laboratory rearing, consistent with patterns previously documented in other tephritids.

Gene Ontology enrichment analysis revealed that both strains shared a core functional signature centered on cytoplasmic translation and cytosolic ribosome terms, indicating the maintenance of essential biosynthetic machinery. However, each strain also displayed distinct enrichment patterns. The Bisexual strain showed 31 enriched GO terms, including humoral immune response, antibacterial humoral response, peptide biosynthetic process, and mRNA metabolic process, suggesting modulation of immune-related transcripts in head tissue. Tap-7, in contrast, showed 27 enriched terms with unique representation of melanin biosynthetic process, tyrosine metabolic process, catechol oxidase activity, and tyrosinase activity, pointing toward oxidative and defensive processes that may impose energetic costs under prolonged rearing conditions.

Kyoto Encyclopedia of Genes and Genomes pathway analysis further differentiated the two mass-reared strains. Both shared enrichment of the Ribosome pathway, with extraordinarily high statistical significance, along with Thermogenesis, Oxidative phosphorylation, and Spliceosome, all associated with energy metabolism and RNA processing. The Bisexual strain uniquely enriched the Toll and Imd signaling pathway, Mitophagy, Carbohydrate digestion and absorption, Phototransduction, and Axon regeneration, suggesting signatures of stress response, immunity, mitochondrial quality control, and neuronal maintenance. Tap-7 uniquely enriched Endocrine and other factor-regulated calcium reabsorption, Salivary secretion, Drug metabolism, Vitamin digestion and absorption, and One carbon pool by folate, indicating ion regulation, secretory activity, detoxification, and metabolic support as distinguishing features.

Perhaps the most compelling findings concern the chemosensory gene families. Both mass-reared strains exhibited upregulation of ionotropic receptors including Ir25a, Ir68b, and Ir85a, along with Or74a-like and SNMP1-like, suggesting conservation of a generalist environmental sensory core potentially relevant to monitoring artificial rearing conditions. In stark contrast, odorant-binding proteins showed convergent downregulation across both strains. The Bisexual strain displayed particularly strong repression of Obp56a-like at two distinct loci, with log2 fold change values of -11.24 and -9.66, while Tap-7 showed a broader reduction across six OBP genes with more uniform downregulation ranging from -2.01 to -5.81. Members of the Obp99 clade, including Obp99a, Obp99b-like, Obp99c, and Obp99d, were consistently repressed in both comparisons.

This convergent downregulation of peripheral odorant-binding proteins, combined with the preservation of central signal transduction components such as SNMP1 and Ir25a, suggests a compensatory transcriptional pattern in which the basic olfactory architecture remains intact while the fine-scale chemical discrimination capacity may be reduced. In tephritids, OBPs are closely linked to host localization and sexual recognition, predominantly expressed in antennal tissues. The authors propose that the restricted chemical spectrum of artificial rearing environments, where homogeneous diets and controlled conditions reduce the need for complex host-cue detection, may favor this chemosensory remodeling. However, they caution that behavioral and electrophysiological assays would be required to confirm whether these transcriptional changes translate into measurable functional differences in host orientation or mating competitiveness after release.

The implications for the sterile insect technique are substantial. The sexual competitiveness of released sterile males depends on the integration of sensory perception, physiological condition, and metabolic homeostasis. Previous research has documented reduced mating success in mass-reared A. ludens males compared with wild individuals, and the current study provides candidate molecular mechanisms that may help explain these differences even in the absence of evident morphological changes. The authors suggest that biological quality in SIT programs may depend not only on physical parameters such as flight ability and emergence rate but also on the functional integrity of sensory and metabolic networks, highlighting the potential value of incorporating molecular markers based on GO and KEGG analyses as complementary tools for strain evaluation.

The researchers acknowledge several limitations. The comparison between wild and mass-reared groups involves multiple confounding factors including larval host or diet, colony history, rearing background, genetic background, and sampling conditions, meaning the observed transcriptomic differences should be interpreted as correlative patterns rather than causal effects exclusively attributable to mass rearing. Because RNA was extracted only from adult male heads, the identified GO and KEGG categories should be considered head-associated transcriptional signatures rather than direct evidence of whole-body physiological changes. Furthermore, the RNA-seq results were not independently validated by RT-qPCR, behavioral assays, electrophysiological assays, or physiological assays, so the identified genes and pathways remain candidate molecular markers requiring further validation. Nevertheless, this study establishes a critical framework for understanding the molecular consequences of mass rearing in one of the world’s most important fruit fly pests and opens new avenues for optimizing the quality of insects used in one of agriculture’s most environmentally friendly pest control strategies.

Subject of Research: Comparative transcriptomics of mass-reared and wild Anastrepha ludens fruit flies

Article Title: Comparative transcriptomic analysis of functional and metabolic changes in mass-reared and wild Anastrepha ludens Loew (Diptera: Tephritidae)

Article References: Aguilar-Castillo, C. E., Segura-León, O. L., Cibrián-Tovar, J., González-Hernández, H., Núñez-Farfán, J., & Torres-Huerta, B. (2026). Comparative transcriptomic analysis of functional and metabolic changes in mass-reared and wild Anastrepha ludens Loew (Diptera: Tephritidae). Frontiers in Insect Science, 6, Article 1913379. https://doi.org/10.3389/finsc.2026.1913379

Image Credits: AI Generated

DOI: 10.3389/finsc.2026.1913379

Keywords: Anastrepha ludens, sterile insect technique, transcriptomics, chemosensory genes, odorant-binding proteins, mass rearing, Comparative, transcriptomic, analysis, functional, metabolic, changes