Appetite regulating genes may contribute to herbivory versus carnivory trophic divergence in haplochromine cichlids

Background

At the onset of exogenous feeding, most marine and freshwater fish species are known to be planktivorous and have a limited ability to detect, capture, ingest and digest prey (Nunn, Tewson & Cowx, 2012). Improvements in vision, swimming performance, gaping abilities and the functions of digestive tract during ontogeny can lead to shifts in diet composition and preference. Importantly, differences in feeding behaviour between fish species both at early stages of exogenous feeding and during later ontogeny can influence diet preference at inter- and intraspecific levels (Nunn, Tewson & Cowx, 2012). Little is known about the role of central nervous system in determining the diet preference and related feeding behaviours required for using specific food resources in natural ecosystems. Repeated evolution of herbivorous and carnivorous trophic specialization in closely related teleost fishes is a prominent example for such a divergence in diet preference, and so far, most studies were mainly focused on related changes in digestive system (morphological and enzymatic differences) (Kapoor, Smit & Verighina, 1976; Chakrabarti et al., 1995; Chan et al., 2004; German et al., 2010). Besides morphological changes in the feeding apparatus, divergence of feeding behaviour might be another key feature of differential trophic adaptation since the two diet habits require foraging on different quantities of food to balance nutritional requirements due to the unequal quality of these diets. Since adult herbivorous and carnivorous fish have distinct appetite and feeding behaviours, these differences might be pre-determined or influenced by early changes in activity of genes controlling feeding behaviours at the onset or prior to exogenous feeding. In the latter case, one would expect to observe differences in expression of these genes at the end of the larval stage, which reflects the feeding behaviour of the adults, by differentiating herbivorous and carnivorous species.

One of the major events preceding differences in feeding behaviour can be changes in activity of appetite-regulating genes in the central nervous system, which later translate into distinct desires for food intake and related locomotory activities (e.g., food searching) (Volkoff et al., 2005). Thus, an immediate approach to investigate such molecular mechanisms is expression analysis of genes involved in regulation of feeding behaviour through the central nervous system in fish (Volkoff et al., 2005). To date, only one study in grass carp (a species of Cypriniformes), which shows the transition from carnivory to herbivory during ontogeny, has addressed gene expression changes in the brain between the two contrasting feeding habits (He et al., 2015). Interestingly, the authors found that few appetite-regulating genes that inhibit food intake (anorexigenic genes) had reduced expression in the brain at the herbivorous life stage, whereas few other genes with opposite effects (orexigenic genes) had increased expression at this stage (He et al., 2015). This finding was consistent with the notion that herbivory requires prolonged insatiety and more active feeding behaviour compared to carnivory in order to compensate for the relatively poorer nutritional quality of a plant-based diet (He et al., 2015). Although, a comprehensive list of potential appetite-regulating genes has been provided mainly from studies on cyprinid model species, such as zebrafish and goldfish, it has turned out that the regulatory function of many of these genes can vary across the orders of teleost fishes (Volkoff, 2016). In addition, only a small subset of the genes are confirmed to have similar appetite-regulating functions in other fish orders including Cichliformes and Perciformes (Volkoff, 2016).

Cichlids of the East African Great Lakes Tanganyika, Malawi and Victoria are well known for their remarkable rates of speciation and adaptive radiation (Fryer & Iles, 1972; Kocher, 2004). Lake Tanganyika, the oldest of the three lakes, shows the most diversity in ecomorphology, behaviour and genetics compared to Lake Malawi, the intermediate, and Lake Victoria, the youngest of the three lakes (Young, Snoeks & Seehausen, 2009; Salzburger, Van Bocxlaer & Cohen, 2014). The Haplochromini are the most species rich tribe, having seeded the entire species flocks of Lake Malawi and Victoria and also recolonized Lake Tanganyika, giving rise to the tribe Tropheini (Salzburger et al., 2005). It is hypothesized that similar trophic ecomorphologies evolved in all three lakes in response to similar selection pressures as they were derived from a common generalist riverine ancestor (Kocher et al., 1993; Salzburger et al., 2005; Cooper et al., 2010).

