Steroid profiles of wild terror green ( 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 in Ecuador are represented by Andionoacara rivulatus and 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; ; 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 fish, locally known as “ azul” in can be affected by environmental, endocrine, paracrine, Ecuador. The 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 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. Finally, five fish were found in the characteristics throughout the tissue and is located next to spawning stage with the presence of a little amount of free the swim vesicle of the fish. Histological analyzes of the sperm and occasional spermatogonia in the lumen of the tests show the presence of all stages of gonadal development testis. (Table 1). B. Gonad morphology and histology in female fish TABLE . I The ovaries of A. rivulatus are paired organs, elongated HISTOLOGICAL CLASSIFICATION OF GONADAL MATURITY and with bilobed sacs, located around the swim vesicle. The STAGE OF WILD TERROR GREEN (A. RIVULATUS). wall of the ovaries is transparent and thin and contains oocytes in different stages of development, characteristic of Females Males an asynchronous fish (Fig. 3). Stages (n=23) (n=20) Immature 3 7 Proliferation (spermatogenesis) 8 2 Growth (spermiogenesis) 4 2 Maturation (spermiation) 2 4 Spawning 6 5

Seven immature individuals present longitudinal and width testes of 18 ± 1 mm and 2 ± 0.3 mm, respectively, with germ cells in testicular lobes and primary spermatogonia, Sertoli cells bordering the spermatogonia, spermatocytes, and fibroblasts in the interlobular interstitial tissue (Fig. 2A). Two individuals were found in the spermatogenesis stage. The presence of different generations of spermatogonia and spermatocytes of secondary spermatogonia that proliferate mitosis is observed. The Fig. 3. Asynchronous ovary of A. rivulatus. (4x) (1500 µm). length of the testis was 20 ± 2 mm and width 2 ± 0.3 mm. Each spermatogonium gives rise to an isogenic germ cell The fusion of the ovaries with an extended and short clone which in turn will give rise to spermatozoon oviduct can be observed, which is directed towards the spermatocytes (Fig. 2B). urogenital pore. In the early stages, the ovaries show a yellowish coloration, increasing their coloration according to the advance of the different stages of development with an absolute fecundity of 1884 ± 250 oocytes per fish. Histological analysis reports that the ovaries of all the sampled fish are at different stages of development (Table 1). Three individuals were found in an immature state with the formation of follicular cells, where ovarian lamellae are observed with a fold of the germinal epithelium that covers the internal part of the ovary. Also, the tunica albuginea and various oogonia immersed in the ovarian stoma can be observed, giving rise to the perinuclear follicles (Fig. 4A). Eight individuals were found in the proliferation, first growth stage, and show the presence of previtellogenic oocytes in different stages of primary follicular development and the beginning of secondary growth. The ovarian cavity can be visualized, with several groups of oogonia, follicular layers, and zona radiata (Fig. 4B). Four individuals were found in the early vitellogenesis phase, which contained oil Fig. 2. Cross sections of A. rivulatus testes showing different maturity stages. A) Testis in immature stage (40x), B) testis in proliferation stage droplets and cortical alveoli. Second growth phase follicles (40x), C) testis in the growth stage (40x), D) testis in the maturation stage with lipid inclusions were also detected in the ooplasm, (40x). located around the nucleus with numerous nucleoli. The cortical nucleoli arranged in the peripheral ooplasm, below Two individuals were found in the spermiogenesis stage, the zona radiata (Fig. 4C) are observed. with a dominance of spermatids and a considerable quantity

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Fig. 5. Plasma steroidal profiles of A. rivulatus males during maturation cycle (Mean ± Standard Error) (P < 0.05).

17β-estradiol The estradiol concentrations in A. rivulatus plasma shows significant differences between the gonadal stages of development (p <0.05). These differences occur between the developmental phases and the vitellogenesis period. The average ovaries length of the females was 13.13 ± 1.45 cm, with a body weight of 171.12 ± 40.9 g. The gonadal immature stage maintains a concentration of 94.99 ± 5.10 pg mL-1 and the proliferation stage with 124.78 ± 16.48 pg mL-1. After this behavior, a significant increase (p <0.05) in the vitellogenesis stage is seen, reaching concentrations of 668.68 ± 436.93 pg mL-1. Subsequently, estradiol levels decrease significantly (p <0.05) at concentrations of 58.76 ± 2.31 pg mL-1 and 36.91 Fig. 4. Cross sections of A. rivulatus ovaries showing different maturity ± 8.56 pg mL-1 corresponding to the maturation and stages. A) Ovary in mature stage (40x), B) Ovary in proliferation stage ovulation stages respectively (Fig. 6). On the other hand, (10x), C) ovary in vitellogenesis stage (10x), D) Oocite in vitellogenesis estradiol concentrations were detected in the immature stage (40x), E) Ovary in the maturation stage (4x), F) Ovary in the spawning stage (4x). male's plasma, with concentrations that fluctuated from 10.4 to 32.75 pg mL-1, and according to the histological samples Two individuals were found in the maturation stage, with analyzed (Fig. 2A). a notable increase size due to hydration, with lipid inclusions and yolk granules distributed as a crown in the peripheral ooplasm, with the area radiata thickened and surrounded by follicular layers (Fig. 4D, 4E). Finally, six fish were found in the spawning phase, since they present a large number of post-ovulatory follicles, atresic oocytes, and oocytes in the early stages of oogenesis (Fig. 4 F).

C. Plasma sex steroid hormonal profile 11-keto-testosterone The concentrations of 11-KT in plasma show significant differences (p <0.05) between the males found in the proliferation stage and other stages of development. The Fig. 6. Plasma steroidal profiles of A. rivulatus females average size of the males was 20.54 ± 2.04 cm with a during maturation cycle (Mean ± Standard Error) (P < 0.05). bodyweight of 214.00 ± 64.33 g. In the immature stage, the fish maintained an average of IV. DISCUSSION 11-KT in plasma of 215.75 ± 39.48 pg mL-1. However, during spermatogenesis periods, the levels of this hormone The presence of the Cichlidae family is common on the increased significantly (p <0.05), reaching high levels up to Ecuadorian coast, with several species such as Andinoacara 1357.2 ± 121.75 pg mL-1. According to gonadal rivulatus, Cichlasoma festae, sapayensis [15], development, the concentrations of 11 KT began to decrease Aequidens coeruleopunctatus [16,17], as well as exotic progressively from 849.56 ± 200.98 pg mL-1, 464.28 ± species such as the Nile tilapia (Oreochromis niloticus), 120.30 pg mL-1, and 103.33 ± 51.55 pg mL-1 in the tilapia hybrids (Oreochromis sp.) among others [18]. spermatogenesis, spermiation, and spawning stages Studies concerning reproductive anatomy and physiology respectively (Fig. 5). are important to understand the reproduction of these species in natural environments. This study represents the first approximation with histological details of the gonadal development in different stages of development, as well as

