Are there differences in the frequency of micronuclei in Astyanax lacustris in relation to sex , mass and length ?

Lucilene Finoto Viana ORCID: https://orcid.org/0000-0002-6654-0355 Universidade Estadual de Mato Grosso do Sul, Brasil E-mail: lucilenefinoto@hotmail.com Bruno do Amaral Crispim ORCID: https://orcid.org/0000-0003-0606-4905 Universidade Federal da Grande Dourados, Brasil E-mail: brunocrispim.bio@gmail.com Alexeia Barufatti ORCID: https://orcid.org/0000-0001-8789-2117 Universidade Federal da Grande Dourados, Brasil E-mail: alexeiabarufatti@ufgd.edu.br Sidnei Eduardo Lima-Junior ORCID: https://orcid.org/0000-0001-7906-9737 Universidade Estadual de Mato Grosso do Sul, Brasil E-mail: selimajunior@hotmail.com


Introduction
Freshwater ecosystems are under great anthropogenic pressures, mainly due to changes in their surroundings, with the decrease in native vegetation cover replaced by large agricultural areas Pandey et al., 2018). Each year, agricultural boundaries expand and the scenery near water bodies becomes a fragmented vegetation presenting only mosaics or stretches of vegetation, causing several damages to the aquatic environment, such as fragile slopes, erosions, siltation and consequently habitat degradation and ecological imbalance (Dar et al., 2016;Lima et al., 2018). Thus, several contaminants are carried into the aquatic environment which can compromise survival, physiology of organisms and induce genetic damage, changes in cell cycle, chromosomal breakage and loss, and cell spindle malfunction, and compromising changes in DNA content (Darzynkiewicz et al., 2017;Corduk et al., 2018;Monteiro et al., 2018).
The environmental health assessment of aquatic ecosystems using fish as biomarkers (DNA damage) has been increasingly recommended due to responses to the effects of contamination on the aquatic environment by genotoxic agents (Viana et al., 2017;Lima et Research, Society and Development, v. 9, n. 8, e181985151, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5151 3 al., 2018Hussain et al., 2019). The micronucleus test, for example, detects aneugenic and clastogenic effects and has the ability to identify genotoxicity of a wide range of contaminants, which can lead to reduced local aquatic diversity, such as endemic species (Hariri et al., 2018;Hussain et al., 2019).
A. lacustris, known as 'yellow tail lambari', has a great ecological importance for the conservation of riparian forest, acting as a seed disperser helping to contribute to the balance in Brazilian hydrographic basins (Lima et al., 2011;Chehade et al., 2014;Siqueira-Silva, 2015;Milanin et al., 2018;Pinheiro et al., 2019;Abdalla et al., 2019). Besides this, shows characteristics that make it particularly useful as a bioindicator species, because it is an omnivorous, opportunistic species, and also has high plasticity for different environmental conditions that is abundant has a wide distribution in the Neotropical region (Siqueira-Silva, 2015; Milanin et al., 2018). Despite this, there is no information in the literature on the variation in genotoxic responses due to variables such as sex and size of individuals. In this background, the study aimed to assess genotoxic effects in the Astyanax lacustris and to test whether there are differences in the frequency of micronuclei in relation to sex, mass and length of individuals.

Data collection and analysis
The specimens of A. lacustris were sampled in the diurnal period with a rectangular metal sieve measuring 0.8 x 1.2 m, with a mesh of 2 mm. Soon after capture, the fish were immersed in cold water to reduce their activity, to obtain blood samples through caudal puncture, with heparinized syringes. For each fish sample, a drop of blood was placed on two slides, to form a thin layer. The smears were dried in air for 15 min, fixed in absolute alcohol for 10 min and then stained with Giemsa solution at 10% for 20 min (Schmid, 1975;Jesus et al., 2016). 2000 blood cells per slide were analyzed, resulting in a total of 4000 cells for everyone, using an optical microscope with magnification of 1000x. The micronuclei were identified following the criteria proposed by Fenech et al. (2003). The collection procedures were approved by the Ethics Committee on the Use of Animals at UEMS (011/2014) and Research, Society and Development, v. 9, n. 8, e181985151, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5151 authorized by IBAMA (SISBIO 11156-1).
In the laboratory, some biometric data were obtained for everyone: standard length (mm), total length (mm), total weight (g) and sex.
For statistical analyses of total weight, total length and micronuclei frequency in relation to sex we used a t-test (α=0.05), after to verify the assumptions of normality and homoscedasticity. To assess the micronuclei frequency in relation to mass and length of the individuals we calculated the correlation by Spearman's coefficient. All tests were performed using the R software (R Development Core Team, 2019).

Results and Discussion
In total, 37 specimens of A. lacustris were collected: 23 females and 14 males ( Table   1). The Figure 2 shows the frequency of micronucleus observed in males and females of A.
Several studies in the literature report positive responses of the species A. lacustris (including its synonyms species) as an environmental model to assess mutagenic and genotoxic effects of various contaminants through bioassays (Dourado et al., 2016;Viana et al., 2018;Pinheiro et al., 2019), but there is no information on the effect of sex and size of individuals on this biomarker. Another species of Characiformes (Prochilodus argenteus), for example, shows differences in the response of histopathological biomarkers in relation to the sexes, since males showed greater foci of chronic inflammation in the gills compared to females (Procópio et al., 2014). This result reinforces the need to know whether individuals of different sexes or different sizes of the A. lacustris have different genotoxic responses when exposed to the same environmental conditions.

Final Considerations
In our study we found that the species showed the same relative frequency of micronuclei for both sexes, and for individuals of different masses and lengths. As this species has been widely used in recent years as an bioassays model for different mutagenicity and genotoxicity tests, our results contribute to these assessments, so we demonstrate that there is no need to control the variables of sex, length and weight when carrying out these bioassays. The results of this study answered the proposed objectives and in addition to showing that both males and females of individuals of the species A. lacustris can be used as animal models for in situ and ex situ studies.