Abstract
Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays.
Highlights
The lipophilic toxin okadaic acid and structurally similar dinophysistoxins are produced by several marine dinoflagellates, notably species of Prorocentrum and Dinophysis, and cause diarrhetic shellfish poisoning (DSP) in humans (Tachibana et al 1981; Yasumoto et al 1985; Trainer et al 2013)
Solutions of okadaic acid were prepared in 0.2 μm filter-sterilised phosphate-buffered saline (PBS) pH 7.4 containing 5% dimethyl sulfoxide (DMSO; v/v)
Administering single doses of either okadaic acid via gavage (Fig. 1) or E. coli via intrahaemocoelic injection (Fig. 2) led to significant declines in larval health in a dose-dependent manner (X2(4) = 110.7, P < 0.0001 [Fig. 1a] and X2(5) = 157.9, P < 0.0001 (Fig. 2a); Table 1)
Summary
The lipophilic toxin okadaic acid and structurally similar dinophysistoxins are produced by several marine dinoflagellates, notably species of Prorocentrum and Dinophysis, and cause diarrhetic shellfish poisoning (DSP) in humans (Tachibana et al 1981; Yasumoto et al 1985; Trainer et al 2013). DSP toxins are the most common output from harmful algal blooms in European, South American, and Asian waters, thereby representing the leading cause of aquaculture harvesting bans and site closures in Europe (Reguera et al.2014; Dhanji-Rapkova et al 2018; Bresnan et al 2021). Okadaic acid inhibits serine/threonine phosphatases (PP1, PP2A; Bialojan and Takai 1988), disrupting cellular homeostasis due to hyperphosphorylation and leading to downstream dysregulation of the gastrointestinal mucosa. The broad symptomology of okadaic acid-induced poisoning cannot be attributed to phosphatases alone and likely involves the enteric nervous system (e.g., neuropeptide Y; Louzao et al 2015). Recent experiments carried out on Swiss female mice suggest that serotonin (5-hydroxytryptamine) levels are a
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