4 the profile of sex steroids related to the reproduction of A. rivulatus. The monitoring of environmental parameters such rivulatus. as temperature, oxygen, pH, salinity, light and conductivity Histological evaluation was able to detect that germ cells affects the reproductive cycles in fish, showing the increase present all stages of development, clearly determining an in the number of oocytes per laying and therefore the asynchronous ovary and testicular development. The steroidogenic behavior in asynchronous fish. Furthermore, it histology of the testes shows the 4 relevant stages such as should be taken into consideration that the enzymatic spermatogenesis, spermiogenesis, spermatocytes, as well as machinery in asynchronous fish has direct effects on gonadal the release of sperm into the lumen. In this species, maturation and steroidogenesis [29]. morphological and physiological changes of the testicle can It was evidenced that A. rivulatus has a buccal incubation, be observed and that are directly associated with the same ones that present a specific gonadal structure in the spermatogenesis with the characteristics described in each spawning stages, primary oogonial formation, and stage of development [19]. The majority of organisms show previtellogenic proliferation. Baroiller et al. [30] describe the the release of sperm in the tubular lumen and from there to enzymatic activity 3β-hydroxysteroid dehydrogenase is the vas deferens, which suggests that the organisms are in a over-expressed in buccal incubation periods in commercial continuous spawning process. About the presence of cichlids, in addition to detecting differences in the enzymatic estrogens in male plasma samples, it is related to the renewal activity of cytochrome P450c17, 17α hydroxylase, and 17,20 of primary spermatogonia, but not to spermatogonial lyase, after spawning and before ovulation. These enzymes proliferation [20,21], therefore, the samples analyzed in are important, since they regulate the pathway for E2 immature stages of A. rivulatus show the behavior of these synthesis and the maturational hormone (DHP), having a characteristics. However, low concentrations of 11-KT direct effect on the proliferation, vitellogenesis, maturation, would also be an incidence in primary spermatogonia as in and spawning stages in asynchronous fish. the proliferation phase [22]. The androgen 11-ketotestosterone is the most V. Conclusion representative hormone that regulate the behavior of spermatogenesis, from spermatogonial proliferation to This study reports the serological fluctuations in natural spermiogenesis [23,24]. The concentration of 11-KT in the conditions of estradiol and 11-ketotestosterone during the spermatogenesis phase is high compared to the other stages gonadal maturation of A. rivulatus in the Baba River of development, demonstrating the importance of this reservoir. These hormones are responsible during the periods hormone in this phase of development. In other fish such as of oogonial proliferation and vitellogenesis in adult females, Neotropical cichlids there is a direct effect on the behavior as well as in the processes of spermatogenesis, and the concentrations are different [25], however, it should spermiogenesis, and spermiation in males respectively. It is a be noted that the samplings were carried out in cold seasons, kind of asynchronous characteristics where gonadal so the low concentrations of the steroid in A. rivulatus, they development is closely related to environmental conditions could be affected by the average temperature during the and where the effect of temperature affects gonadal sampling period [26]. development under natural conditions. In future studies, it is The gonadal development and histology for the A. important to monitor the maturational hormone 17α, 20β-DP, rivulatus females are similar to that of other commercial as well as the vitellogenin concentrations and thus complete cichlids such as tilapia zilli and other tilapias [27], exhibiting the understanding of the reproductive physiology of A. the same characteristics at each stage of oocyte development rivulatus. with asynchronous characteristics. Relative fertility in the study by Jamali et al. [6] shows ranges from 8.5 to 28.6 Acknowledgment oocytes g-1 of female, and that are comparable with those obtained in A. rivulatus under natural conditions by 9.54 to This work was supported by the following grants: PIC-18- 12.4 oocytes g-1 female. INE-ESPE-001 (INEDITA - SENESCYT), Universidad de Studies related to the reproductive cycle of commercial las Fuerzas Armadas - ESPE (Vice-Rector's Office for cichlids detect parallel fluctuations in FSHβ and LHβ Research and Innovation), funds administration PNUD promoter genes after spawning and ovulation. This suggests (Program for the United Nations Development), and the that FSH and LH play an important role in asynchronous fish, authors gratefully acknowledges for financially supported by both for vitellogenesis and for maturation and spawning the Korea Research Fellowship Program through the [8,9]. In the present study, elevated concentrations of E2 are National Research Foundation of Korea (NRF) funded by detected in vitellogenesis of A. rivulatus, but much lower the Ministry of Science and ICT (KRF Grant No. than the concentrations detected in commercial cichlids 2016H1D3A1938252). To the local fishermen Guayas rivers, ranging from 7.7-9.7 to 20.8-23.1 ng mL-1 [28,11]. In this Buena Fé - Ecuador, especially Mrs. Angela Rodríguez and sense, it could be speculated that the response generated in Mr. Oscar Andrade for your entire helps in the recollection follicular cells of the ovary does not depend only on the sample fish. availability of circulating peptide hormones, but also on nearby factors such as light and temperature and that it REFERENCES would affect the seasonality and concentration of E2 and [1] Z. Musilová, O. Říčan, K. Janko, J. Novák. Molecular phylogeny and therefore the concentrations Vitellogenin (Vtg) in plasma, biogeography of the Neotropical cichlid fish tribe prolonging gonadal development during this period [10] and (Teleostei: Cichlidae: ). Molecular Phylogenetics that are related to the low concentrations detected in A. and Evolution, 46(2), 659–672, 2008. doi:10.1016/j.ympev.2007.10.011.