Interestingly, haplochromine cichlids are mostly maternal mouthbrooders so the fry start feeding independently at a more mature stage, at the end of larval development (stage 26), compared to non-mouthbrooders (Fujimura & Okada, 2007; Fujimura & Okada, 2008). At stage 26, the larvae leave the buccal cavity of the mother and are exposed for the first time to the environment as they start foraging independently. Due to the high trophic phenotypic plasticity in haplochromine cichlids (Gunter et al., 2013; Schneider et al., 2014), it is important to decipher whether gene regulatory circuitry of appetite-regulating genes that triggers feeding behaviour can be already observed upon completion of the larval development prior to the onset of food intake or is activated once the larvae start feeding. The dietary plasticity, mouthbrooding and swimming behaviours (e.g., locomotory activities), and immense diversity of trophic specializations and foraging in cichlid fishes of East African species flocks provide an excellent opportunity to investigate the role of appetite-regulating genes in differential trophic adaptations associated with species divergence.

Here, we hypothesize that appetite-regulating genes might be already differentially regulated in the brain of distinctly adapted haplochromine cichlids at the end of larval development, thus, affecting feeding behaviour (i.e., food intake and related locomotory activity), before the fry is released from the mother’s mouth to forage on their own. Our hypothesis might advocate for low plasticity and high genetic wiring of feeding behaviour in these fish, although studies investigating potential effects of appetite-regulating genes on foraging tactics are required to make such a claim. Therefore, we selected 16 appetite-regulating genes and analysed their expression level in the brain in a set of three herbivorous and three carnivorous haplochromine cichlid fish species at stage 26 (Fujimura & Okada, 2007; Fujimura & Okada, 2008), which marks the end of larval development and the initiation of exogenous feeding. The selected candidate genes are known to have brain expression in fish and are involved in regulation of feeding behaviour by enhancing or inhibiting food intake in teleost fishes (Table 1). The functions of most of these genes have been investigated in Cypriniformes (on which most studies have been conducted) and fish species with evolutionary closer relatedness than Cypriniformes to cichlids, such as members of Perciformes and Cichliformes. Five of these genes are also playing a role in food habit transition from carnivory to herbivory in grass carp (He et al., 2015) (Table 1). The study species belong to two major trophic niches in the three Great East African Lakes; Lake Tanganyika (LT), Lake Malawi (LM) and Lake Victoria (LV). We test whether the differential expression of appetite-regulating genes in the brain predicts the divergence in trophic specialization in differentially adapted species pairs prior to the actual searching for food resources. The study also addresses this possibility in the context of parallel trophic specialization across three independent adaptive radiations. This study reports the results of a first step by validation of stably expressed reference genes in the brain at the end of the larval stage, which allows us to accurately compare inter-species expression of the appetite regulating-genes in haplochromine cichlids. Our results suggest that expression differences of the candidate genes might predict the feeding behaviour of herbivore versus carnivore species before the onset of plastic molecular responses emanating from contrasting feeding diets.

Methods

Fish husbandry and sampling

Six haplochromine cichlid species belonging to two major trophic niches from Lakes Tanganyika (LT), Malawi (LM) and Victoria (LV), were chosen for studying brain gene expression. All samples were obtained from lab stocks, which have been previously acquired from aquarium trade suppliers. In order to compare divergent trophic niches, we used one carnivorous species (a piscivore/insectivore) and one herbivorous species (an algae-grazer) for each lake (Fig. 1A), based upon previous phylogenetic studies (Koblmüller et al., 2008; Irissari et al., 2018). The parental fish were reared under standardized aquarium conditions and diet (Spirulina flakes with average protein content) until sexual maturation. The spawning pairs were closely observed and 24 h after mating their eggs were collected from the mouth of the females through exerting mild manual pressure to their cheeks. Then, the eggs of each species were placed in a standard glass jar with constant gentle shaking during the incubation period until hatching stage. After hatching, larvae were transferred to small floating tanks and kept until stage 26, the time of yolk sac absorption, marking the end of larval development (Fujimura & Okada, 2007; Fujimura & Okada, 2008). The rearing and incubation temperature was kept constant at 25.8 degrees centigrade. For each species six larvae were euthanized in water containing 0.2 gram MS-222 per litre, and the entire brain was carefully dissected using a stereomicroscope. The brain tissue from each individual represents one biological replicate, and therefore, six biological replicates per species were used for further analysis of gene expression. Moreover, by the end of the study the parents of the six haplochromine species were sacrificed in water containing 0.8 gram MS-222/litre.

