Gut barrier stability, metabolic reprogramming, and microbiota shifts support toxin tolerance in Anastrepha acris (Diptera: Tephritidae) larvae

Plant phenolics/xenobiotics impose strong selection pressure on phytophagous insects and the mechanistic basis of tolerance in extreme specialists remains poorly understood in the case of true fruit flies (Tephritidae). Anastrepha acris is a monophagous tephritid fly infesting the Apple of Death ( Hippomane mancinella ), considered one of the most toxic fruits worldwide because it is rich in ellagitannins (e.g., Hippomanin A) and other highly toxic allelochemicals (e.g., phorbol type diterpene esters, sapogenins and furanocoumarins). We combined a high dose reconstituted lyophilized H. mancinella pulp (RLHMP) dietary challenge in conjunction with antibiotic treatments to dissect how midgut structural integrity in the larvae, detoxification-related metabolism, and the gut microbial community composition jointly support A . acris ´s survival in such a toxic host. A multitrait larval performance index (ω, 0–1) measuring a variety of survival parameters declined markedly with RLHPM diet (50% concentration) and the reduction was amplified by antibiotics during larval rearing. Across all groups, RLHMP decreased ω by ~ 0.28 on average; the decrease was ~ 0.38 with antibiotics versus ~ 0.17 without them. Antibiotics improved fitness on the standard diet but conferred no benefit under RLHMP. Whole-larva RNA-seq across a RLHMP gradient identified a coordinated induction of carbohydrate and lipid catabolism, xenobiotic-processing enzymes (including cytochrome P450s and oxidoreductases), and large cohorts of serine-type endopeptidases, together with enrichment of antibacterial humoral signaling. Additionally, we found that microtubule-associated transcripts were selectively down-modulated. Importantly, transmission electron microscopy showed that A. acris larvae maintained an intact midgut peritrophic matrix, densely packed microvilli, preserved mitochondrial membranes, and characteristic cytoplasmic vacuoles and lipid droplets even under high RLHMP concentrations. In sharp contrast, larvae of the phylogenetically closely related species A. ludens exhibited early mitochondrial and microvillar collapse at much lower RLHMP diet concentrations in midgut tissue. 16S rRNA amplicon profiling of gut bacteria revealed Proteobacteria-dominated communities structured by a small set of core genera that survived despite exposure to the xenobiotic and antibiotic treatments. RLHMP consistently reduced Achromobacter and enriched Phyllobacterium and Ralstonia regardless of antibiotic history. Neutral models showed that Achromobacter and Phyllobacterium occurred more often than expected in laboratory treatments, whereas taxa occurring less often than expected accounted for a larger proportion of reads in wild larvae and the combined RLHMP–antibiotic treatment. Anastrepha acris larvae withstand extreme xenobiotics loads via an integrated three-component strategy: preservation of midgut barrier architecture, metabolic reallocation that sustains energy-demanding detoxification and proteostasis, and immune-mediated pruning that fosters a phenolic-tolerant core microbiota. Experimental exposure to antibiotics removed microbial allies precisely when the host needed them most, exacerbating performance costs under RLHMP exposure. Targeted manipulation of the identified core taxa and pathways offers testable levers to improve larval survival on phenolic rich hosts.