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[2] FISHBASE, 2020. https://www.fishbase.de/summary/Aequidens- Growth and Differentiation, 38(3), 257–262, 1996. rivulatus.html). doi:10.1046/j.1440-169x.1996.t01-2-00004.x. [3] G. Alvarez-Mieles, K. Irvine, A. V. Griensven, M. Arias-Hidalgo, A. [20] T. Miura, C. Miura, T. Ohta, M. R. Nader, T. Todo, K. Yamauchi. Torres, A. E. Mynett. Relationships between aquatic biotic Estradiol-17β stimulates the renewal of spermatogonial stem cells in communities and water quality in a tropical river–wetland system males. Biochemical and Biophysical Research Communications, (Ecuador). Environmental Science & Policy, 34, 115–127, 2013. 264(1), 230–234, 1999. doi:10.1006/bbrc.1999.1494. http://doi:10.1016/j.envsci.2013.01.011. [21] M. A. Amer, T. Miura, C. Miura, K. Yamauchi. Involvement of sex [4] M. Arias-Hidalgo, G. Villa-Cox, A. V. Griensven, G. Solórzano, R. steroid hormones in the early stages of spermatogenesis in Japanese Villa-Cox, A. E. Mynett, P. Debels. A decision framework for huchen (Hucho perryi )1. Biology of Reproduction, 65(4), 1057–1066, wetland management in a river basin context: The “Abras de 2001. doi:10.1095/biolreprod65.4.1057. Mantequilla” case study in the Guayas River Basin, Ecuador. [22] T. M. Kortner, E. Rocha, A. Arukwe. Previtellogenic oocyte growth Environmental Science & Policy, 34, 103–114, 2013. and transcriptional changes of steroidogenic enzyme genes in http://doi:10.1016/j.envsci.2012.10.009. immature female Atlantic cod (Gadus morhua L.) after exposure to [5] Food and Agriculture Organization, FAO, 2020. the androgens 11-ketotestosterone and testosterone. Comparative http://www.fao.org/figis/servlet/SQServlet?file=/usr/local/tomcat/8.5. Biochemistry and Physiology Part A: Molecular & Integrative 16/figis/webapps/figis/temp/hqp_4763616801375773071.xml&outty Physiology, 152(3), 304–313, 2009. doi:10.1016/j.cbpa.2008.11.001. pe=html. [23] T. Ohta, H. Miyake, C. Miura, H. Kamei, K. Aida, T. Miura. [6] H. Jamali, N. Ahmadifard, F. Noori, E. Gisbert, A. Estevez, N. Agh, Follicle-stimulating hormone induces spermatogenesis mediated by N. Lecithin-enriched Artemia combined with inert diet and its effects androgen production in Japanese eel, Anguilla japonica1. Biology of on reproduction and digestive enzymes of Aequidens rivulatus. Reproduction, 77(6), 970–977, 2007. Aquaculture, 511, 734253, 2019. doi:10.1095/biolreprod.107.062299. doi:10.1016/j.aquaculture.2019.734253. [24] T. Guellard, H. Kalamarz-Kubiak, E. Kulczykowska. Concentrations [7] A. Neissi, A., Rafiee, G., Nematollahi, M., Razavi, S. H., & Maniei, of melatonin, thyroxine, 17β-estradiol and 11-ketotestosterone in F. (2015). Influence of supplemented diet with Pediococcus round goby (Neogobius melanostomus) in different phases of the acidilactici on non-specific immunity and stress indicators in green reproductive cycle. Reproduction Science, 204, 10–21, 2019. terror (Aequidens rivulatus) during hypoxia. Fish & Shellfish doi:10.1016/j.anireprosci.2019.02.014. Immunology, 45(1), 13–18. doi:10.1016/j.fsi.2015.04. [25] M. R. Ramallo, A. Birba, R. M. Honji, L. Morandini, R. G. Moreira, [8] A. H. Berg, L. Westerlund, P. E. Olsson. Regulation of Arctic char G. M. Somoza, M. Pandolfi. A multidisciplinary study on social (Salvelinus alpinus) egg shell proteins and vitellogenin during status and the relationship between inter-individual variation in reproduction and in response to 17β-estradiol and cortisol. General hormone levels and agonistic behavior in a Neotropical cichlid fish. and Comparative Endocrinology, 135(3), 276–285, 2004. Hormones and Behavior, 69, 139–151, 2015. http://doi:10.1016/j.ygcen.2003.10.004. doi:10.1016/j.yhbeh.2015.01.008. [9] J. Bobe, T. Nguyen, B. Jalabert. Targeted gene expression profiling [26] R. J. Wilkinson, R. Longland, H. Woolcott, M. J. R. Porter. Effect of in the Rainbow trout (Oncorhynchus mykiss) ovary during elevated winter–spring water temperature on sexual maturation in maturational competence acquisition and oocyte maturation. Biology photoperiod manipulated stocks of rainbow trout (Oncorhynchus of Reproduction, 71(1), 73–82, 2004. mykiss). Aquaculture, 309(1-4), 236–244, 2010. doi:10.1095/biolreprod.103.025205. doi:10.1016/j.aquaculture.2010.08.023. [10] J. Ortiz Tirado, L. Valladares, D. Munoz, J. Caza, B. Manjunatha, R. [27] K. Coward, N. Bromage. Spawning frequency, fecundity, egg size R. Kundapur. Levels of 17β estradiol, vitellogenin, and and ovarian histology in groups of maintained upon two distinct food prostaglandins during the reproductive cycle of Oreochromis ration sizes from first-feeding to sexual maturity. Aquatic Living niloticus. Latin American Journal of Aquatic Research, 45(5), 930– Resources, 12(1), 11–22, 1999. doi:10.1016/s0990-7440(99)80010-2. 936, 2017. http://doi:10.3856/vol45-issue5-fulltext-8. [28] P. J. Babin, J. Cerdà, E. Lubzens. (Eds.). (2007). The fish oocyte. [11] B. Levavi-Sivan, J. Biran, E. Fireman. Sex steroids are involved in doi:10.1007/978-1-4020-6235-3. the regulation of gonadotropin-releasing hormone and dopamine D2 [29] M. Carrillo, S. Zanuy, A. Felip, M. J. Bayarri, G. Molés, A. Gómez. receptors in female tilapia pituitary 1. Biology of Reproduction, Hormonal and environmental control of puberty in perciform fish. 75(4), 642–650, 2006. doi:10.1095/biolreprod.106.051540. Annals of the New York Academy of Sciences, 1163(1), 49–59, 2009. [12] E. Bonnet, A. Fostier, J. Bobe. Microarray-based analysis of fish egg doi:10.1111/j.1749-6632.2008.03645.x. quality after natural or controlled ovulation. BMC Genomics, 8(1), [30] J.-F. Baroiller, Y. Guiguen, A. Fostier. Endocrine and environmental 2007. doi:10.1186/1471-2164-8-55. aspects of sex differentiation in fish, Cellular and Molecular Life [13] J. Aizen, H. Kasuto, M. Golan, H. Zakay, B. Levavi-Sivan. Tilapia Sciences, 55(7), 910, 1999. doi:10.1007/s000180050344. Follicle-Stimulating Hormone (FSH): Immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion1, Biology of Reproduction, 76(4), 692–700, 2007. doi:10.1095/biolreprod.106.055822. [14] E. M. Donaldson. Manipulation of reproduction in farmed fish. Animal Reproduction Science, 42(1-4), 381–392, 1996. doi:10.1016/0378-4320(96)01555-2. [15] Regan. A revision of the fishes of the South-American cichlid genera Acara, Nannacara, Acaropsis, and Astronotus. Annals and Magazine of Natural History, Ser. 7, 15: 329-347, 1905. [16] R. Kner. Eine Uebersicht der ichthyologischen Ausbeute des Herrn Professors Dr. Mor. Wagner in Central-Amerika. Sitzungsber. Königlich Bayerische Akademie der Wissenschaften zu München 2:220-230, 1863. [17] R. Samaniego. Evaluación de parámetros de crecimiento e inmunidad en vieja azul Andinoacara rivulatus utilizando dietas con diferentes niveles proteicos y suplementadas con Astaxantina, 2015. (Tesis pregrado, Escuela Politécnica del Litoral). Recuperado de https://www.dspace.espol.edu.ec/bitstream/1234567 89/ 34064/1/20151SFMAR052801_2.PDF. [18] G. Marcillo, J. Landivar. Tecnología de producción de alevines monosexo de Tilapia, Primera edición. IEPI: 029318. ESPOL, 2008. [19] C. Miura, T. Miura, M. Yamashita, K. Yamauchi, Y. Nagahama. Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis. Development,

