Abstract
Social animals with hierarchical dominance systems are susceptible to changes their environment. Interactions with conspecifics can greatly affect individual’s behavior and reproductive success. This review will show how social behavior modulates gonadal steroidogenesis and spermatogenesis in African and Neotropical cichlid fish with different social systems and how this modulation regulates reproductive capacity. Social behavior and aggressiveness are strongly linked to sex steroids, glucocorticoids and neuropeptides. The challenge hypothesis suggests that behavioral interactions increase androgen levels in response to social instability, but there is little evidence regarding estradiol levels. It has been recently demonstrated that in male Cichlasoma dimerus, a Neotropical cichlid fish, the challenge hypothesis could also be extended to estrogens. In C. dimerus, dominant males have higher gonadosomatic index than subordinated; the percentage of spermatocytes and spermatids is higher in subordinates, while dominants show a greater percentage of spermatozoa. In other species of African cichlids, socially suppressed subordinate males are not reproductively incompetent maintaining some activity at every level of their reproductive axis. Axis reactivation upon social ascent is similar to the initiation of puberty in mammals, as well as the reoccurrence of puberty observed in seasonally breeding animals. In conclusion, social behavior and reproductive strategies in females cichlids are still understudied, and Neotropical cichlids still constitute a group that deserves more attention, considering cichlids’ diversity in mating systems, reproductive behavior and parental care. This review highlights the importance of performing further studies and additional research in these two areas, which still remain to be addressed.
Introduction
The social environment can have profound effects on the behavior and physiology of each animal (Galhardo & Oliveira 2014). Besides social cues and external factors, individual’s physiology and motivational states also contribute to determine the behavioral output (Tudorache et al. 2013). As a consequence, the hormonal profile of each individual modulates reproductive and social behavior (e.g. aggression) which, in turn, is affected by social interactions with conspecifics. However, steroid levels are usually a result rather than a cause of the social position as social and reproductive displays influence hormone levels.
These intertwined and usually bidirectional effects are of particular importance in social species in which hierarchical dominances are established. In such cases, social status can greatly affect an individual’s behavior and physiology through interactions with its conspecifics (Sapolsky 2005). Social interactions that determine a position within a hierarchical system have profound and diverse effects over animals’ reproductive behavior and physiology (Fernald 2012, Fernald & Maruska 2012, Maruska et al. 2013, Ramallo et al. 2015). Cichlid fish constitute ideal models to study reproductive physiology and behavior because they show diverse forms of parental care, such as substrate guarding, delayed and immediate mouthbrooding. Moreover, they also show variation in which sex provides parental care, whether it is biparental, female-only or male-only care. As a consequence, during last decades they have become a group of growing interest to study social control of reproduction. In this regard, cichlid fish dominance and social rank are usually associated with distinct sex steroid hormone profiles. The most studied species to date are the African cichlids, while Neotropical species are understudied. African and Neotropical cichlids are both monophyletic and sister groups (Farias et al. 2000), and results based on mitochondrial DNA suggest that there are significantly higher rates of genetic variation in Neotropical than in African taxa (Farias et al. 2000).
The most popular cichlid models in which social control of reproduction has been assessed are African species: Astatotilapia burtoni and Neolamprologus pulcher, from Lake Tanganyika, and Oreochromis mossambicus, inhabitant of the Limpopo and Zambezi rivers (Neat et al. 1998, Oliveira 2009). A. burtoni is a species with a lek-like social system in which dominant territorial males aggressively defend a spawning territory and actively court females, while subordinate non-territorial males present a submissive behavior, resembling and schooling with females (Fernald & Hirata 1977). Females of this species also school and feed with other females and juveniles. Once gravid, a female will spawn with dominant males, after which she incubates the fertilized eggs in her buccal cavity for several weeks, known as maternal mouthbrooding behavior. After releasing the fry, female can defend the territory and exhibit maternal care for a short period (Fernald & Hirata 1977). Interestingly, even if females of this species do not present social hierarchies when there are males present, in all-female communities they acquire dominance phenotype and male-typical behavior such as aggressive territorial defense and courtship behavior (Renn et al. 2012). On the contrary, N. pulcher is a cichlid species with cooperative breeding in which subordinate members of the group (so-called ‘helpers’) assist the breeding pair in caring of offspring, defense of the territory against intruders and digging shelters (Taborsky & Limberger 1981, Balshine et al. 2001).
