The subterranean environment is often described as “extreme” and food poor. Laboratory experiments have shown that blind Mexican tetra Astyanax mexicanus (De Filippi, 1853) cavefish are better at finding food in the dark than surface fish. Several morphological and behavioural attributes that could foster this obvious adaptive response to cave environments have been described. Nonetheless, it is currently unknown what young cavefish actually eat in their natural cave environment. Our results from the Pachón cave in México during the dry and rainy season show that fry are efficient predators in their natural cave environment. Their primary food item is aquatic crustaceans. The guts of post-larval, pre-juvenile stage individuals (n=9) contained an average of 17.9 water fleas (Cladocera), copepods, ostracods, and isopods. Thus, the fry in this cave are well-fed. The Pachón cave environment does not appear to be “food poor” for juvenile cavefish. Food regimes change between post-larval and adult stages to become more dependent on partially decomposed material, guano, or detritus from the mud. We discuss the data with regards to our current developmental and genetic understanding of cavefish morphological and behavioural evolution, particularly regarding its enhanced Vibration Attraction Behaviour (VAB).

Predation, gut contents, troglomorphy, VAB, neuromast

The blind Mexican tetra Astyanax mexicanus (De Filippi, 1853) has become a well-established model system in evolutionary developmental biology (EvoDevo). This species has been the main contributor in the understanding of the genetic and developmental controls of troglomorphic features. There are over 30 known caves harbouring Astyanax cavefish populations in México (Mitchell et al. 1977; Espinasa et al. 2001). Their conspecific surface-dwelling morph lives in nearby surface streams throughout most of México. The eyed and pigmented surface morph and the eyeless, depigmented cave morph are inter-fertile, making the species well-suited for experimental manipulations (Jeffery 2012; Elipot et al. 2014).

Literature often states that the cave morph is more efficient at finding food in darkness. Multiple morphological and behavioural attributes have been described to support this statement, such as a higher number of taste buds (Yamamoto et al. 2009; Varatharasan et al. 2009), higher chemosensory capabilities (Protas et al. 2008; Bibliowicz et al. 2013; Hinaux et al. 2016), an enhanced number of cranial neuromasts (Yoshizawa et al. 2012), modulation in early developmental signalling pathways influencing brain development and organization (Yamamoto et al. 2004; Pottin et al. 2011), and a behaviourally more efficient posture with respect to the substrate when bottom feeding (Schemmel 1980).

Increased efficiency in food finding has been supported by five controlled observations or experiments in which cavefish directly outcompeted surface fish for a limited amount of food. Three of these observations were in adult fish (Hüppop 1987; Yoshizawa et al. 2010; Wilkens and Hüppop 1986), and two supported that the enhanced skills were already in place as early as 25 days after birth (Espinasa et al. 2014; Hinaux et al. 2016). For example, Hüppop (1987) showed that under competitive conditions, cavefish found about 80% of all pieces of beef-heart muscle provided in the dark whereas the epigean fish were successful at finding only 20%. This suggests enhanced smell and chemical detection in cavefish. Direct measurements of the size of the olfactory pits have shown that eyeless cavefish have a wider olfactory pit than eyed surface fish. The width of the olfactory pit is on average 12.9% larger due to the eye-dependent developmental processes (Yamamoto et al. 2003; Hinaux et al. 2016). An enlarged olfactory pit could result in an enhanced sense of smell, which could directly correlate with the feeding skills of eyeless fish (Bibliowicz et al. 2013). Indeed, 30 day-old juvenile cavefish were recently shown to have outstanding olfactory capabilities at a magnitude 105 higher than surface fish for food-related amino-acid odorant cues (Hinaux et al. 2016).

In two studies (Yoshizawa 2010; Espinasa et al. 2014), food finding efficiency was tested using competition assays where fish had to capture live Artemia larvae (nauplii). The results suggest that both adult and young Astyanax have enhanced abilities for prey capture. For example, in the Espinasa et al. (2014) study, 25 day-old cavefish and surface fish raised under light/dark conditions were paired to compete for Artemia nauplii in the dark. On average, cavefish captured more Artemia than surface fish in each paired match. Both studies suggest that other complex skills, apart from smell, may be enhanced in cavefish to allow for efficient predation.

Small crustaceans such as copepods disturb the water at 30–40 Hz when swimming (Montgomery and Macdonald 1987). Vibration attraction behaviour (VAB) is the ability of fish to swim toward the source of such vibrations and has been shown to be advantageous for Artemia feeding competition assays in the dark by adult cavefish (Yoshizawa et al. 2010). In cavefish, the detection range of the superficial neuromasts in the lateral line system is tuned to 35 Hz (Yoshizawa et al. 2010). The cupulae of cavefish superficial neuromasts are about 300 μm in length compared to about 42 μm in surface fish (Teyke 1990). Neuromasts within the eye orbit and in the suborbital region are about twice as sensitive in young adult cavefish as in surface fish (Yoshizawa et al. 2014). VAB is typically seen in cavefish, but rarely in surface fish. While in the laboratory VAB is not statistically evident before cavefish reach three months of age (Yoshizawa et al. 2010), results by Espinasa et al. (2014) suggest it is possible that some of the physiological bases behind the behaviour are active in young fish.

