Steroid profiles of wild terror green (Andinoacara rivulatus) associated with gonadal histology in the Baba river, Ecuador Juan Ortiz Tiradoa,1, Manjunatha Bangeppagarib,1, Alfonso Benavidesa,2, Daysi Muñoza,3, Pedro Romeroa,4, Sang Joon Leeb,2 a Department of Life Sciences, Universidad de las Fuerzas Armadas-ESPE, Sangolquí - P.O.BOX: 171-5-231B, Ecuador E-mail: 1 [email protected] , 2 [email protected],3 [email protected], 4 [email protected] b Center for Biofluid and Biomimic Research, Pohang University of Science and Technology, Pohang-37673, South Korea E-mail: 1 [email protected], 2 [email protected] Abstract— The native cichlids in Ecuador are represented by Andionoacara rivulatus and Cichlasoma festae mainly. These aquatic species are a fundamental part of the food chain of rural communities in the western subtropical of Ecuador. For the first time, a detailed study of the gonadal development in A. rivulatus males and females were carried out, as well as the characterization of the sex hormones present in the blood plasma during the period May 2019 to January 2020. The fish was captured in the multipurpose dam of the Baba river upstream of the Guayas River, Ecuador. Gonad and blood samples were collected for histological analysis and steroidal hormones such as 17β-estradiol, 11-ketotestosterone. The quantification of sex steroids was performed by enzyme immunoassay (ELISA). The results reflect that the fish has asynchronous characteristics and samples showed all development stages in testes and ovaries. The serological fluctuations detected statistical differences (p <0.05) between the development stages, with low concentrations of estradiol and 11-KT during the period 2019. These results relate to the hydrological periods of capture, where the temperature, luminosity, and rainfall play a fundamental role in the processes of oogenesis and spermatogenesis in fish. The highest concentrations of E2 were detected in female vitellogenesis stages and the highest concentration of 11-KT in the male spermatogenesis stage, confirming the fundamental role of these hormones in key periods of gonadal development. It is important that in the future, studies related to the determination of the maturational hormone concentrations, as well as the detection of plasma vitellogenin for gonadal development, are developed, and in this way contributes with basic information on the reproductive physiology of A. rivulatus. This information will be useful for the controlled management of the reproduction of this species, with a focus on the conservation and sustainability of local fisheries.. Keywords— Andinoacara rivulatus; green terror; gonad maturation; sex steroids; Baba river; Ecuador. reproductive endocrinology, which remain undisclosed [6]. I. INTRODUCTION The hypothalamic-pituitary-gonad (HPG) axis is the central Terror green (Andinoacara rivulatus) is a freshwater paradigm for the study of the biology of reproduction, which benthopelagic cichlid fish, locally known as “vieja azul” in can be affected by environmental, endocrine, paracrine, Ecuador. The genus Andinoacara is originally from north- autocrine, and intracrine factors [8-10]. western South American species found between Esmeraldas, Wild cichlids can naturally spawn in the tank when Ecuador and Tumbes, Peru, Figure 1 [1,2]. The global environmental conditions are favorable; however, several landings of A. rivulatus included caught and harvest from species of fish are inhibited from reproductive processes aquaculture and other fish farming with historical peaks of during captivity [11]. In general, inhibition of reproductive about 329 ton between 2000 and 2018, but there were processes occurs in females and is associated with final intensely decreasing catches in later years due to oocyte maturation, ovulation, and spawning [11]. These overexploitation, environmental pollution and loss of dysfunctions are most likely the result of a combination of habitats and refuges for spawning [3-5]. captivity-induced stress, the lack of a suitable environment Few studies related to nutritional behavior and taxonomy for natural spawning, and the social stimulation of a have been developed for the green terror [6,1,7]. However, compatible mate [12]. Endocrine regulation strategies are there are many relevant questions about reproductive widely used to synchronize fertility and improve biology, especially about the cycle of sexual maturation and 1 reproductive processes in fish, but the success of treatment classified according to their maturation status as immature, varies by species. In this regard, carp pituitary extracts and proliferation, growth, maturation and spawning. After the gonadotropin-releasing hormone (GnRH) analogs are widely maturation stage was determined, the samples were used in the critical stage of fish reproduction [13]. However, correlated with the levels of serological steroid. these treatments are not effective in the quality of C. Analysis of hormonal steroids reproduction in many species of cichlids and can affect the quality of the oocytes