As most of these studies were performed in African species and considering that African and Neotropical cichlids are monophyletic and sister groups in which Neotropicals present higher genetic variation than Africans, it is important to increase research on understudied Neotropical cichlids. Variable reproductive physiology and behavior of the cichlid family can be better understood if new studies increase knowledge on different cichlid species from diverse habitats and with different social systems. In order to search for the characteristics of social control of reproduction in Neotropical cichlids, since 2010 we have been studying the behavior and endocrinology of Cichlasoma dimerus, a South American species locally known as chanchita (reviewed in Ramallo et al. 2014, Silva & Pandolfi 2018). This substrate breeding fish shows biparental care of the eggs and larvae (Meijide & Guerrero 2000) and undergoes multiple spawning events within a single reproductive period (November–March) (Vázquez et al. 2012). C. dimerus presents a dominance hierarchy that determines access to breeding territories among males and females (Alonso et al. 2011, 2012, Ramallo et al. 2015) (Fig. 1). Considering there is only one dominant pair per social tank, both dominant male and female aggressively defend their territory and brood. If conditions and social hierarchy are maintained, the same dominant pair mates repeatedly. Non-territorial individuals shoal in subordinate mixed-sex groups, perform few and sporadic aggressive displays toward the dominant pair, and are subject of highly aggressive displays performed by dominant fish (Ramallo et al. 2015). In social tanks, aggressive behavior is related to body size and social hierarchies tend to be stable, being dominant males the largest animal of the tank (Alonso et al. 2011). This way, if there are no new intrusions or removal of individuals, social hierarchies are maintained. However, if social context changes, an individual can transition from dominant to subordinate, and vice versa. If a dominant male is removed from his original tank and is placed in another tank where there is already a social hierarchy, this dominant will be an intruder in this new scenario, and he will become a subordinate because resident fish will display aggressive behavior, leading even to death. On the contrary, if the dominant male is removed from the tank (resembling a potential capture by predator in natural environment), the highest ranked non-territorial male will acquire dominant position (personal observations). In view of its highly social behavior and effortless maintenance under laboratory conditions, chanchita has emerged as a suitable biological model to investigate the intertwined relationship between hormones, social context, and behavior.
(A) Dominant pair of Cichlasoma dimerus guarding the eggs and defending the spawning site from other conspecific. (B) Schematic representation of different social status fish on the experimental aquaria. Dom, dominant (female or male); sub 1, 2 or 3, Subordinate of different ranking (female or male). Modified from Alonso et al. (2011).
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
Cichlid fish are interesting species to understand how social environment can modulate molecular, cellular and behavioral outcomes that influence reproductive success. Even if social control of reproduction in cichlid fish has been already reviewed (Maruska 2014), future studies can take advantage of the extreme diversity in mating systems, reproductive tactics, and parental care strategies within this group. The goal of this article is to review evidence on the social control of spermatogenesis and gonadal steroidogenesis in fish and how this modulation regulates reproductive capacity. Even though our focus is on cichlid fish species, we begin by introducing gonadal histology and regulation of spermatogenesis and steroidogenesis in teleost fish. Next, we review the relationship between sex steroids and social behavior, focusing on the challenge hypothesis and different cichlid species with diverse complex social structures. Finally we refer to how social behavior (agonistic encounters, dominance/subordination and parental care) can modulate spermatogenesis, steroidogenesis and reproductive capacity in African and Neotropical male cichlid fish species, focusing on the case of C. dimerus. Collectively, these studies reveal a core set of evidence summarizing the relationship between social behavior and reproduction in male cichlid fish.
Steroidogenesis in fish
The core pathway of steroidogenesis in teleost fish is similar to mammals and includes the conversion from cholesterol to steroids with 21 carbon atoms (C21), while pathways involving steroids with 18 and 19 carbon atoms (C18 and C19) present more differences (reviewed by Tokarz et al. 2015) (Fig. 2). The cytochrome p450 cholesterol side chain cleavage enzyme (cyp11a1), 17a-hydroxylase/lyase (cyp17a) and the aromatase (cyp19a1) are the most studied genes in the steroidogenic pathway because they constitute three key steps in steroidogenesis: cyp11a1 converts cholesterol to pregnenolone as the first step of the steroidogenic pathway, cyp17a is responsible for the conversion of C21 steroids to C19 steroids, and cyp19a1 is responsible for the formation of C18 steroids and is the key enzyme-regulating sexual development in teleost fish (Rashid et al. 2007, Mills et al. 2014). Moreover, 11-beta hydroxysteroid dehydrogenase type 2 (hsd11b2) is also a key enzyme in the steroidogenic pathway since it has two main functions: it regulates the production of 11-ketotestosterone (11-KT) (Lokman et al. 2002) and also converts the active ligand cortisol to cortisone, an inactive form unable to bind to glucocorticoid receptors (Zachayus et al. 1994, Nematollahi et al. 2009). As a consequence, this enzyme could play a key role during social situations such as social challenges, in which both androgen and glucocorticoid concentrations are altered and deserves to be taken into account when studying social hierarchies in fish (Filby et al. 2012). Besides these key genes, which have been characterized in different teleost fish species, there are others that have received less attention. This is the case of 17b-hydroxysteroid dehydrogenases type 3 and type 1, which are essential for the biosynthesis of 11-KT (Mindnich et al. 2005) and for converting estrone to E2 (Zhou et al. 2005), respectively.
Steroidogenic pathway in teleost fishes. Gene names of supposed enzymes are denoted on each arrow. Steroids are grouped according to the number of carbon atoms in the steroid nucleus (C18, C19, C21). cyp, cytochrome P450; hsd, hydroxysteroid dehydrogenase; OH–, hydroxy–.