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DEVELOPMENT, PURIFICATION, AND CHARACTERIZATION OF IGY ANTIBODIES AGAINST VITELLOGENIN OF THE "VIEJA COLORADA” FISH (Cichlasoma festae), A SPECIES NATIVE TO THE GUAYAS RIVER, ECUADOR. 1 1 1 1 1 Juan Ortiz Tirado* , Jairo Caza , Andrea Tufiño , Henry Pardo , Daysi Muñoz & Bangeppagari

Manjunatha 2

Address (es): 1 Universidad de las Fuerzas Armadas-ESPE, Department of Life Sciences, Laboratory of Water Resources and Aquaculture, Sangolquí – P.O.BOX: 171-5-231B, Ecuador, South America. 2 Center for Biofluid and Biomimic Research, Pohang University of Science and Technology (POSTECH), Pohang - 37673, South Korea.

*Corresponding author: [email protected]

ABSTRACT

Vitellogenin (Vtg) is a specific phospho glycoprotein used as an environmental and steroidogenesis biomarker in fish. The present study shows the techniques to isolated IgY anti vitellogenin (Vtg) in Cichlasoma festae fish. Vtg was purified from adult males previously treated with estradiol and isolated from blood plasma using ion-exchange chromatography. Subsequently, Lohmann Brown hens were immunized at different doses, including three antigen booster doses. At day 45 post-immunization, posture eggs were collected, and successfully optimized the extraction of anti-Vtg -IgY antibody. The purified antibodies were validated with SDS-PAGE, detecting heavy and light chains of 70 kDa and 30 kDa, respectively. Further, we developed ELISA to estimate the Vtg specificity. Commercial anti-Vtg antibodies of Dorada (Sparus aurata) were used as a control, and immunoglobulins Y were validated for native species by Western Blot. The IgY anti-Vtg obtained by local conditions can be successfully used for programs of environmental monitoring and reproduction control in native aquatic species in Ecuador.

Key words: Cichlasoma festae . Vitellogenin . IgY . Western Blot . ELISA

INTRODUCTION

Neotropical cichlid Cichlasoma festae is a Guayas River´s native species, locally known as “vieja colorada” in Ecuador. C. festae is considered a unique group inside the tribe with more than 150 species where many species of this clade hasn´t been described completely, Figure 1 (López- Fernández et al., 2010; Tubert et al., 2012).

Fig.1 Cichlasoma festae “vieja colorada” female

In the Guayas River there is a dramatic decrease in fisheries groups, where native cichlids as Andinoacara rivulatus (Gunther 1859) and Cichlasoma festae (Boulenger 1899) are the most affected by different factors: i) environmental pollution caused by chemical and organic discharges from the agricultural sector; ii) loss of habitats and refuges for spawning, due to the dam's construction; iii) high fisheries extractions due to the demand for fishery products (Alvarez - Mieles et al., 2013; Arias- Hidalgo et al., 2013). The Statistical data from the National Fisheries Institute (INP) in the Chongón reservoir show a dramatic reduction in the extraction of "Vieja Azul" fish (Andinoacara rivulatus) and other native cichlids fish with 2 ton at 2016 and nothing in 2017 to 2018 (FAO, 2018). The same situation can find with other freshwater fish as Pseudo curimata boulengeri (Eigenmann 1907), Brycon alburnus (Gunther 1860) , Hoplias microlepis (Gunther 1864), Dormitator latifrons (Richardson 1844) and Ichthyocelephas humeralis (Gunther 1860), with the high risks to lost much native fish germplasm (Alvarez - Mieles et al., 2013). With this background, conservation strategies of native species should be established in Ecuador, where the reproduction management and repopulation of native species into hydrographic basins are too important for a sustainable fishery. These processes are related to the reproductive health of female fish and where the Vtg production is a marker of oocyte growth. Vtg is a complex high molecular weight phospho-lipo-glycoprotein that binds calcium and is produced by liver hepatocyte in response to 17β-estradiol action by the interaction with intracellular receptors alpha type (ERα) (Patiño, 2003). The Vtg genes are highly conserved and their number depends on the species and it generates multiple Vtg specifics isoforms, with a different role in vitellogenesis periods, oocyte maturation and subsequent embryonic development, and in the surface mucus of females, Vtg could have a pheromonal function (Garstka and Crews, 1981; Berg et al., 2004 ). On the other hand, Vtg measurements could provide a presence of endocrine disruptors in aquatic environments that have a high probability to bind to hepatocytes males ERα intracellular receptors and provoked a high Vtg production without a specific function (Moncaut et al., 2003). The Vtg concentrations can be estimated by immuno-enzymatic assays, but the availability of IgG anti-Vtg is scarce in the market due to the specificity of Vtg by species. IgY is more suitable for diagnostic purposes than mammalian antibodies, and immunoglobulins Y anti 1 tilapia Vtg have been used to evaluate the reproduction in commercial cichlids in Ecuador (Ortiz et al., 2017). The IgY antibody can bind the specific sites of proteins, in response to a foreign stimulus and wide recognition of different epitopes of the same biomarker (Gassmann et al., 1990; Pereira et al., 2019). In this context, the IgY antibody production technology offers versatile advantages compared to mammalian IgG, because the production of antibodies in mammals can be challenging due to the fact that some antigens elicit weak immune responses or are even completely non-immunogenic. Moreover, the production of antibodies in mammals involves procedures that cause pain and distress to ; such as immunization, blood sample collection, and sacrifice (Pereira et al., 2019). The aim of this paper is to develop egg yolk antibodies (IgY) anti Vtg protein of C. festae, and generate useful and transferable information for native fish reproduction in Ecuador. Such information provides an initial tool to identify Vtg protein into the spawning cycle of Cichlasoma festae , which can lead to support local fisheries communities, in developing sustainable activities and the assurance of aquatic environments quality in upstream Guayas river.