Results

Expression differences between herbivores and carnivores

At first, we used the relative expressions of all 16 target genes in each species in order to construct a dendrogram cluster representing the similarities between species in brain expression of appetite-regulating genes (Fig. 1C). The results showed that the similarities between the species are mainly determined by evolutionary relatedness by which species from the same lake (for Malawi or Victoria) are paired together. However, an interesting difference was observed for the LT species where the carnivore species (C.h) was clustered with the LM species and the herbivore species (P.p) branched distantly from the other clusters (Fig. 1C). This might indicate that the LT species with their much older evolutionary divergence have more distinct expression pattern of appetite-regulating genes prior to foraging, as outlined in more detail in the discussion. It also appears that the herbivore brain might have more diverged gene expression patterns for appetite-regulating genes in LT.

When the overall expression levels of the appetite-regulating genes were compared between herbivores and carnivores across the lakes six genes, cart, drd1, gabra1, npy2r, pyy and trh appeared to have differential expression (Fig. 2). Among these, cart, gabra1 and npy2r displayed strong expression differences. Also, all of these genes, except pyy, had shown higher expression in the carnivores than herbivores (Fig. 2). These results demonstrate expression differences of certain appetite-regulating genes in herbivorous versus carnivorous haplochromine cichlids prior to initiation of their feeding. This also suggests that feeding behaviour can be already determined in the brain by differential expression of appetite-regulating genes before exposure to available food resources. However, considering the opposing appetite-regulating functions of these genes, i.e., cart, drd1 and npy2r are anorexigenic whereas gabra1 and trh are orexigenic genes (Table 1), it is difficult to interpret the feeding behaviour outcome of such contrasting expression differences.

Next, we compared the expression levels of each gene between the herbivorous and carnivorous species within the lakes. All of the genes, except cck and npy , showed differential expression between the two trophic niches in at least one lake (Fig. 3). Out of the 16 tested genes, 7, 12 and 13 genes were differentially expressed in LM, LV and LT, respectively. The effect sizes, as indicated by the calculated Cohen’s d values (Supplementary data 1), further demonstrated that all the differentially expressed genes had large effect sizes (d values >0.8), indicating that the results have not been influenced by low statistical power. In LT, all of the 13 differentially expressed genes showed higher expression in the carnivore species, but this number declined by the age of divergence between the trophic niches in each lake, i.e., four out of the seven genes for LM and four out of the 12 genes for LV (Fig. 3). When comparing the lakes, four genes, cart, hcrt, npy2r and trh, showed similar expression difference between LT and LM, four genes, agrp2, cart, crh and npy2r, did between LT and LV, and four genes, cart, drd1, gabra1 and npy2r between LM and LV. Among these, only cart and npy2r (both anorexigenic genes) had similar differences of higher expression level in carnivorous than herbivorous species in all the three lakes (Fig. 3). The differential expression of cart appeared to be increased in the carnivore brains according to the age of divergence between the contrasting species of each lake (i.e., LT >LM >LV). Moreover, cart displayed the strongest herbivorous versus carnivorous expression difference among the identified differentially expressed genes. The expression results of genes like cart and npy2r suggest that carnivory versus herbivory and possibly their related feeding behaviour in Haplochromine cichlids might be linked to divergence in brain expression of the anorexigenic genes prior to initiation of feeding.

Discussion

Diversity in cichlid diet and foraging behaviors is thought to be a key factor facilitating their rapid divergence by enabling effective trophic specialization and ecological speciation (Liem, 1973). Plasticity in trophic morphology and physiology, manifested in jaw shape, intestine length and enzymatic activities, are believed to have played an important role in the adaptation to new habitats and the optimization of feeding during ontogeny (Sturmbauer, Mark & Dallinger, 1992; Takahashi & Koblmüller, 2011). But little is known about the link between the brain and foraging behavior. In particular, the appetite-regulating genes in the brain that might contribute to different dietary and swimming habits, prior to the onset of feeding have not been studied. Here we investigated the expression of appetite regulating genes in the brains of cichlids adapted to herbivorous and carnivorous trophic niches and identified two appetite-regulating genes, cart (or cartpt1) and npy2r, to have higher expression in the carnivore brains prior to the initiation of feeding across at least two of the lakes. Cart and npy2r genes are indicated to have anorexigenic function in both distant and closely related groups of fishes to cichlids (i.e., Cypriniformes and Perciformes, respectively) (Matsuda et al., 2012; Babichuk & Volkoff, 2013; Wang et al., 2014; He et al., 2015; Volkoff, 2016; Porter, Roberts & Maruska, 2017). This suggests potential involvement of the two anorexigenic genes in parallel trophic radiations of the cichlids used in this study. Furthermore, the increased expression of cart and npy2r in carnivorous species at the end of larval development can be reminiscent of the adult feeding behaviour based on which herbivorous species have an increased level of appetite compared to carnivorous species in order to compensate their nutritionally poorer diets. This may indicate heterochrony in brain expression of these genes prior to emergence of herbivorous versus carnivorous feeding behaviours during later ontogeny. Heterochrony in gene expression is already suggested as a potential molecular mechanism in divergent adaptive radiation of teleost fish (Albertson et al., 2010; O’Quin et al., 2011; Shkil & Smirnov, 2015).

