Where are my nematodes? Labelling and visualising entomopathogenic nematodes In Vivo using carbon quantum dots

Identifying single or groups of animals has significantly advanced our understanding of animal biology and ecology. However, labelling is extremely difficult in small animals, like soil invertebrates. Due to the complexity of current methods, the dynamics and interactions of these populations are often studied indirectly. Labelling nematodes or microarthropods such as collembolans or soil acari has been challenging due to the high cost, potential toxicity, genetic modification requirements, cellular processes interference, and photobleaching. In this scenario, no methods can be applied to large numbers of microorganisms at once due to their mentioned practical and biological limitations and cost. In this work we show that quantum carbon dots (Cdots) are effective for labelling infective juveniles (IJs) of entomopathogenic nematodes (EPN). In in vitro assays the IJs gradually acquired fluorescence, as Cdots accumulated in the lysosome-related organelles from their intestine cells, peaking at day 14, and with no lethal or sub-lethal effects on IJs. Fluorescence was clearly distinguishable from the natural auto-fluorescence of non-labelled IJs and persisted for months in IJs transferred to water. We and non-experts easily differentiated between similar species of EPNs and between two strains of S. feltiae placed in the same matrix (soil or water). We demonstrated for the first time the feasibility of labelling large numbers of IJs (hundreds of thousands/millions) with Cdots at minimal cost without any adverse effects for over a year. Our findings could be the starting point for detailed and large-scale field investigations on nematodes and other small organisms, allowing deeper understanding of their roles in soil ecosystems. This method provides a cost-effective and reliable tool for advancing research in the ecology of soil invertebrates, such as the interactions occurring in communities or between specific organisms, movement and dispersal, population dynamics or ecosystem services in a cryptic environment difficult to study.