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
When comparing steroidogenesis in fish and in mammals, three main differences emerge (Miller & Auchus 2011, Tokarz et al. 2015). First of all, teleost fish seem to lack aldosterone, since this hormone has not been detected and an enzyme responsible for aldosterone synthesis has not been identified yet (Baker 2003). Also in teleost fish, corticosterone is an endpoint of the steroidogenic pathway, while in humans, it is an intermediate for the biosynthesis of aldosterone. Another main difference between these groups is regarding the maturation inducing steroids 17a,20b-dihydroxt-4-pregnen-3-one and 171,20b,21-trihydroxy-4-pregnen-3-one, which induce oocyte maturation in teleost fish and do not exist in humans (Nagahama & Yamashita 2008). Finally, the most important difference in both steroidogenic pathways refers to androgens. Besides testosterone, in teleost fish the main ligand for androgen receptor is the 11-KT (Kime 1993). This hormone is synthesized via 11b-hydrox-androgens, which do not occur in the human androgenic pathway that is focused on 51- and 31-reductions (Miller & Auchus 2011). As a consequence, while in humans the active androgens are testosterone and 5a-dihydrotestosterone (DHT) (Mooradian et al. 1987) in fish the major androgens are testosterone and 11-KT. Despite this, DHT with androgenic potency has been detected in plasma of fathead minnow (Pimephales promelas) (Margiotta-Casaluci & Sumpter 2011), so further research is needed to disentangle the importance of this androgen in other teleost species.
Fish and other vertebrates are also divergent in the genes that regulate steroidogenesis. One of the major differences is that fish underwent genome duplication (Taylor et al. 2003), and this may have resulted in mutations and silencing, neofunctionalization or coexisting genes with different regulation (Hughes 1994, Glasauer & Neuhauss 2014). For example, in some teleost species such as Nile tilapia (Oreochromis niloticus) two genes for steroidogenic acute regulatory protein (StAR) have been identified (Yu et al. 2014). StAR is one of the key enzymes of steroidogenesis, since it transports cholesterol to the inner membrane of mitochondria, which is then substrate for steroid synthesis. However, in Nile tilapia two isoforms were detected in different tissues, suggesting that StAR1 is involved in cortisol production in kidney, StAR2 is involved in estrogen production in ovary and that both of them could participate in androgen production in the testes (Yu et al. 2014). This suggests that both isoforms are involved in common but also in differential functions. Moreover, the aromatase gene also is duplicated and both forms differ in their regulation (Callard et al. 2001) and on their localization: while cyp19a1a is mainly ovarian, aromatase and cyp19a1b is mainly expressed in the brain (Chiang et al. 2001). As a consequence, when studying steroidogenesis and the physiological role of steroids in teleost fish, it is important to take into account gene duplication and to study the regulation of all steroidogenic enzymes, regardless of their localization.
Spermatogenesis and gonadal histology in fish
As in all vertebrates, fish testes are organized in two compartments: the interstitial one, where steroidogenesis takes place in Leydig cells, and the germinal tissue, where spermatogenesis occurs. During these process, single undifferentiated spermatogonia I passes through numerous species-specific mitotic divisions (Schulz & Nobrega 2011a ,b ), two meiosis and a morphological differentiation to originate the final male gamete, the spermatozoon. In amniotes, such as mammals, spermatogenesis occurs in tubular testes with seminiferous tubules (Lombardi 1998). Sertoli cells form an epithelium with tight junctions that forms the ‘blood–testis barrier’ and separate the tubule into basal compartment, with mitotic spermatogonia, and adluminal compartment, in which meiotic spermatocytes continue spermatogenesis until spermatozoa (Hess & França 2005). This way, in the amniotes testes each Sertoli cell provides different environments that allow germ cells to differentiate from the basal to the apical compartment. Conversely, in fish and amphibians spermatogenesis occurs in cystic testes. In anamniote vertebrates, each Sertoli cell encloses a primary spermatogonium with its cytoplasmic processes forming a spermatocyst, where spermatogenesis occurs (Pudney 1998). As a consequence, each cyst contains a group of germ cells that share a single syncytium, and synchronously divide and differentiate (Schulz et al. 2010).
Fish with cystic testes are classified according to the organization of the germinal epithelium, as anastomosing tubular or lobular types (reviewed by de Siqueira-Silva et al. 2019). In the anastomosing tubular type, the germinal epithelium is branched and forms anastomoses that do not blind end in the gonadal periphery. On the other hand, Neoteleostei present the lobular testis type, in which the germinal epithelium ends blindly at the periphery of the testes. Furthermore, two types of arrangements have been described in teleost fish testes regarding how spermatogonia are distributed in the germinal compartment (Grier 1981; reviewed by Schulz et al. 2010, de Siqueira-Silva et al. 2019). In the restricted spermatogonial distribution, which can be found in higher teleosts such as Atheriniformes, Cyprinodontiformes and Beloniformes (Parenti & Grier 2004), Sertoli cells surround undifferentiated spermatogonia in the distal regions of the germinal compartment. On the other hand, in the unrestricted spermatogonial distribution, which can be found in more primitive taxonomic groups such as Cypriniformes, Characiformes, and Salmoniformes (Parenti & Grier 2004), spermatogonia are spread throughout the germinal compartment along the testes and cysts do not migrate during their development (Grier 1981). In general, in the anastomosing tubular testis the distribution of spermatogonia is unrestricted, while in the lobular testis they are restricted to the distal part of the lobule, at the blind end (reviewed by de Siqueira-Silva et al. 2019). Despite this general pattern, there are some cases in which lobular testis have unrestricted spermatogonial type (e.g. Synbranchus marmoratus, Cichla kelberi, Cichla intermedia, reviewed by de Siqueira-Silva et al. 2019). Moreover, intermediate forms between restricted and unrestricted spermatogonial distribution can be found in other species, such as tilapia Oreochromis niloticus (Vilela et al. 2003).
