Micro‐CT imaging of Onchocerca infection of Simulium damnosum s.l. blackflies and comparison of the peritrophic membrane thickness of forest and savannah flies

Abstract

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro‐computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34–48 min after a blood‐meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro‐computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education.

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana

“…In addition, iodine staining sometimes results in blurry edges of some key tissues and structures [19]. To increase X-ray absorption of lowdensity tissues and enhance overall contrast, subsequent sand flies were stained for one week by immersion in a solution of 1% phosphotungstic acid (PTA) in 80% ethanol, with continual swirling on a laboratory mixer and a change to fresh staining solution mid-period [16]. Unlike the iodine stain, PTA staining penetrates more slowly into the tissues, but it binds permanently and results in sharp tissue differentiation [18,19], so material could be held for a lengthy period before scanning without loss of stain.…”

“…Many of those studies have been of larger Diptera: to achieve sufficient resolution in the scanning of small Diptera is more challenging due to staining issues and the increased scan times needed. However, a recent study of the infection of Simulium blackflies with Onchocerca nematodes demonstrated the feasibility of using micro-CT to scan small flies, revealing a parasite in the flight muscles of the fly [16].…”

Dynamic monitoring of vital functions and tissue re-organization in Saturnia pavonia (Lepidoptera, Saturniidae) during final metamorphosis by non-invasive MRI

“…With this technique internal morphological alterations can be visualized at very high-resolution 14 – 17 and by time resolved X-ray imaging also dynamic processes, such as movement of air trapped in an “air bubble” from inside the pupae to the space between the adult insect and the puparium 18 . Modern micro-CT systems can yield virtual sections of the insect brain and the optical lobe 19 , 20 and even visualise infectious nematodes in its vector, black flies 21 .…”