The first gene, cart, or cocaine- and amphetamine-regulated transcript, encodes a pre-proprotein which proteolyzes to multiple active peptides and participates in biological processes related to regulation of appetite, energy balance, stress response, and reward and addiction responses (Volkoff, 2006; Volkoff, 2016; Koylu, Balkan & Pogun, 2006; Vicentic et al., 2007; Rogge et al., 2008). In most teleost fish including Perciformes, Salmoniformes and Gasterosteiformes only one cart isoform has been found (Murashita et al., 2009; Figueiredo-Silva et al., 2012; Striberny et al., 2015), whereas, in two model fish species; medaka and zebrafish (Beloniforme and Cypriniforme, respectively) more than one cart isoforms have been characterized (Murashita & Kurokawa, 2011; Akash et al., 2014). In a Haplochromine cichlid, Astatotilapia burtoni, six cart isoforms have been described and among them cart/cartpt1 show the greatest similarity to mammalian CART gene (Hu et al., 2016). The brain expression pattern of cart appeared to be similar to its orthologs in other teleosts in the lateral posterior part of the hypothalamus (or lateral tuberal nucleus), which is also similar to the expression of mammalian CART in a comparable region called arcuate nucleus (Porter, Roberts & Maruska, 2017). Studies of Cypriniformes have demonstrated that cart induction inhibits food intake and increases locomotion and responsiveness to different sensory stimuli, and thus affecting feeding behavioural activity (Volkoff & Peter, 2000; Woods et al., 2014). It has been long known that predatory behaviour is directly influenced by the ability to respond to a range of sensory stimuli mediated by vision, olfaction and the lateral line in fish (Adams & Johnsen, 1986; Gehrke, 1988; Carr et al., 1996; Montgomery & Hamilton, 1997; Liao & Chang, 2003; Del Mar Palacios, Warren & McCormick, 2016). In addition, the decrease in brain expression of anorexigenic genes has been linked to the transition from carnivory to herbivory feeding behaviour in grass carp (He et al., 2015).

In our study, the increased cart expression in the carnivore brains prior to feeding may indicate less appetite and a predisposition for more environmental responsiveness in the carnivores, which may be a favorable behaviour for predatory-based trophic specialization. Furthermore, the conserved anorexigenic role of CART peptides in teleost fish has been demonstrated in a wide range of species during fasting and re-feeding experiments (reviewed in Volkoff, 2016). Interestingly, we found that the difference in cart expression level between the herbivorous and carnivorous species in each lake to be associated with the age of divergence in each lake, i.e., the older divergence had the highest difference in cart expression levels (Fig. 3). In the youngest lake (LV), on the other hand, only slight expression difference between the herbivorous and carnivorous species was found. This is especially interesting as the cichlids from older lakes have longer larval developmental periods as they have larger yolk sacs that provide nourishment for longer, so food intake may need to be inhibited for longer (Dreo and Gallaun, 2018, unpublished data). We also found the similar pattern of increased carnivorous expression in the two older lakes for trh, thyrotropin-releasing hormone, which is characterized as orexigenic factor in Cypriniformes through increasing feeding and locomotor behaviours (He et al., 2015; Volkoff, 2016). However, such a function has not been identified in Cichliformes or Perciformes, and its induced expression in carnivorous species of LT and LM can be the result of strong induction of cart in these species. This is due to the fact that trh is at downstream of cart in vertebrates and it can function as negative regulatory feedback for cart signal (Fekete & Lechan, 2006). Based on this negative feedback, prolonged expression of the anorexigenic cart gene induces the transcription of orexigenic trh which in turn can control cart expression through a complex molecular cascade (Fekete & Lechan, 2006). Therefore, the increased expression of trh in carnivorous species can be in fact a confirmation for functional activation of cart signal in their brain.