According to the habitat of each fish species, spermatogenesis can present seasonal or continuous activity. In testis from tropical fish species, spermatogenesis usually occurs throughout the year. However, in several species from habitats at higher latitudes, spermatogenesis can be cyclic and present seasonal variations according to environmental cues (reviewed by Schulz et al. 2010). In this sense, in some species spermatogenesis is active during summer, such as trout and carp; in others it occurs in spring, such as sea bream and tench, while in others it can begin in autumn and finish in spring, such as stickleback and killifish.
The progression of spermatogenesis is regulated by a delicate balance between different pituitary hormones, and steroid hormones such as estrogens, androgens and progestins (reviewed by Schulz et al. 2010). In male fish both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate Leydig cell steroidogenesis, while Sertoli cells are mainly regulated by FSH but also by LH when high concentrations of this hormone can cross-activate the FSH receptor (So et al. 2005). In turn, FSH also stimulates early stages of spermatogenesis by enhancing spermatogonial proliferation (Ohta et al. 2007). Furthermore, steroid hormones play a central role controlling fish spermatogenesis. Despite the fact that 17b-estradiol (E2) has been historically considered a ‘female’ hormone, it is present in male fish (Amer et al. 2001, Chaves-Pozo et al. 2007, Scaia et al. 2018b ) as well as in other vertebrates (Schlinger & Arnold 1992, Scaia et al. 2013, 2019). Estrogens have several effects on fish testes and they are a topic of growing interest. In gilthead sea bream (Sparus aurata) they inhibit the proliferation of spermatogonia in early stages, induce apoptosis of undifferentiated spermatogonia, accelerate spermatogenesis events and induce infiltration of acidophilic granulocytes, which usually occur during post-spawning (Chaves-Pozo et al. 2007). Moreover, in rainbow trout (Oncorhynchus mykiss) E2 plasma levels show an increase at the beginning of the reproductive cycle when initial phases of spermatogenesis take place (Gomez et al. 1999). High levels of this hormone reduce the seminal fluid volume, increase the percentage of locally motile spermatozoa and could eventually lead to sterility (Lahnsteiner et al. 2006). Androgens (testosterone, 11-KT) play a central role in spermatogenesis. For example, they stimulate spermatogonial proliferation and spermatocyte formation in guppies (Poecilia reticulata) and spermatocyte maturation in killifish (Fostier et al. 1983). They are also especially important for hydration and for sperm final maturation in seminiferous tubules and efferent ducts (Rolland et al. 2013) and can induce spermiation in some species such as the amago salmon (Oncorhynchus rhodurus) (Ueda et al. 1985). However, spermiation is mainly regulated by progestins such as 17a,20b-dihydroxy-4-pregnen-3-one or 17a,20b,21-trihydroxy-4-pregnen-3-one (Ueda et al. 1985). Progestins are also central in milt production and stimulate spermatozoa motility (Baynes & Scott 1985).
Relationship between sex steroids and behavior: the challenge hypothesis
As already mentioned, social behavior and dominance in cichlid fish are usually related to sex steroid hormone profiles. For example, there is a positive association between dominance and androgen and estrogen levels in males of A. burtoni (Maruska et al. 2013, O’Connell et al. 2013). In C. dimerus even though circulating androgen levels are higher in dominant males, plasma E2 levels show the opposite tendency (Ramallo et al. 2015). Moreover, higher plasma androgen levels are associated to dominant reproductive status in males of O. niloticus (Pfennig et al. 2012) and Pundamilia nyererei (Dijkstra et al. 2007). In N. pulcher androgens are also associated to dominant reproductive status in both social context and after short periods of instability, since dominant males present higher 11-KT but similar testosterone than helpers (Desjardins et al. 2008, Taves et al. 2009).
Taking into account that steroid levels are usually a result rather than a cause of the social position, behavior influences hormone levels (e.g. aggression). In this regard, the challenge hypothesis suggests that male–male aggressive interactions increase testosterone levels above the breeding baseline which, in turn, stimulate aggressive behavior and further testosterone production (Wingfield et al. 1990) (Fig. 3). This hypothesis has been originally proposed for song sparrows (Melospiza melodía) (Wingfield 1985), but it has been later extended to other bird species (Lacava et al. 2011), mammals (Rincon et al. 2017) and fish (Desjardins et al. 2006, Almeida et al. 2014, Teles & Oliveira 2016, Scaia et al. 2018b ). Even though this hypothesis originally suggested that androgens are expected to increase due to male–male interactions, a more recent revision discusses how androgens are also expected to fluctuate in response to season, to male–female interactions and to non-social environmental cues (Goymann et al. 2007).
