Echoentomography: a novel non-destructive imaging of soft-body insects through ultra-high frequency ultrasonography (UHFUS)

Developing non-destructive tools for soft tissue imaging is crucial to shed light on insect morpho-biology and morpho-physiology, including the impact of detrimental cues on behaviour and locomotion. In this regard, major advances in entomological research have been recently achieved using micro-computed tomography (micro-CT) technology. However, micro-CT is characterized by high costs and slow data processing. Recently, ultra-high frequency ultrasonography (UHFUS) - to date used for preclinical and clinical research - was adopted for the first time to study insect morphology and physiology. This highly pioneering and promising technique was named echoentomography. A non-destructive imaging approach was adopted for studying five insect species. Successful imaging was achieved analysing the larvae of two species of high agricultural importance, i.e., the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae) and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Results showed that UHFUS allowed for high-resolution imaging on both soft-bodied larvae, finely characterizing the insect circulatory organs and midgut morpho-physiology. L. botrana larvae were characterized by a dorsal vessel corresponding to the heart with 55-bpm frequency and an apparent monophasic heartbeat. A sectorized digestive system was also observed. Moreover, with a spatial resolution of up to 30 mu m, some anatomical features such as the moth pyloric valve and the ventral ganglion chain were observed. The pyloric valve had two degrees of freedom, a rotating movement on the transversal plane and a rotating movement on the sagittal plane. The ganglion chain appeared as a homogeneously hypoechogenic structure characterized by regular repetitions of nodular elements. C. capitata larvae were characterized by a dorsal heart with a 77-bpm frequency and a triphasic heartbeat. Overall, the UHFUS-based approach proposed here is non-destructive, allowing in vivo analysis of both anatomical and physiological features on soft-bodied arthropods of value (including hard-to-collect and endangered species), providing a valuable alternative to classic destructive dissections, as well as to the use of expensive and slower technologies, such as micro-CT and micro-MRI.