due to the difference in the structures Blood was collected from each fish by caudal vein via heparinized syringe and directly placed on ice, where they of the glycosidic residues present in the heterodimers that were allowed to clot for 3-6 h. Blood samples were later constitute the follicle-stimulating hormone (FSH), especially centrifuged for 15 min at 1500 g (Scilogex Centrifuge, in the β subunit, which can alter the response of the D3024 R) and serum stored at -80°C for further sex steroid steroidogenic signal when they bind to its specific membrane hormone analysis. receptor. In green terror farming, an alternative for An enzyme-linked immunoassay technique (ELISA) was reproductive management can be the use of tilapia pituitary, used by the commercial kits, protocols in 11- which has a homology and provides interesting results in in ketotestosterone (11-KT) and 17 β-estradiol (E2) (Cayman vitro and in vivo cultures in tilapia [10], also, the use of Chemicals Company, MI, USA). ELISA was performed for fortified foods to achieve greater productivity and controlled all samples in duplicates and a separate standard curve was spawning of captive wild breeders [6]. run for each ELISA plate. Different serial dilutions of the Considering the present problem, the analysis of steroid steroid were prepared and its levels were validated by levels in the blood has been used to clarify the optimal time running in parallel to the relevant standard curve. The limit to induce the hormonal spawning in fish, which can help to of steroid detection was 10 pg mL-1 by estradiol and 11-KT. obtain higher quality gametes, and therefore, strong larvae and viable for production, as well as preventing over D. Statistical analysis maturation and follicular atresia of gametes [14]. Strictly, the Data from steroids samples were monitored for similarity use of quantitative analysis of steroidal hormones in the of alteration and familiarity with the Levene and Shapiro- blood, as a method to predict the maturation stage of green Wilk tests, respectively. An analysis of variance (ANOVA) terror, implies less manipulation of the reproducers was achieved to determine the existence of significant compared to the current method of gonadal biopsies, which differences among treatments. Differences in mean values is invasive and requires large samples. On the other hand, were determined by Tukey´s test. Also, data were analyzed understanding of the concepts of reproductive organization, by the Kruskal-Wallis nonparametric test followed by the such as the maturation cycle, reproductive endocrinology, Mann-Whitney test. The results were considered significant and gonadal development is unclear in green terror. Thus, to if p≤0. 05. The data were expressed as mean ± SEM with improve the small-scale aquaculture with a focus on the Info-stat 5.0 statistical software. sustainability of local fisheries, research with native cichlids in Ecuador is transversal. Therefore, the objective of the present study was to recognize the developmental stages of gametes and characterize the plasma sex steroidal hormones during the maturation cycle of green terror in the wild. II. MATERIALS AND METHODS A. Biological samples The specimens were captured by shortline gear in 2-3 m depth at the Baba river multipurpose dam upstream Guayas river (0° 39’ 08.47’’ S; 79° 25’ 06.90’’ W, Buena Fé, Ecuador). Fishing activities were carried out from May 2019 to January 2020. Throughout this period, fishes were collected every three or four weeks depending on weather conditions. Immediately upon reaching the surface, fishes were sacrificed and the samples (blood and gonads) were collected and stored for further examination. Fig. 1 Andinoacara rivulatus “vieja azul” (a) male; (b) female. B. Gonadal histology For histological analysis, we used forty three samples of III. RESULTS gonads in different maturation stages from 23 females and The average temperature from May to October was 23.92 20 males of A. rivulatus were collected. After that the tissue ± 0.69 °C and from November to January 25.75 ± 0.49 °C, were fixed in 5% formalin for 24 h, then the fixed tissue was with rainfall of 34.10 ± 54.56 and 376.31 ± 265.69 mm, and subsequently dehydrated and embedded in the paraffin heliophany of 62.96 ± 25.93 and 69.04 ± 37.11 hours, blocks. The embedded tissue was longitudinally sectioned (7 respectively. Under these environmental conditions, the μm) and stained with hematoxylin and eosin. The fish sections were examined under a light microscope and 2 natural spawning frequencies detected were different (p of sperm, spermatocytes, and spermatogonia (Fig. 2 C). The <0.05). length of the testis was 25 ± 2 mm, width 4.00 ± 0.2 mm. Four individuals were found in the spermiation stage, with A. Gonad morphology and histology in male fish the exclusive presence of free semen in the lumen of the The testes of A. rivulatus males are pairs, of similar size lobular testicle (Fig. 2D) with a testis length of 30 ± 3 mm, and color. The organ is lobular with the same morphological width 6 ± 0.2 mm.