Evidence on the challenge hypothesis in the most studied cichlid species. (A) The challenge hypothesis suggests that male–male aggressive interactions increase testosterone levels above the breeding baseline, and this stimulates aggressive behavior and testosterone production even more. Most studies determine testosterone and 11-ketotestosterone (11-KT), which is produced by the cyp11b/cyp11c1 and the hsd11b2. However, considering that testosterone can also be converted to estradiol (E2) by the aromatase cyp19a1a/b, recent studies also extend this hypothesis to estrogens. (B) Most studies assessing predictions of the challenge hypothesis refer to African cichlids and, more recently, to one Neotropical species. According to existing evidence, three different patterns can be described. In Astatotilapia burtoni males, 11-KT, testosterone and E2 levels increase after social ascent (Maruska & Fernald 2010, Huffman et al. 2012). In females of this species there is an increase in testosterone after territorial intrusion, but there is no evidence referring 11-KT and E2 levels after this social challenge (Renn et al. 2009). In Neolamprologus pulcher, a cooperatively breeding species, when analyzing sex steroid levels as a response to territorial intrusion, in males there is an increase in 11-KT levels, but not in testosterone and E2, while in females there is an increase in 11-KT and testosterone levels, but not in E2 (Desjardins et al. 2006). In the Neotropical cichlid Cichlasoma dimerus recent evidence suggests that in males there is an increase in 11-KT, testosterone and E2, after social challenge, while in females no changes in sex steroids were detected (Scaia et al. 2019).
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
The challenge hypothesis has been widely studied in male cichlid fish with different reproductive strategies and parental behavior. The challenge hypothesis predicts that androgen levels should increase as a consequence of social instability (e.g. territorial intrusion), preparing the animal for future competitive situations (Oliveira 2004). In A. burtoni, after social ascent males show an increase in androgen levels, both 11-KT (Maruska & Fernald 2010) and testosterone (Huffman et al. 2012). In N. pulcher, a cooperatively breeding cichlid fish, males displayed an increase in 11-KT levels, but not testosterone levels as response to territorial intrusion (Desjardins et al. 2006). This increase in 11-KT level but not in testosterone level has also been demonstrated in other African cichlid species with different breeding systems. Such is the case of Lamprologus callipterus, in which territorial males defend a collection of shells that are used by females as shelters in which they lay their eggs and take care of the clutch and brood (Mitchell et al. 2014). It is also the case of Tropheus moorii, a monogamous species with maternal mouthbrooding (Egger et al. 2006), and Pseudosimochromis curvifrons, species in which males present a sneaking mating behavior, and the breeding strategy is maternal mouthbrooder (Kuwamura 1987). Using a simulated territorial intruder protocol as the ethological challenge trial, an increase in 11-KT, but not in testosterone levels, was observed in males of these species after the intrusion (Hirschenhauser et al. 2004). It is also worth mentioning that, even though this androgen responsiveness could not be explained by the degree of parental care, it is related to the reproductive behavior since it is greater among males of monogamous species and also in species with more intense pair bonding such as T. moorii (Hirschenhauser et al. 2004). Prediction of the challenge hypothesis has also been tested in Oreochromis mossambicus males with different approaches, since social manipulation in that case was tested in dominant males that were swapped between groups, and also after territorial intrusion, in which a male was introduced into the aquaria. Interestingly, only testosterone and not 11-KT increased after social manipulation in socially stable groups but not in unstable ones, in which dominant males were swapped between groups for five consecutive days, while neither androgen increased after territorial intrusion (Almeida et al. 2014). Authors discuss that this lack of increase in androgens could be because of the existence of a certain androgen threshold, above which social challenge no longer induces androgen response. This way, in those cases that basal levels are below that threshold, androgen levels increase as a consequence of social challenge, but if basal levels are above that threshold, this increase does not take place. This way, males that have reached this threshold of androgen response due to social stimulation could become unresponsive to further social challenge.
Not much is known about endocrine regulation of reproductive behavior and social challenge in Neotropical South American cichlid species compared with African cichlid species. Considering cichlids show diverse forms of parental care, substrate guarding is mostly represented by Neotropical and Madagascar cichlids, while mouth brooding behavior is mainly represented by African species. Moreover, even if biparental behavior can be found in Neotropical and African species, female care behavior is mainly represented in African cichlids (Goodwin et al. 1998). Taking into account these variable characteristics and considering that predictions of the challenge hypothesis can differ according to the parental care behavior (Wingfield et al. 1990), surprisingly Neotropical cichlids have been understudied in this regard. In this sense, the challenge hypothesis has been recently tested in C. dimerus. In a trial involving intrasexual dyadic encounters in neutral arenas, an increase in both testosterone and 11-KT was observed in males (Scaia et al. 2018b ). Considering that this difference with the before mentioned species could be due to phylogenetic distance, geographical or environmental constrains, or to differences in reproductive and parental behavior, further studies in other Neotropical cichlid species are necessary.
Even though aggression has been historically analyzed in males, females also show high levels of aggressive behavior in different groups and ethological contexts (Elekonich & Wingfield 2000, Langmore et al. 2002, Davis & Marler 2003). Taking into account that the challenge hypothesis has been initially proposed in males, very few studies have assessed this hypothesis in female fish despite the fact that in some species they can be as aggressive, and even more aggressive than males depending on the context (Renn et al. 2009, 2012, Scaia et al. 2018a ). In cichlid fish, female aggression varies according to the mating and parental behavior of each species. For example, C. dimerus is a monogamous and biparental species with a hierarchical social system in which dominant females aggressively defend their territory together with males (Pandolfi et al. 2009, Alonso et al. 2011, Ramallo et al. 2014). Moreover, it has been recently determined that, when exposed to dyadic intrasexual encounters in neutral aquaria, females are as aggressive as males and show the same aggressive displays toward the opponent (Scaia et al. 2018a ). The challenge hypothesis has been also assessed in females of C. dimerus, but no increase in androgen levels were detected after the challenge trial (Scaia et al. 2018b ). Female aggression has been also studied in A. burtoni, a polygamous and maternal mouthbrooder cichlid species in which females usually do not present social hierarchies and they only adopt aggressive behavior and male-typical courtship displays toward other females in the absence of males (Renn et al. 2012, O’Connell et al. 2013). They also show aggressive displays toward male intruders when they are caring for their brood (Renn et al. 2009), but unfortunately female aggression after territorial intrusion in this species has only been assessed regarding maternal aggression and not in neutral aquaria. Moreover, the challenge hypothesis has also been assessed in females of this species, suggesting that testosterone increases in challenged females when compared to controls (Renn et al. 2009). Regarding N. pulcher, which has a cooperative breeding system, circulating levels of 11-KT and testosterone increased in females after simulated territorial intrusion (Desjardins et al. 2006). The fact that A. burtoni and N. pulcher females, but not C. dimerus, support the challenge hypothesis could be explained considering that it has been suggested that predictions of this hypothesis can differ according to the parental care behavior (Wingfield et al. 1990). However, further studies in cichlid species with different reproductive and parental strategies are needed, which will disentangle the relationship between social challenges, reproduction and sex steroids in females.