MATERIALS AND METHODS

Biological samples Twelve males adult specimens of C. festae were catches from wild populations in Baba river multipurpose dam upstream Guayas river ( 0° 39’ 08.47’’ S;79° 25’ 06.90’’ W, Buena Fé, Ecuador). Fish were transferred to Aquaculture IASA - ESPE laboratory in Sangolquí and housed in an aquaculture recirculating systems (RAS) in tanks of 4 m3 capacity with aquatic plants and stones, under conditions mimicking their natural reproductive habitat: 24,35°C ± 1,02 °C, and 14:10 light: dark cycle, external filtration, and constant aeration. Everyday fish were fed ad libitum with trout pellets (Biomix ®). All experiments were conducted in accordance with international standards on animal welfare (Guide for the care and use of laboratory animals, 2011), and were previously approved by the local Ethical Committee (DCVI, Life Sciences Department, Universidad de las Fuerzas Armadas - ESPE). For induction of Vtg in males fish (250 g ± 24), synthetic estradiol (Sigma, USA) was used at a dose of 5 μg/g BW. From 8th day post- inoculation, blood collection and plasma collection were performed at 1372 g for 10 minutes at 4°C. Plasma samples were preserved with protease inhibitors (PMSF) at -80 ºC for further analysis (Denslow et al., 1999).

Vtg Purification The Vtg was purified from blood plasma by using Sartobind MA Q-15 anion exchange columns (Sartorius, Germany). Previously a 1: 4 plasma (plasma: buffer) dilution was performed, and transferred through 0.22 μm membrane filters. Subsequently, NaCl concentration gradients of 0.27; 0.33; 0.37; 0.40 M , used for later elution in the corresponding columns (Shi et al., 2003). The best concentration fraction of Vtg (100 kDa) was centrifuged at 448 g for 4 minutes at 4 ° C and aliquoted into Millipore tubes (100000 MWCO) for further analysis.

Vtg validation by SDS PAGE The Vtg protein concentration was quantified with the Qubit ® Protein Assay kit (Invitrogen, USA) and validated by SDS PAGE on 8% polyacrylamide gel. Previously, the samples were mixed with the loading buffer 5X [glycerol 20%; 63 mM TRIS-HCl pH 6.8; 2% SDS, 0.1% Bromophenol Blue; β-mercaptoethanol] in a 1: 1 ratio, and denatured at 95 °C for 10 minutes and cooled to 4 °C for 10 minutes. A molecular weight marker (Novex ® ) was used to determine the protein size. The electrophoretic run was performed at 110V for 30 minutes. The gel was stained with Coomassie R-250 for 30 minutes, constant stirring at room temperature, and finally destained with methanol until the clear bands were visible.

Immunization of chickens against Vtg Adult laying hens of Lohmann Brown (Gallus gallus domesticus)(n= 4) were immunized intramuscularly with four doses of purified Vtg protein. The treatment included the first inoculation of 125 μg and three post inoculation booster dose of 75 μg for intervals of 8 days and control treatment in which no Vtg was inoculated. The first dose of treatment was mixed with complete Freund's adjuvant and the subsequent booster doses with incomplete Freund's adjuvant in a ratio of 1:1, and a final inoculation volume was adjusted to 1 mL. The birds were fed with the balanced feed at 23% protein, spirulina, and vitamin E as a compliment enrichment feed. The hens were maintained with the respective biosafety measures in the IASA - ESPE Aviculture Laboratory. For the IgY antibody extraction and purification process, the eggs were collected every day before and after immunization. For this trial, 5 eggs after immunization were selected to verify the IgY antibody titer compared to the control.

Extraction and purification of IgY-anti-Vtg antibodies At 60 days, post-immunization eggs were selected and separated. Prior washing and disinfecting each egg, the yolk was extracted and diluted in pectin (0.1%, pH 4), was kept in a shaker with continuous stirring all night at 4 ° C. The mixture solution was adjusted at pH 5.2 and centrifuged at 11652 g, the supernatant was collected at 4 °C. 35% ammonium sulfate was used to isolate IgY antibodies and added the supernatant with constant agitation all night. The mixture was centrifuged at 11652 g for 25 minutes at 4 ° C and the pellet was suspended in 0.025 M phosphate buffer (pH 8.0). Subsequently, the IgY antibodies purification was carried out by ion-exchange chromatography (Shimizu et al., 1988). For further purification and to eliminated salts, the eluted fractions were ultrafiltrated in Millipore tubes. Five IgY purified fractions were quantified for total protein by the Bradford method, and verification by SDS –PAGE.

Western blotting test After the electrophoresis process (SDS - PAGE), the gel and the nitrocellulose membrane were transferred to the western blotting unit. For the transfer process, the following conditions were used in the Mini Protean II electrophoresis chambers (BIO- RAD Technologies): transfer buffer (Tris, Glycine, methanol, pH 7.4), previously cooled to -20 °C, transfer time for 1 hour, at 100 V and 0 ° C. Ponceau Red was used for visualization and marking the standard bands on the membrane. After washing the membrane with TTBS (TBS and 0.1% tween), the blocking of specific sites was carried out for one hour, with skim milk (5%), Tween-20 (0.1%), and TBS. 5 mL of diluted IgY-anti Vtg primary antibody (1:2000) were added in the blocking solution and incubated overnight at 4 °C. The membrane was washed and then incubated with 5 mL of the diluted anti-chicken HRP secondary antibody (1:5000) in blocking solution for 2 hours under constant stirring at room temperature. The blot was developed using 0.5 mL of hydrogen peroxide solution containing 0.5 mL of luminol substrate (LumiGLO, BIORAD) for 1 minute at room temperature and the blot was read using C-Digit Scanner.

