Ichthyoplankton studies provide valuable information on the biology of a species, making significant contributions to various fields of science. Specifically, they contribute to reproductive biology (Ribolli et al., 2020; Pachla et al., 2020; Frantine-Silva et al., 2022), ecology (Soares et al., 2022; Sulzbacher et al., 2023), systematics (Ticiani et al., 2022), aquaculture and fisheries (Loeb and Rojas, 1998; Lozano and Houde, 2013), and conservation (Pachla et al., 2020; Reynalte-Tataje et al., 2020a). Furthermore, these survey techniques are highly useful in inventory and diagnostic research, enabling the identification of breeding locations and periods (Pachla et al., 2020; Soares et al., 2022), as well as nursery areas (Ávila-Simas et al., 2014; Sulzbacher et al., 2023), migration routes (Sanches et al., 2006; Pachla et al., 2020), and factors affecting recruitment (Kemp and Froneman, 2004; Ferraz et al., 2022). They can also be used to evaluate the influence of anthropogenic factors, including the introduction of species (Vásquez-Yeomans et al., 2011; Reinas et al., 2022), pollution (Lima et al., 2014; Santos et al., 2022), and habitat fragmentation, among others (Reynalte-Tataje et al., 2012; Ziober et al., 2015; Silva et al., 2017; Rosa et al., 2022; Souza et al., 2023), on the ecosystem.
However, identifying eggs and larvae from both marine and freshwater environments has presented a significant challenge in conducting ichthyoplankton studies since Sars initiated such research in 1865 (Bialetzki et al., 2016). This obstacle makes ichthyoplankton research highly challenging (Bialetzki et al., 2016; Reynalte-Tataje et al., 2020b). In Neotropical environments, larvae identification is commonly performed by experienced taxonomists. However, owing to the high morphological similarity among species of the same genus, identification is often made at the genus or family level and rarely at the species level (Reynalte-Tataje et al., 2020b). At the same time, practical egg identification techniques have been overlooked based on the belief that such identification is impossible by the high level of similarity. Moreover, it is claimed that sampling procedures would complicate the challenge for several reasons. For example, organisms would undergo structural loss and/or damage from excessive sampling time and the substantial amount of suspended material accompanying the eggs, as well as the risk of improper sample handling. Importantly, the fixation process that alters the characteristic coloration of each egg could modify its membrane structure (Reynalte-Tataje and Zaniboni-Filho, 2008).
The remarkable biodiversity of Neotropical freshwater fish comprises over 6,200 species, representing the greatest diversity of vertebrate fauna in continental environments globally (Albert et al., 2020). Among them, potamodromous species are highly valued for fishing and aquaculture based on their size and zootechnical features (Carolsfeld et al., 2003; Reynalte-Tataje and Zaniboni-Filho, 2008). Furthermore, potamodromous species exhibit elevated levels of functional and taxonomic diversity. Moving between diverse habitats within a river, they play a pivotal role in the circulation of energy throughout the basin (Barthem et al., 2017; Pachla et al., 2022). Thus, these fish possess both ecological and socioeconomic importance and serve as valuable indicators of long-term habitat variations. At the same time, however, they are relatively long-lived and mobile, making them highly susceptible to anthropogenic impacts (Pompeu et al., 2012; Pelicice et al., 2015).
Riverine environments have undergone extensive degradation and fragmentation that have led to the decline, or even collapse, of many species in certain basins (Pelicice et al., 2015). Consequently, understanding the reproductive dynamics of potamodromous species, particularly the identification of spawning sites, is essential for establishing guidelines to promote their long-term maintenance (Reynalte-Tataje et al., 2012; Pachla et al., 2020; Reynalte-Tataje et al., 2020a). In recent years, interest in ichthyoplankton studies has been growing. These studies focus on the early forms of migratory fish, such as fish eggs and larvae. These studies also involve sampling the ichthyoplankton community to gather information on important aspects of fish biology, including spawning and nursery areas, reproductive period, and other relevant factors (Ávila-Simas et al., 2014; Pachla et al., 2020; Soares et al., 2022).
In the literature, studies that have sought to identify the eggs of Neotropical potamodromous fish have employed either of two methods. First, semi-dense eggs are separated with a large, non-adhesive perivitelline space (Nakatani et al., 2001; Soares et al., 2022). In this case, a given set of eggs would represent the number of eggs of potamodromous species present in the environment, but without any classification by species or genus. A second, alternative method, uses genetic tools. The genetic method has shown promise in identifying eggs and larvae, as well as providing a way to quantify the identified organisms (Frantine-Silva et al., 2015; Mariac et al., 2018; Lima et al., 2020; Lira et al., 2022; Teixeira et al., 2023). However, this technique is expensive and therefore inaccessible to most researchers. It also requires the establishment of species-specific primers in advance for identification, and it is a destructive method.
Here we report a novel technique, termed as the Live Ichthyoplankton Identification Technique (LIIT), which has been developed, applied and improved over the course of a decade to enable the identification of eggs of potamodromous species.