Morphological and temporal variations in the gametogenic development of the killifish Melanorivulus rossoi (Cyprinodontiformes, Rivulidae) in an area under crop influence

Melanorivulus rossoi has a restricted distribution and, due to land modifications for agricultural use, may be on the verge of extinction. So, understanding its gametic development and reproductive cycle can expand the knowledge of the species and possible reproductive and/or ecological interventions. Specimens were collected in swamps adjacent to crop areas in Campo Grande, Mato Grosso do Sul, Brazil. Individuals were fixed in Karnovsky’s solution, gonadal samples were extracted and submitted to the histological preparation protocol. 221 individuals were analyzed, with the highest abundance of individuals collected from February to September. Larger fish were observed in the last months of the year, especially in November, while the smallest ones were collected in June. For females, all individuals presented ovarian follicles in different periods of maturation and gonadal differentiation. The same was observed for males, with constant proportions of spermatic cells. Thus, for M. rossoi, it has been found that the production of gametic cells in both male and female gonads is constant, characterizing a synchronous cell development in males and females. The data observed for Melanorivulus rossoi compared to M. punctatus, previously studied by our research group, showed that despite the species being related, the gametogenesis data have some differences.


Introduction
Rivulidae fish are exclusively freshwater (Hrbek and Larson 1999) and are distributed over a wide area from southern Mexico to the pampas of Argentina (Parenti 1981).Altogether, there are 39 genera included (Fricke et al. 2023a), and its greatest diversity can be found in Brazil, with 60% of the total species of the family (Costa 2008).
Among these representatives, Melanorivulus is a nonseasonal fish genus (Costa 2011), with greater species richness and widely distributed (Costa et al. 2015).There are currently 61 species described for this genus (Fricke et al. 2023b).They are present in different types of habitats, with the greatest diversity found in the Paraná and Alto Araguaia River basins (Costa et al. 2015;Nielsen et al. 2016), including the model species of this study-Melanorivulus rossoi (Costa 2005).
Melanorivulus rossoi has a restricted distribution on a set of wetlands in the Anhanduí river drainage, Alto Paraná basin, Mato Grosso do Sul state (Costa 2005;Severo-Neto and Volcan 2018).The species presents sexual dimorphism, represented by differences in the morphology of the fins (Costa 2005), in addition to color characters, such as the presence of a brownish eye spot on the caudal fin of females and the absence of red spots on its dorsal fin (Severo-Neto and Volcan 2018).Rivulidae is the Brazilian fish family with the largest number of species in some degree of threat of extinction (Castro and Polaz 2020).Due to land modifications for agricultural use, M. rossoi is one such species (Severo-Neto and Volcan 2018).Thus, studies related to the reproductive biology of this species are necessary.
The biology of reproduction is an area that involves several fields of research, from behavior, hormonal regulation, and gametogenesis.Gametogenesis can be defined, briefly, as the process of formation of sperm or oocytes in the gonad.Although briefly described, gametogenesis is a process that involves highly complex steps.It comprises successive periods of germ cell proliferation, cell cycle pauses and gametic differentiation, with strong variations between females and males (Brown-Peterson et al. 2011).
Studies regarding the gametogenesis of Melanorivulus species are scarce in the literature.Cassel et al. (2013), in one of the few existing works, described for Melanorivulus aff.punctatus lobular testicles, like elongated pouches converging on the urogenital papilla, and spermatogenesis with a continuous cycle.They have a cyclic and annual reproduction, as in most Teleosts (Wildner et al. 2013), with a continuous reproductive cycle for the male and discontinuous for the female (Cassel et al. 2013).In addition, it should be noted that this species has a high tolerance for extreme environments, with little dissolved oxygen, acidic pH, and high concentration of ammonia (Taylor 2012).
Among the studies of the reproductive strategies of a species, as well as the tactics developed, understanding the reproductive cycle is what makes it possible to recognize the period of reproduction and the gonadal morphological changes that occur (Suzuki et al. 2004;Vazzoler 1996).In this way, understanding the gametic development and reproductive cycle of M. rossoi can expand the knowledge of the species and possible reproductive and/or ecological interventions.This is even more important when considering that this species is in an area of agricultural interest and, consequently, on the verge of extinction.Thus, emphasis is given in this study to the morphological and temporal variations found for this species, based on other studies of this group of fish.

