The Sepioteuthis lessoniana species complex, oval squids, are emerging model organisms in diverse scientific fields including neuroscience, behavior, ecology, and aquaculture, driving increased interest in their care under artificial environments such as laboratories, aquaria, and industrial aquaculture facilities. There is limited information on multiple aetiological agents such as pathogenic bacteria species and their associated diseases affecting squid under intensive aquaculture conditions, highlighting the need for further investigation. The objective of this study was to identify Vibrionaceae species associated with disease symptoms in captive oval squid and to compare the effectiveness of bacterial-culture/DNA-based, and metatranscriptomic diagnostic approaches. A total of 62 distinct Vibrionaceae species were identified. Comparative analyses showed that metatranscriptomic approaches provided enhanced resolution of actively infecting Vibrio species compared with bacterial-culture- and DNA-based methods alone. Among these, Vibrio harveyi and V. chagasii showed the highest relative transcript abundance and were consistently associated with clinically unhealthy squid. The relative abundance of Vibrionaceae transcripts was higher in moribund individuals and coincided with increased morbidity and mortality in captive squid stocks. Infected squids were characterized by clinical signs including lethargy, anorexia, cachexia, skin erosions, vision loss, and the development of “white arms”, characterized by opaque whitening of the arm-base musculature. Histopathological examination revealed eosinophilic exudate and increased mucus secretion in the arm epidermis, indicative of an innate immune response to Vibrionaceae infection. In addition, fin epidermal detachment and ulceration were observed in affected individuals, while necrotic lesions in the digestive gland and kidney suggested hepatic and renal damage. Together, these findings provide the first integrative evidence of spontaneously occurring Vibrionaceae-associated disease conditions in captive oval squids and demonstrate the added diagnostic value of a newly developed and highly accurate metatranscriptomic method of active microbial taxa identification and quantification in cephalopods. This work establishes a foundation for future studies investigating Vibrionaceae-related disease dynamics within a pathobiome framework and for health management strategies in cephalopod aquaculture.