The second gene, npy2r, encodes a receptor of Neuropeptide Y (npy), and interestingly, an ortholog of the same receptor has been identified to have reduced expression during the transition from carnivory to herbivory in grass carp (He et al., 2015). The ligand of this receptor, npy, is expressed in different tissues, particularly in brain and intestine, and its encoded peptide (NPY) has been one of the first studied appetite-regulating factors in fish (Volkoff, 2016). In this study we found reduced expression of npy2r in the brain of herbivores which is consistent with the suggested anorexigenic role of npy2r in grass carp (He et al., 2015). Although, the ligand of npy2r, NPY peptide, acts as an orexigenic factor in most teleost fish species (reviewed in (Volkoff, 2016), npy2r is among the NPY receptors in vertebrates that functions as inhibitory auto-receptor, and thus playing an opposite role to NPY in appetite regulation (Chen et al., 1997; Naveilhan et al., 1999).

Overall, most of the selected appetite-regulating genes showed no consistent expression differences between herbivores and carnivores across the three lakes indicating that most of these genes probably do not contribute to determination of feeding behaviour prior to foraging in the selected cichlid species. Moreover, their expression differences between the two trophic niches showed the most discrepancies between the species of the youngest and oldest lake adaptive radiations (LV versus LT). This might reflect more extensive divergence in evolutionary trajectories of the LV and LT lineages in expression of the appetite-regulating genes. Although, the consistently increased expression of the two anorexigenic genes, cart and npy2r, in carnivores in all the lakes could imply on their potential role in determination of the feeding behaviours prior to foraging in the cichlid species in this study. Further functional investigations are required to confirm such a role for appetite regulating genes in fish. The number of species used in this study to represent each trophic niche is not sufficient to conclude that signals mediated by cart and npy2r are potential drivers for divergent trophic specialization across the entire Haplochromini tribe. In addition, it is not clear if the peptides encoded by these genes interact with other appetite-regulating factors and whether they override the effects of the other differentially expressed factors in the brain.

Other behavioural factors that can affect or being affected by gene expression in the brain are the divergent habitat use and swimming strategies by species with contrasting trophic specialization. Although, all the species in this study (except C. horei) are reported to occupy somewhat similar habitats in the three lakes: M. lutea; rock-dwelling (Seehausen et al., 1998), H. thereuterion; rock-dwelling with surface oriented swimming (Seehausen, 1996), S. fryeri; rocky and intermediate habitats (Konings, 1993), P. sp. chitimba; rock-dwelling (Johnson et al., 2019), P. polyodon; rock-dwelling (Kohda, 1998), and C.horei; rocky-sandy or muddy and sediment-rich habitats (Wanek & Sturmbauer, 2015). The difference in swimming strategies, however, can be more consistent between the two trophic niches, i.e., carnivorous species expected to have more surface-oriented or free-range swimming in the water column, and it is already known that most appetite-regulating genes can affect swimming, locomotor activity and food searching behaviour in fish (Volkoff, 2016). It is worth noting that the observed expression differences could be originated from proportional anatomical discrepancies in the brain regions expressing these genes and not necessarily the result of transcriptional changes. This is particularly important topic for future histological investigations, since anatomical variations in brain regions have been already demonstrated between East African cichlids with divergent ecological adaptation (Sylvester et al., 2010). Finally, we should emphasize that the time to reach to stage 26 for haplochromine cichlids varies between the lakes, i.e., the length of this time is positively correlated with the age of the lakes, and therefore, this limits such gene expression studies to only comparisons of the species within each lake .

Conclusions

Diet is a major factor mediating adaptive divergence in the adaptive radiation of cichlids fishes. Here we present the first step towards delineating the genes involved in regulating appetite in herbivorous and carnivorous cichlids prior to the onset of independent feeding. We identified two anorexigenic genes, cart and npy2r, to be differentially expressed between the two trophic categories in parallel cichlid radiations of three East African Lakes, which is suggestive of their role in controlling satiety in these species. It might also imply that appetite gene regulation is genetically hardwired. However, future studies investigating potential links between the functions of appetite-regulating genes and specific foraging tactics are essential to conclude that these results advocate low plasticity in regard with herbivory and carnivory in cichlids. Also, large-scale studies using transcriptomic approach with more species (representing carnivorous and herbivorous trophic niches) from each lake are required to test whether appetite-regulating genes are among the divergent genetic signals in brain participating in trophic niche specialization during cichlid adaptive radiation. In conclusion, we present a first glimpse into an important aspect of feeding in cichlids that is the regulatory control of appetite in the context of contrasting trophic niche adaptation.