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Indirect ELISA An indirect ELISA was developed according to the method of (Swart and Pool, 2009) with minor modifications. Standard antigens (Vtg) a concentration of 5 μg/mL were diluted in citrate buffer pH 8, added to Nunc-Immuno Maxisorp F8 plates (Nalge Nunc, Denmark ® ) at 100 μL volume by each well. The incubation period was overnight at 4 ° C. Plates were washed 5 times with TBS-T and antigen unbound sites were blocked with 200 μL of blocking solution (TBST with 2% BSA) and incubated for 1 hour at 37°C. The plate was washed 5 times. IgY-anti Vtg were prepared at a specific dilutions (1:500 to 1:10000), mixed with the pre-diluted blocking solution at 100 μL volume and added to the wells. Incubation was for one hour at 37 ° C. The plate was washed 5 times again and performed with anti-chicken HRP conjugate diluted in blocking solution (1:10000) at 100 μL volume for each well. The incubation period was for one hour at 37 ° C and followed by a series of washes. Finally, 100 μL of 3,3',5,5'-Tetramethylbenzidine (TMB) in developing solution (dimethylsulfoxide; phosphate-citrate buffer pH 5; 1% hydrogen peroxide) was added in the plate and incubated for 30 minutes. The reaction was stopped by sulfuric acid (2 M). The absorbance was measured at 450 nm in the spectrophotometer Biorad.

Assay performance and validation Assay precision, sensitivity, and specificity were determined by conventional methods (Maltais et al., 2010; Wang et al., 2015). The intra -assays precision was assessed by the coefficient of variation and using eight replicates of Vtg standard dilutions on the same plate. The inter-assay precision was determined by six separate assays. The detection limited (LOD) corresponded to the mean absorbance value of twelve zero standards replicates and two standard deviations.

RESULTS

Protein levels in male plasma by estradiol treatment was 24.4 ± 1.94 mg/mL and control with 15 ± 1.5 mg/mL (p <0.05; n = 12). The best fraction elution corresponded at 0.37 M of NaCl, and detected 541.5 and 624.5 µg Vtg/mL , with a molecular weight of 150 kDa (Figure 2).

Fig. 2 SDS-PAGE 8% of purified Vtg. Samples tested include male plasma treated with pure estradiol (lane 1 to 6), 20 -270 kDa protein marker (lane 7)

IgY antibodies production was different by the time. The maximum concentration was at 45th day with 7.86 mg/mL, and the control was 2.54 mg/mL. The BCA calibration curve maintained an r2 = 0.9962. IgY molecule has a structure with two heavy chains (H), each one with a molecular weight of 70 kDa, and two light chains (L), with 30 kDa (Figure 3).

Fig. 3 SDS-PAGE 10%, IgY by ion exchange chromatography. (MW= molecular weight marker; 1-3= purified samples of IgY (CP= heavy chain; CL= light chain); IgY antibodies production (mg/mL) during two months.

Western blot results showed that anti-Vtg antibodies recognized the Vtg purified protein at different concentrations. IgY antibody specificity was optimal to detect Vtg monomer at a 150 kDa band height and optimal dilution at 1:2000 (Figure 4).

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Fig. 4 Western Blot control samples. Vtg at 624.5 μg/mL (lane 1); Vtg at 563 μg/mL (lane 2); Vtg at 281 μg/mL (lane 3); Vtg at 140 μg/mL (lane 4); control (lane 5)

The Vtg antigen dilution (1:200) and anti-Vtg IgY dilution (1: 2000) were chosen to optimize the ELISA assay. These test concentrations gave an absorbance value close to one (Figure 5). The Vtg standard curve had a working range within the linear zone of 10-1280 ng / mL (y = 0.0021x + 0.3759, R² = 0.9938), and the ELISA LOD was at 5.71 ng / mL. Intra an inter assays were a CV of 0.17 - 1.40% and 0.18 - 4.1% respectively.

Fig. 5 Optimal Vtg concentrations plasma and anti-Vtg IgY antibodies for ELISA validation assay.

DISCUSSION

The detection of Vtg is being studied intensively because the specific association between Vtg synthesis and estradiol stimulation can be influenced by other compounds such as endocrine disruptors that exhibit estrogenic activity, for example, organochlorine pesticides, biphenyl polychlorinated, among others. This environmental contamination generates changes in reproductive physiology such as impaired gonad function, low amount of gametes, various cellular alterations, and decreased gonadosomatic index in Cichlasoma dimerus males (Moncaut et al., 2003). In this study, we have detected Vtg in plasma of the South American cichlid, C. festae, after 8 days of hormonal treatment with 17β- estradiol (5 μg/g BW). Using anion exchange columns at a gradient of 0.37 M and its validation by SDS-PAGE and Western blot, we obtained the same profile of Vtg in plasma samples with a molecular mass of 150 kDa. We used estradiol as a Vtg inducer in C. festae males in order to detect and purify the Vtg molecule according to conventional methodologies (Denslow, 1999). Vtg identification by SDS-PAGE under reducing and denaturing conditions is preponderant to obtain highly readable bands and permitted to visualize a band above 150 kDa in C.festae. In C. dimerus the molecular mass of Vtg in plasma corresponded to two major bands of 180 and 120 kDa, while in the surface mucus the estimated molecular masses were 120 and 110 kDa (Moncaut et al., 2003). Similar studies showed in Oreochromis niloticus Vtg at 185 KDa (Buerano et al., 1995), in mossambicus tilapia above 200 KDa (Swart et al., 2009). Other authors in Cyprinus carpio obtained Vtg at 150 KDa (Fukada et al., 2003), in medaka (Oryzias latipes) at 220 KDa (Shimizu et al., 2002). In this sense, the Vtg molecular weight by each species fish is unique and depend on different physiological intrinsic and extrinsic factors as fish genetic, age, kind of food and environmental conditions (Moncaut et al., 2003; Babin et al. 2007; Ortiz et al., 2017). The maximal IgY concentrations obtained at 45th days post-immunization was 7.86 mg/mL. It should be noted that the peak IgY antibody concentration can occur within 30 days after the first immunization and maintained until day 70, so it is possible to keep high titers of egg yolk antibodies for more than 150 days (Gassmann et al., 1990; Meenatchisundaram et al., 2011). The use of Freund´s adjuvants together antigens dilutions enhanced the immunogenicity status in hens without severing inflammation and increased the production of IgY anti-Vtg of C. Festae. Though Freund's Complete Adjuvant (FCA) is the most potent to induce antibodies in laboratory animals and is more tolerated by birds than by mammals. On the other hand, Freund's Incomplete Adjuvant (FIA), which, unlike FCA,