Historically androgens have been suggested as main mediators of aggressive behavior. However, even though testosterone modulates aggression, aggressive behavior can also be observed in individuals with low circulating androgen levels (Caldwell et al. 1984, Demas et al. 1999, Pinxten et al. 2003). As a consequence, androgens have a limited explanatory power to understand regulation of aggressive behavior, and there is evidence suggesting the aromatization of testosterone to E2 plays an important role in this sense (reviewed by Trainor et al. 2006). Estrogens have been mainly associated with female-typical behavior, but several authors suggest that they are key factors regulating male aggression in mammals (Ogawa et al. 1998, Toda et al. 2001), birds (Soma et al. 2000a ,b , Silverin et al. 2004) and fish (Filby et al. 2010, Huffman et al. 2013). In spite of this growing evidence suggesting E2 as a key mediator of aggression, and despite the fact that the challenge hypothesis is intrinsically related to aggressive interactions, there is almost no evidence exploring the potential link between this hypothesis and estrogens. To our knowledge, the first study relating the challenge hypothesis to E2 is in cichlid fish. In N. pulcher, there is no increase in E2 levels as a consequence of social instability (Desjardins et al. 2006). However, there is an increase in E2 levels after social ascent in A. burtoni males (Huffman et al. 2012). These experiments, in which dominant males are removed creating an opportunity for reproductively suppressed males to ascend in social hierarchy and status, resemble a common situation in the natural environment (e.g. dominant males being captured or predated). Finally, also recent evidence in C. dimerus suggests that E2 increases after a challenge trial in males, but not in females (Scaia et al. 2018b ). Despite this difference between N. pulcher and C. dimerus, which could be related to different mating and parental systems or to taxonomical distance, the challenge hypothesis could be extended to estrogens and this evidence encourages further research in species with diverse social behavior.
Social control of spermatogenesis in African and Neotropical cichlid fish
Even though the brain–pituitary–gonad axis (BPG) controls reproduction in all vertebrates, vast evidence suggests that in different groups of fish this axis is socially regulated (Filby et al. 2010, Tubert et al. 2012, Almeida et al. 2014, Jalabert et al. 2015). In the African cichlid A. burtoni, testicular morphology and physiology were examined in males after protocol of social instability (Maruska & Fernald 2011). After removing the dominant male, changes in gene expression and testicular morphology were measured at different times during social ascension (0.5, 6, 24, 72 and 120 h). This species showed a rapid upregulation of mRNA levels of FSH receptor (0.5 h) and different androgen receptor (ARα, ARβ) and estrogen receptor (ERα, ERβa and ERβb) subtypes in the testes. LH receptor was not elevated until 72 h after ascent, but this increase coincided with elevated circulating 11-KT and early stages of spermatogenesis, suggesting a role in steroidogenesis (Maruska et al. 2011). The spermatogenic potential of the testes, as measured by cellular composition, was also elevated before the overall increase in testes size during social ascent. Moreover, the presence of cysts at all stages of spermatogenesis, coupled with lower levels of gonadotropin and steroid receptors (ARα, ARβ, ERα, ERβ) in subordinate males, suggests that the BPG axis and spermatogenesis are maintained at a sub-threshold level in anticipation of the chance to gain a territory and become reproductively active (Fig. 4).
Spermatogenesis in testes of Astatotilapia burtoni subordinate, dominant and ascending male (0.5, 6, 24, 72, 120 h after perception of social opportunity). The scheme illustrates a progressive increase in absolute spermatogenic potential during ascent, represented by a gradual increase in the percentage of advanced stages of spermatogenesis (spermatocytes, spermatids and spermatozoa).
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
Differences between dominant and subordinate males regarding testicular physiology and histology have been also detected in the Neotropical cichlid fish C. dimerus. Dominant males show proportionally larger testis than subordinated males as evidenced by their higher gonadosomatic index (GSI) (Ramallo et al. 2015). Within dominant male testes, spermatozoa dominated the cellular landscape with an estimated relative abundance of 34.5%, whereas spermatocytes were the major cellular component within subordinated males’ testes (39.1%). The comparison between males of different social status revealed that the percentages of spermatocytes and spermatids were higher in subordinated males, while dominant males showed a greater percentage of spermatozoa (Fig. 5). As a consequence, social scenario in this species not only shapes the endocrine landscape as already mentioned in section 4 (e.g. dominant males with higher androgens and lower estrogens), but also affects testicular morphology and composition.