While it is evident that Astyanax have undergone significant modifications in feeding skills, the actual food sources of Astyanax remain unclear in their natural cave environment. Some authors have mentioned that their food consists almost completely of bat guano rather than live and mobile organisms (Kasumyan and Marusov 2015; Wilkens 1972; Mitchell et al. 1977; Hüppop 1987). If this was the case, it would be difficult to explain the selective value of VAB found in multiple cave populations. The purpose of this study is to assess the actual food source of Astyanax cavefish. Stomach and intestine contents were analysed from nine fry and five adults captured during both dry and rainy seasons from the Pachón cave, which is where the most commonly studied population of Astyanax lives.

Our results show that post-larval fry from Pachón cave appear to be well-fed and are efficient predators. This is evident in the guts of the nine individuals that contained an average of 17 specimens of microscopic crustaceans. Our results show that arthropods are the main source of nourishment for 1-2 cm long Astyanax fry in Pachón cave, with 89.8% of their stomach contents being readily identifiable arthropods. Data suggests that they are active hunters of aquatic water fleas, ostracod, copepod and isopod crustaceans, which constitute 60.6% of the total food volume found in their stomachs. Only 10.2% of the stomach content by volume belonged to the type of unidentifiable gunk in fry. It is likely that during this stage, for the Pachón cave population and perhaps other cave populations, arthropods constitute most of their nourishment through active predation.

While all fry specimens were well-fed with their guts containing considerable amount of contents, it appears that those specimens collected during the dry season (March) were proportionally better-fed than those collected in the rainy season (August). The first had an average of 23 readily identified food items in their guts, while the second had an average of 7.7 readily identified items. It is also noteworthy that the gunk content found in the stomach increased from 6.2% to 18.2% in the rainy season. While intestine gunk may represent digested prey, stomach gunk is likely to be indicative of the fish eating guano, decomposed detritus, or mud. It may be that during the rainy season, fry had less live prey available for sustenance and relied on other nourishing items to supplement their consumption. An example supporting this idea is that in one fry, a pellet of stool gunk was largely comprised of insect scales, most likely from guano droppings originating from a bat that ate moths.

It may seem counterintuitive that there are more stomach contents in the fry during the dry season than the rainy season. One would expect that more food items become available during the wet season. Our experimental protocol was not designed to provide an explanation and we can only report observed results. Future studies will document ecological parameters as well as environmental conditions throughout a full year. Nonetheless, while the idea of seasonal flooding bringing items into the caves is an appealing one, one should remember that each cave is a unique case. Pachón cave does not have a stream flowing into it during the rainy season, nor does it experience flash flooding in ways similar to Rio Subterraneo, Tinaja, Sabinos or other caves. Pachón cave has a sump pool where more or less filtrated water trickles in. Water level of the pool increases or decreases, without the influx of large debris being flushed in flash floods. One of the many possibilities is that microscopic crustaceans that come from the epikarst are actually diluted during the rainy season. Only future longitudinal studies will be able to resolve this conundrum.

There is a plethora of literature on Astyanax cavefish discussing the adaptations that allow them to be very skilful at locating nourishment in an environment where food is often scarce (Espinasa et al. 2014; Rétaux and Elipot 2013; Soares and Niemiller 2013; Yoshizawa 2015). Our results confirm that they are indeed excellent hunters, but suggest that, at least in this cave and for this particular stage in their ontogeny, food is not that limiting.

There are very few published materials regarding what Astyanax cavefish actually eat in their natural environment. To our knowledge, this has been restricted to adult fish. Wilkens and Burns (1972) studied the gut content of 17 specimens from Río Subterraneo cave in the Micos area, whose total length varied from 3-10 cm. Their stomachs and intestines were filled with a brown substance, which was interpreted as guano from insectivorous bats that cover the floor in parts of this cave. Examination of this brown substance showed that it was mostly fragments of chitin originating from the exoskeleton of insects and considerable amounts of scales from wings of nocturnal Lepidoptera. Some large specimens of the Micos fish also contained partly digested fish bodies, suggesting cannibalism or carrion feeding on their conspecifics. In the five Pachón adults we examined, their gut contents were also primarily gunk. Identifiable structures were also pigmented chitin fragments originating from the exoskeleton of arthropods that inhabit the cave or as part of the guano of bats. Contrary to the post-larval fish where they averaged 17 ingested preys, the five adult specimens did not exhibit evidence for active hunting of crustaceans, and only showed the consumption of a single fly and a single beetle, leading to an average of 0.4 prey ingested per individual.