4 does not contain mycobacteria is recommended the use since the first immunization without any prejudice to the antibody titer. In spite of this, the first inoculation is generally performed using FCA, while the subsequent inoculations are performed using FIA, without the occurrence of inflammation at the injection site (Pereira et al., 2019). The maximum concentration of Igy anti-Vtg in hens obtained in the present study was by the first inoculation of 125 μg Vtg/mL and three post inoculation booster dose of 75 μgVtg/mL for intervals of 8 days. (Denslow et al., 1999; Li et al.,2016) obtained polyclonal anti tilapia Vtg with high immunogenicity in hyperimmunized rabbits and mice, with at the first dose of 100 μg/mL and reinforcements of 50 μg/mL. Similar concentrations of antibodies were obtained by (Buerano et al., 1995) in gray tilapia with inoculum ranges of 200- 1000 μg/mL. (Tada et al., 2004) produced polyclonal anti-Vtg of Chinese turtle (Chinemys reevesii) at inoculation doses of 500 μg/mL. Two bands could be seen in each lane of purified IgY samples. The first band (heavy chain) was located at 70 kDa, and the second band (light chain) at 30 KDa. The IgY molecule has a structure similar to that of IgG, with two heavy chains (H), each one with a molecular weight of 67 to 70 kDa, and two light chains (L), with 25 kDa (Pereira et al., 2019).The light chains have one constant region (CL) and one variable region (VL), similar to IgG. The major difference between IgY and IgG is found at the heavy chains. IgG has three constant regions at the heavy chains (CH1-CH3), while IgY has four constant regions (CH1-CH4). A further constant domain, with the corresponding carbohydrate chains, gives IgY a higher molecular weight in comparison to IgG (Pereira et al., 2019). The use of commercial IgG the anti-Vtg Sparus aurata with cross-reactivity anti-Vtg of tilapia and C.festae in immunosorbent assay (ELISA) was important for the validation of the enzymatic assay (Swart and Pool, 2009). This situation showed the enzyme-linked and antigen-antibody reaction specificity (Cevallos et al., 2017). In the present paper, this reaction recorded a maximum optical density at 450 nm by 2.59. Treatment A presented the best absorbance result at 1.42 with a Vtg concentration as the antigen of 10 μg/mL and an IgY concentration of 2 μg/mL. On the other hand, treatment B exhibited the best absorbance at 1.41 under conditions of 5 μg/mL Vtg and 2 μg/mL IgY as antigen and primary antibody respectively. These results are comparable to those obtained by (Swart and Pool, 2009), who standardized an ELISA for Vtg detection of Oreochromis mossambicus. Similarly, (Sotiropoulou et al., 2012) has also tested the antigen- antibody binding at a concentration of 1 μg/mL IgY. This result is comparable with the present study in ranges of sensitivity and concentration of IgY anti-Vtg from 1 to 4 μg/mL. The specificity of anti-Vtg at a dilution of 1:2000 was validated by Western Blot, where the Vtg monomer was detected in dilution ranges from 140 μg/ml and with a molecular weight of approximately 150 kDa. These data were earlier documented by (Caza et al., 2019) using a 1:1500 dilution of primary anti-Vtg antibodies in tilapia.

CONCLUSIONS In sum, we have detected Vtg in plasma of the South American cichlid, C. festae, after 8 days of hormonal treatment with estradiol (5 μg/g BW). Using anion exchange columns at a gradient of 0.37 M and its validation by SDS-PAGE and Western blot, we obtained the same profile of Vtg in plasma samples with a molecular mass at 150 kDa. In the same sense, we detected by ELISA the Vtg concentration from 10-1280 ng / mL with IgY anti-Vtg at dilution of 1: 2000 and 450 nm as a maximal optical density. This is the first report to address to develop immunoglobulins Y to detect Vtg synthesis in Neotropical fishes as a C. festae in Ecuador. Considering all these results, this tool could be used as an effective mechanism for environmental conservation programs with many endemic oviparous fishes that are unknown about their reproduction cycle in Ecuadorian rivers.

Acknowledgement We especially thank Dr. Rodolfo Fernández and Dr. Luis Valladares, reviewers for their pertinent comments and suggestions that helped to improve the manuscript. This work was supported by the following grants: PIC-18-INE-ESPE-001 (INEDITA - SENESCYT), Universidad de las Fuerzas Armadas - ESPE (Vice-Rector's Office for Research and Innovation), and funds administration UNDP (a program for the United Nations Development).To Mr. Alfonso Benavides (Socio-Tilapia) and the local fishermen Guayas rivers, Buena Fé - Ecuador, especially Mrs. Angela Rodríguez and Mr. Oscar Andrade for your entire helps in the recollection sample fish.

REFERENCES

Alvarez-Mieles, G., Irvine, K., Griensven, A. V., Arias-Hidalgo, M., Torres, A., & Mynett, A. E. (2013). Relationships between aquatic b iotic communities and water quality in a tropical river–wetland system (Ecuador). Environmental Science & Policy, 34, 115–127. http://d oi:10.1016/j.envsci.2013.01.011. Arias-Hidalgo, M., Villa-Cox, G., Griensven, A. V., Solórzano, G., Villa-Cox, R., Mynett, A. E., & Debels, P. (2013). A decision framew ork for wetland management in a river basin context: The “Abras de Mantequilla” case study in the Guayas River Basin, Ecuador. Enviro nmental Science & Policy, 34, 103–114. http://doi:10.1016/j.envsci.2012.10.009 . Babin, P. J., Cerdà, J., & Lubzens, E. (Eds.). (2007). The Fish Oocyte. http://doi:10.1007/978-1-4020-6235-3 . Berg, A. H., Westerlund, L., & Olsson, P. E. (2004). Regulation of Arctic char (Salvelinus alpinus) egg shell proteins and vitellogenin du ring reproduction and in response to 17β-estradiol and cortisol. General and Comparative Endocrinology, 135(3), 276–285. http://doi:10. 1016/j.ygcen.2003.10.004 . Buerano, C. C., Inaba, K., Natividad, F. F., & Morisawa, M. (1995). Vitellogenins ofOreochromis niloticus: Identification, isolation, and biochemical and immunochemical characterization. Journal of Experimental Zoology, 273(1), 59–69. http://doi:10.1002/jez.1402730108. McMahan, C. D., Geheber, A. D., & Piller, K. R. (2010). Molecular systematics of the enigmatic Middle American genus Vieja (Teleoste i: Cichlidae). Molecular Phylogenetics and Evolution, 57(3), 1293–1300. http://doi:10.1016/j.ympev.2010.09.005. Cevallos, W., Calvopiña, M., Nipáz, V., Vicente-Santiago, B., López-Albán, J., Fernández-Soto, P., Muro, A. (2017). Enzyme-linked im munosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans. Memórias Do Instituto Oswaldo Cruz, 112(5), 364 –369. http://doi:10.1590/0074-02760160426. Denslow, N. D., Chow, M. C., Kroll, K. J., & Green, L. (1999). Ecotoxicology, 8(5), 385–398. http://doi:10.1023/a:1008986522208 Food and Agriculture Organization. Revised September 14, 2019 (http://www.fao.org/figis/servlet/SQServlet?file=/usr/local/tomcat/8.5.16/figis/webapps/figis/temp/hqp_4763616801375773071.xml&out type=html)