Comparison of gonadosomatic index (GSI) and cellular composition (spermatozoa, spermatid and spermatocyte) in testis from subordinate and dominant males of Cichlasoma dimerus. Dominant males present higher GSI than subordinate and a predominance of spermatozoa, while spermatocytes are the predominant cellular type in testis from subordinate males.
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
This association between GSI and social status has also been found in other cichlid species, such as O. niloticus (Golan & Levavi-Sivan 2013), O. mossambicus (Oliveira & Almada 1998) and Amatitlania nigrofasciata (Chee et al. 2013), since dominant males also present higher GSI when compared to the physiologically suppressed subordinated males. Accordingly, in C. dimerus subordinate males show higher GSI in correlation with a higher dominance index (Alonso et al. 2012). Also larger testes are in agreement with steroid hormone profiles, as higher 11-KT levels observed in dominant males are probably associated with stimulation of Sertoli cells, which in turn promote spermatogonial proliferation, meiosis and spermiogenesis (Miura et al. 1991). These similarities among species could imply a common evolutionary response to social suppression in cichlids, resulting in a reduced reproductive investment in subordinates. Moreover, this regulation between social suppression and reproductive physiology has also been detected in some mammal species. In the naked mole rats breeding males have larger reproductive tract masses and higher urinary testosterone concentrations when compared to non-breeders, even if spermatogenesis is active in both cases (Faulkes et al. 1991). Moreover, in primate male sifakas (Propithecus verreauxi) dominant males have higher testosterone levels than subordinates, and this difference is even more robust during breeding season (Kraus et al. 1999). Similarly, it has been suggested that in the gray mouse lemur (Microcebus murinus), which is a prosimian primate, testosterone from subordinate males decreases after they are exposed to urine from dominant males (Schilling et al. 1984). Considering that testosterone levels are an indirect method to estimate testicular physiology, these studies imply a suppression of reproductive physiology in mammal subordinate males.
Interestingly, it has been suggested that the constant intimidation and attacks executed by highly ranked fish over the lowest-ranked males exert a sort of ‘social contraceptive’ which seems to delay, rather than completely impair, spermatogenesis in subordinates (Alonso et al. 2011, Ramallo et al. 2015, 2017). Considering that spermatocytes and spermatids accumulate within subordinated males’ testis, this social constraint is particularly effective at late stages of spermatogenesis during final spermatozoa maturation (Ramallo et al. 2015). However, subordinate males still possess cysts of every spermatogenic cell type, which points for a still ongoing spermatogenesis. As a consequence, even though dominant males were better suited for immediate reproduction, subordinated males still hold reproductive potential. These observations constitute evidence with special ecological importance because in case social structure becomes unstable (e.g. predation or agonistic fights) the opportunity for social ascent emerges. Similarly, evidence in N. pulcher suggest that reproductive capacity of subordinates is merely impaired and not completely suppressed: even if they have relatively smaller gonads, sperm characteristics of high ranked helpers were similar to those of breeders (Fitzpatrick et al. 2006). For example, small helpers have slower swimming sperm, shorter lived sperm after activation and a lower percentage of motile sperm than breeders, suggesting that their sperm would be less competitive. However, as helpers get larger, their sperm is physiologically equivalent to that of breeders. The fact that large helpers have sperm with similar characteristics to those of breeders, but that they have relatively small gonads suggests that helpers are limited in reproductive capacity. This way, it has been suggested that rather than investing in gonadal development, subordinate helpers of N. pulcher may invest in strategic somatic growth, using stored energy only to rapidly enhance gonad development when breeding opportunity arises during social ascent (Fitzpatrick et al. 2006). This is in agreement with the notion by Maruska and Fernald (2011) suggesting that males transitioning from subordinate to dominant status undergo a reactivation of an already functional reproductive axis (brain–pituitary–gonads), similar to pubescent mammals. It would be of great interest to further analyze the dynamics in spermiogenesis reactivation and the endocrine and molecular processes involved, as a subordinated male of different social status can suddenly become territorial or ascend in the hierarchy.
Spermatogenesis and paternal care in Cichlasoma dimerus
Besides social and reproductive behavior, parental care can also modulate reproductive physiology in males. Parental care represents a trade-off between the survival of the offspring and parents’ future reproduction (Huxley 1938). Parental care is defined as any form of parental behavior that appears likely to increase the fitness of the offspring (Trivers 1974). Within the animal kingdom, fish show the greatest variety of parental care especially within cichlids (Goodwin et al. 1998). Fish parental behavior includes activities such as nest construction, tending, guarding and fanning of the eggs and guarding of the brood from predators (Royle et al. 2012). The strategy of parental care adopted by each species is strongly related to the mating system. For example, fish species with lek-breeding or harem systems, for example, show a strong association with maternal mouthbrooding, such is the case for Oreochromis sp. and A. burtoni (Turner & Robinson 2000). However, there is great variability among cichlids and both parental and reproductive strategies could also depend on environmental conditions.