Some authors have suggested that Astyanax food consists almost completely of bat guano rather than live and mobile organisms (Kasumyan and Marusov 2015). It is unlikely that bat guano is the primary food item throughout their development and across the entire range of cave Astyanax. Firstly, the large interconnecting underground system that makes the habitat of cave Astyanax (Espinasa and Espinasa 2015) is composed of thousands of kilometres of underground passages that are beyond the reach of bats. While cave biologists studying the over 30 described localities of cave Astyanax often encounter bats, both human and bat advancement into the cave is stopped by sumps that fish can swim through. The largest proportion of the Astyanax populations undoubtedly inhabits the unreachable, larger sections of the underground system beyond these sumps. Fish in these sections must find nourishment from sources other than bat droppings.

A second reason to doubt that bat guano is their single source for nourishment is based on the presence of other non-smell detecting adaptations that are fine-tuned for locating live prey, including VAB. As mentioned before, many crustaceans in the water column produce 30-40 Hz water fluctuations while swimming (Lang 1980). Most Pachón fish have VAB, while most surface fish lack it. Even in the surface fish that have VAB, the ability to “tune-in” to certain vibration frequencies is absent and they have a broad response range from 5-50 Hz. By contrast, cavefish have a significant peak at 35 Hz brought by changes in the morphology and number of superficial neuromasts within the orbit of the degenerated eye (Yoshizawa 2015). QTL analyses have shown multiple genomic loci for VAB, superficial neuromast number at the eye orbit, and eye size. Importantly, the QTL for all three traits overlap each other in two regions of the Astyanax genome (Yoshizawa 2015).

We do not argue that guano is not a source of food. The five adult specimens examined from Pachón cave, anecdotal comments by colleagues, and our own personal unpublished observations of gut contents from other caves suggest that many El Abra populations have gut contents composed of gunk suggestive of eating guano or detritus from the mud. Astyanax, like many other cave adapted organisms, is probably a generalist and an opportunist. It is likely that its source of nourishment varies greatly not only between caves, but also throughout its ontogeny. Such is the case for surface Astyanax. A surface population studied by Wolff et al. (2009) showed that in fish of less than 5 cm standard length, 75.8% of their diet by volume was made of animal items. In 5.1 to 7.5 cm long fish, animal content decreased to 28.6%, and when longer than 7.6 cm, its animal derived diet was reduced to 20.7% to the increase of plant items.

Here, we also found that the food regime in Pachón cave Astyanax varies greatly between post-larval 1-2cm long individuals and ~4cm long adults. This may occur in other Astyanax cave populations as well. Young fish are highly dependent on their hunting skills and their food is significantly made of nourishing sources such as microscopic crustaceans. As they get older and larger, these microscopic animals become a less effective source of food. They then change their diet to more abundant but perhaps less nourishing sources like guano or mud detritus, as well as the opportunistic insect or carcass that may fall in or brought in during the rainy season. The idea that there is a change in the cavefish’s diet that reduces their dependence for hunting live prey is supported by the observation that in large, mature adult cavefish (> 6 cm long), superficial neuromasts showed reduced sensitivities compared to those in smaller, younger adults (< 4 cm long), corresponding to a significantly attenuated VAB in large Pachón cavefish (Yoshizawa 2015).

Fry had an average of 17 (+/- 14.5 StDev; Range=3-50) prey in their guts while adults had an average of 0.4 (+/- 0.5 StDev; Range=0-1) prey, which is significantly different (P=.001). In conclusion, our results suggest that at a young stage when the yolk has been depleted and young larvae must find food for themselves, Astyanax cavefish’s enhanced skills for prey capture become the primary means for obtaining nourishment. Close to 90% of their food items may derive from arthropods and at least 60.6% by volume are the likely product of active hunting of microscopic crustaceans. These enhanced hunting skills in fry are probably essential for the survival within the cave environment. These skills may be modulated by the enhancement of superficial neuromast activity. Other options may be possible, such as the enhancement of mechanosensors, chemical sensors, or performance of the brain, to name a few. Astyanax diet changes with age, probably as microscopic crustaceans are no longer adequate for larger specimens. Adult cavefish probably feed on a variety of stationary and moving items in cave pools that may progressively rely less on VAB. Stationary objects located at the bottom of cave pools, such as particles of detritus, washed-in cadavers, or bat guano, could be more efficiently detected using olfactory cues and an enlarged olfactory pit.

Abigail Descoteaux helped with the illustrations. We would like to thank Patricia Ornelas-Garcia who obtained a collective collection permit. Thanks to all group members who participated to the March 2016 field trip: M. Blin, D. Casane, L. Devos, J. Fumey, C. Hyacinthe, L. Legendre, S. Père, V. Simon. We thank the two anonymous reviewers for helping improve the manuscript. This study was supported by Marist College and its School of Science (to LE), an ANR grant [BLINDTEST] and a FRM grant [Equipe FRM] (to SR), and a collaborative exchange program [Ecos-Nord] to SR and Patricia Ornelas-Garcia.