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Fukada, H., Fujiwara, Y., Takahashi, T., Hiramatsu, N., Sullivan, C. V., & Hara, A. (2003). Carp (Cyprinus carpio) vitellogenin: purificat ion and development of a simultaneous chemiluminescent immunoassay. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 134(3), 615–623. http://doi:10.1016/s1095-6433(02)00348-3 . Garstka, W., & Crews, D. (1981). Female sex pheromone in the skin and circulation of a garter snake. Science, 214(4521), 681–683. http: //doi:10.1126/science.7292007. Gassmann, M., Thömmes, P., Weiser, T., & Hübscher, U. (1990). Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. The FASEB Journal, 4(8), 2528–2532. http://doi:10.1096/fasebj.4.8.1970792. López-Fernández, H., Winemiller, K. O., & Honeycutt, R. L. (2010). Multilocus phylogeny and rapid radiations in Neotropical cichlid fis hes (Perciformes: Cichlidae: ). Molecular Phylogenetics and Evolution, 55(3), 1070–1086. http://doi:10.1016/j.ympev.2010.02. 020 . Li, X., Jing, K., Wang, X., Li, Y., Zhang, M., Li, Z., … Xu, Y. (2016). Protective effects of chicken egg yolk antibody (IgY) against expe rimental Vibrio splendidus infection in the sea cucumber (Apostichopus japonicus). Fish & Shellfish Immunology, 48, 105–111. http://do i:10.1016/j.fsi.2015.11.024 Maltais, D., Roy, R. L., & Couillard, C. M. (2010). Hybrid ELISAs for vitellogenins of the endangered copper redhorse Moxostoma hubb si and the shorthead redhorse Moxostoma macrolepidotum (Cypriniformes, catostomidae). Ecotoxicology and Environmental Safety, 73( 5), 883–892. http://doi:10.1016/j.ecoenv.2010.03.005 Meenatchisundaram, S., Shanmugam, V., Anjali, V. 2011. Development of chicken egg yolk antibodies against Streptococcus mitis – purification and neutralizing efficacy, J. Basic Clin. Pharm., 109–114. Moncaut, N. (2003). Vitellogenin detection in surface mucus of the South American cichlid fish Cichlasoma dimerus (Heckel, 1840) indu ced by estradiol-17β. Effects on liver and gonads. Aquatic Toxicology, 63(2), 127–137. http://doi:10.1016/s0166-445x(02)00175-3 . Ortiz Tirado, J., Valladares, L., Munoz, D., Caza, J., Manjunatha, B., & Kundapur, R. R. (2017). Levels of 17β estradiol, vitellogenin, an d prostaglandins during the reproductive cycle of Oreochromis niloticus. Latin American Journal of Aquatic Research, 45(5), 930–936. ht tp://doi:10.3856/vol45-issue5-fulltext-8 Patiño, R., Thomas, P., & Yoshizaki, G. (2003). Ovarian follicle maturation and ovulation: an integrated perspective. Fish Physiology an d Biochemistry, 28(1-4), 305–308. http://doi:10.1023/b:fish.0000030565.74702.0a Pereira, E. P. V., van Tilburg, M. F., Florean, E. O. P. T., & Guedes, M. I. F. (2019). Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. International Immunopharmacology, 73, 293–303. http://doi:10.1016/j.intimp.2019.05.015. Shi, G., Shao, J., Jiang, G., Wang, Q., Lu, Y., Liu, J., & Liu, J. (2003). Membrane chromatographic method for the rapid purification of v itellogenin from fish plasma. Journal of Chromatography B, 785(2), 361–368. http://doi:10.1016/s1570-0232(02)00958-3. SHIMIZU, M., FITZSIMMONS, R. C., & NAKAI, S. (1988). Anti-E. coli lmmunoglobulin Y Isolated from Egg Yolk of Immunized Chi ckens as a Potential Food Ingredient. Journal of Food Science, 53(5), 1360–1368. http://doi:10.1111/j.1365-2621.1988.tb09277.x Shimizu, M., Fujiwara, Y., Fukada, H., & Hara, A. (2002). Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen. Journal of Experimental Zoology, 293(7), 726–735. http://doi:10.1002/jez.10178. Swart, J. C., & Pool, E. J. (2009). The Development and Validation of a Quantitative ELISA forin vivoandin vitroSynthesized Vitellogen in from Mossambicus tilapia (Oreochromis mossambicus). Journal of Immunoassay and Immunochemistry, 30(2), 208–223. http://doi:10. 1080/15321810902782905. Sotiropoulou, G., Pampalakis, G., Prosnikli, E., Evangelatos, G. P., & Livaniou, E. (2012). Development and immunochemical evaluatio n of a novel chicken IgY antibody specific for KLK6. Chemistry Central Journal, 6(1). http://doi:10.1186/1752-153x-6-148. Tubert, C., Lo Nostro, F., Villafañe, V., & Pandolfi, M. (2012). Aggressive behavior and reproductive physiology in females of the social cichlid fish Cichlasoma dimerus. Physiology & Behavior, 106(2), 193–200. http://doi:10.1016/j.physbeh.2012.02.002 Wang, J., Shan, R., Zhang, X., Tian, H., Wang, W., & Ru, S. (2015). Development of a lipovitellin-based sandwich ELISA for quantifica tion of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus). Ecotoxicology and Environmental Safety, 120, 80–87. h ttp://doi:10.1016/j.ecoenv.2015.05.010 Tada, N., Saka, M., Ueda, Y., Hoshi, H., Uemura, T., & Kamata, Y. (2004). Comparative analyses of serum vitellogenin levels in male and female reeves´ pond turtles (Chinemys reevesii) by an immunological assay. Journal Comp. Physiol., 13- 20. https://doi.org/10.1007/s00360-003-0384-2 The development, purification and characterization of anti- vitellogenin antibodies raised in hens (Gallus gallus domesticus) against tilapi a (Oreochromis niloticus) vitellogenin protein. (2019). Journal of Microbiology, Biotechnology and Food Sciences, 8(5), 1165–1168. htt p://doi:10.15414/jmbfs.2019.8.5.1165-1168

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