The endocrine system plays an important role in the control of reproductive and parental behavior in vertebrates (Reburn & Wynne-Edwards 1999). In general, it is suggested that 11-KT and testosterone decrease during parental care periods because of an apparent incompatibility of male parental behaviors with aggression (reviewed by Hirschenhauser et al. 2003). However, in N. pulcher (Desjardins et al. 2008), Lythrypnus dalli (Rodgers et al. 2006) and Parablennius parvicornis (Ros et al. 2004) elevated androgen levels do not necessarily decrease parental investment. Moreover, in Lepomis macrochirus males there is no androgen-mediated trade-off between parental aggression and nurturing behavior (Rodgers et al. 2012). Furthermore, males of cichlid species with exclusive maternal care have lower plasma androgen levels than males of closely related cichlid species with biparental care (Hirschenhauser et al. 2004).
Regarding C. dimerus, male parental care period can be divided in four different phases according to the developmental degree of the offspring, as has also been suggested for females of this species: pre-spawning males (Pm, day 0), males guarding eggs (E, 1 day after fertilization (1 DAF)), males guarding hatched larvae (HL, 3 DAF) and males guarding swimming larvae (SL, 8 DAF) (Tubert et al. 2012). Males exhibiting pre-spawning activity showed 8.4 times higher 11-KT and 5.63 times higher testosterone levels than males in the different stages of paternal care (Birba et al. 2015). It is worth mentioning that no differences were observed in E2 and cortisol levels in these males among the different phases.
In C. dimerus, the cellular composition of testes varies during the reproductive and paternal care periods. Even though the GSI is a common metric used to estimate reproductive investment in fish, it has been suggested that in males it does not provide information on cellular changes that could have important functional consequences for sperm and steroid production (Maruska et al. 1996). In the case of C. dimerus, no differences in the GSI were found among males in the four phases already described (Pm, E, HL, SL). Testes cellular composition throughout the reproductive and parental care period was analyzed and the proportion of each cellular type during the four phases was characterized. In this social context, males exhibiting pre-spawning activity presented testes composed of 51.8 ± 4% mature spermatozoa. After spawning, E males exhibited an elevated mean percentage of spermatogonia B and spermatocytes (31.9 ± 5.2%; 35.9 ± 3.1, respectively) and HL males had an elevated mean percentage of SG B (37.9 ± 3.9%), reflecting a high level of germ cell proliferation (Birba et al. 2015). Finally, testes from SL males showed a more homogenous distribution of each cell type with a preponderance of SC. A morphometric analysis of Leydig cells nuclear area revealed that pre-spawning and E males Leydig cells averaged 1.27 times larger than that those of MHL and MSL and was positively correlated with circulating 11-KT and testosterone levels (Fig. 6).
Spermatogenesis in testes of Cichlasoma dimerus in four different parental care phases: pre-spawning males (day 0), males guarding eggs (one DAF), males guarding hatched larvae (three DAF), and males guarding swimming larvae (eight DAF). Testes of pre-spawning males were composed of 50% of spermatozoa, whereas spermatogonia type B and spermatocytes were predominant in the subsequent parental phases. DAF, days after fertilization.
Citation: Reproduction 159, 1; 10.1530/REP-18-0650
These data suggest that C. dimerus spermatogenesis remains active during the parental care periods, which is in concordance with the fact that C. dimerus undergoes multiple spawning events within a single reproductive period (November–March) (Vázquez et al. 2012).
Conclusions and future directions
In this review we have summarized studies suggesting that social environment regulates spermatogenesis and steroidogenesis in cichlid fish, thus modulating the reproductive capacity. Several evidence supports this social regulation of reproduction: (1) agonistic behavior influences hormonal levels, which in turn, modulates reproductive and social behavior, establishing intertwined and bidirectional effects; (2) cichlid fish exhibit a wide range of social behaviors and reproductive strategies and constitute ideal models to study social control of reproduction; (3) the challenge hypothesis constitutes an appropriate framework to understand the relationship between sex steroids and agonistic behavior; (4) the BPG axis is socially regulated; (5) dominant and subordinate males from different cichlid species differ in testicular morphology, circulating hormone levels, spermatogenesis and GSI; (6) parental care can also modulate reproductive capacity, since it affects testicular morphology, androgen levels, testicular cellular composition and the rate of spermatogenesis.
However, this review highlights some questions that deserve more attention and areas that remain to be studied. Even though there is extensive research on African cichlid species, Neotropical cichlids still constitute a group that deserves more attention. Moreover, there is still a lack of comparative approach assessing species with different reproductive behavior and parental care strategies; this will help to disentangle whether differences among fish species could be due to phylogenetic distance, to behavioral differences, or to both.
Finally, it is worth mentioning that social behavior and reproductive strategies in female cichlids are still understudied when compared to males. This could be due to the fact that in A. burtoni, which has been historically the focus in studies referring to social control of reproduction in cichlids, females only present aggressive displays in wild and not in laboratory stocks and they do not present social hierarchies in the presence of males. However, new growing evidence in other species such as C. dimerus suggest that females have interesting agonistic behavior, such as aggressive and submissive displays not only related to the reproductive behavior and maternal role defending the fry, but also in neutral aquaria with no resources. These results, and the fact that this female aggressive behavior is regulated by sex steroids and also modulates sex steroid landscape, suggest that female fish deserve further attention.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was supported by the Agencia de Promoción Científica y Tecnológica (PICT 2016-0086, 2016-1614), the Universidad de Buenos Aires (UBACyT 2016-0038).
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