Evaluation of three methods for biomass estimation in small invertebrates, using three large disparate parasite species as model organisms

Invertebrate biomass is considered one of the main factors driving processes in ecosystems. It can be measured directly, primarily by weighing individuals, but more often indirect estimators are used. We developed two indirect and non-destructive approaches to estimate biomass of small invertebrates in a simple manner. The first one was based on clay modelling and the second one was based on image analysis implemented with open-source software. Furthermore, we tested the accuracy of the widely used geometric approximation method (third method). We applied these three different methods to three morphologically disparate model species, an acanthocephalan worm, a crustacean and a flatworm. To validate our indirect estimations and to test their accuracy, we weighed specimens of the three species and calculated their tissue densities. Additionally, we propose an uncomplicated technique to estimate thickness of individuals under a microscope, a required measurement for two of the three indirect methods tested. The indirect methods proposed in this paper provided the best approximation to direct measurements. Despite its wide use, the geometric approximation method showed the lowest accuracy. The approaches developed herein are timely because the recently increasing number of studies requiring reliable biomass estimates for small invertebrates to explain crucial processes in ecosystems.