Materials and methods
Specimens of Melanorivulus rossoi were collected in swamps adjacent to crop areas in the municipality of Campo Grande, state of Mato Grosso do Sul.Testimonial specimens were fixed in 10% formaldehyde and deposited in the Zoological Collection of the Federal University of Mato Grosso do Sul (ZUFMS).The collections were carried out monthly between the months of April/2015 and March/2016.
All individuals were fixed in Karnovsky's solution and measured using a manual caliper, obtaining data on their standard length (head to the end of the caudal peduncle).
Gonad samples were extracted and submitted to the histological preparation protocol, with dehydration in 70% ethanol for 24 h, followed by 100% ethanol for another 24 h.Afterwards, the samples were infiltrated and included in hydrophobic plastic resin of the glycol methacrylate type.Blocks were cut to 3 μm thick, stained with 1% Toluidine Blue and photographed.Considering that the specimens were very small, the identification of the gonads, and consequently the sex of the specimen, could only be done after histological processing.The analysis followed the morphological and gametogenic cycle parameters described in Cassel et al. (2013).

Results
A total of 221 individuals distributed over 12 months were analyzed.Table 1 shows the sample numbers analyzed monthly.It should be noted that, although the sampling effort was the same in all months, few individuals were collected in January, November, and December, with no individuals in October.Highest abundance of individuals was collected from February to September.
In addition to the collection data, only the standard length was considered for length analysis since many specimens lost their tail, both due to manipulation and possible predation prior to collection.Even so, it was possible to observe that larger fish are observed in the last months of the year, with the largest individuals being collected in the month of November (average of 2.95 cm in length).The smallest individuals were collected in the month of June, with an average of 2.22 cm long (Fig. 1).
As for the histological evaluation of the female gonads, in all individuals sampled, regardless of the time of year, ovarian follicles are observed in different periods of maturation and gonadal differentiation (Fig. 2).Thus, oogonia and early-stage oocytes are observed (Fig. 2A, B), the latter going through two phases of growth.The first phase occurs with the primary oocyte, where alveoli, vesicles and some yolk droplets begin to appear (Fig. 2A, B).In secondary oocytes, however, there is a large accumulation of yolk, responsible for increasing the width of the oocyte and for giving the impression of "covering" the cell nucleus, which has an irregular shape (Fig. 2A, C).Maturation occurs when the alveoli and nucleus migrate to the periphery of the cell (Fig. 2C).Mature ovarian follicles occurred in all months sampled, however, in the rainiest months (November to February) the female gonads seem to be occupied by only these cell types.Thus, these individuals were classified as able to reproduce, as shown in Fig. 3.While for males, all cell types of spermatogenesis were observed, from spermatogonia to spermatozoa (Fig. 4).These cells seem to have similar and constant proportions in all months of the year.

Discussion
The morphological appearance of gametic cells does not vary, which is expected within the same group of vertebrates.Morphological variations in the development and final characteristics of gametes can only be perceived between genera and under an ultrastructural analysis.However, analysis under light microscopy reveals diversities between organisms of the same species that live in different environmental and ecological conditions.The study among the species of the Rivulidae family, which in this case were chosen M. punctatus and M. rossoi, took place in two very different locations.
In the study with M. punctatus, specimens were collected in the locality of the Municipal Reservoir of Quineira, in the municipality of Chapada dos Guimarães, state of Mato Grosso (data published by Cassel et al. 2013).The study, conducted in the Cerrado biome and in a reservoir with perennial water, found that there was a period of ovarian recrudescence and constant testicular gametic production.These data gave indications that, for some reason, the females of the species reduced the energetic investments of their physiology to produce gametes in a short period of the year.This reduction in female gametic production implies periods of asynchronous reproductive fitness for this group of fish in this locality.
For the study with M. rossoi, specimens were collected from a swamp located in a matrix of crops in the Cerrado, with a marked seasonality between rainy and dry seasons, which may dry out in some periods.It has been found that the production of gametic cells in both male and female gonads is constant.There may be a reduction in the proportion of mature ovarian follicles in the first months of the year and right after the rainiest period, but mature gametic cells are observed in all periods of the year.These characteristics confer reproductive fitness in terms of the presence of gametes ready for fertilization in all periods of the year.Thus, the cycle of gametogenic activity in M. rossoi is synchronous between males and females.
The data observed for Melanorivulus rossoi compared to data previously studied by our research group for M. punctatus (Cassel et al. 2013), shows us that despite the species being related, the gametogenesis data analyzed shows some differences.The observed differences regarding the gametogenesis of M. punctatus and M. rossoi are interesting data for future speculations and hypotheses regarding the adaptive processes of fish and their reproductive physiology in locations under different environmental and ecological conditions.

Table 1
Total number of Melanorivulus rossoi specimens collected per month, number of individuals separated by sex and distributed over the months of collection, and the variation in its standard length separated by sex and month of collection

Fig. 1 Fig. 2 Fig. 3
Fig. 1 Box-plot graph with the standard length of specimens and the variation observed each month