Coppola & Espinasa

Cave Astyanax: Hunters or scavengers? Evidence from gut contents

Johnmarco Coppola1 & Luis Espinasa1,2

1 School of Science, Marist College, 3399 North Rd, Poughkeepsie, New York 12601, USA 2 [email protected] (corresponding author)

Key Words: Stygobite, troglobite, Astyanax, predator, prey, troglomorphic, VAB, vibration attraction behavior, Caballo Moro Cave.

Mexican tetra of Astyanax have two morphs: a surface, eyed morph and an eyeless, cave troglomorph. Astyanax has been a particularly helpful model to study the role of evolutionary development and biological processes in caves (Jeffery 2012). To date, scientists have confirmed collections of troglomorphic Astyanax from a total of 32 caves (Elliott 2018; Espinasa et al. 2018). The surface-dwelling morph lives in nearby surface streams throughout most of México. Both morphs are inter-fertile and their entire genome has been sequenced, making the well-suited for experimental manipulations (Jeffery 2012; Elipot et al. 2014).

A key adaptation to survive in caves, an environment that has often been described as extreme and food-poor (Espinasa et al. 2017), is an enhanced ability to find food while in the darkness. Increased efficiency in food finding has been supported by five controlled observations or experiments in which cavefish directly outcompeted surface fish in their search for food (Hüppop 1987; Yoshizawa et al. 2010; Wilkens and Hüppop 1986; Espinasa et al. 2014; Hinaux et al. 2016). Features that could give this adaptive edge include 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 signaling pathways influencing brain development and organization (Yamamoto et al. 2004; Pottin et al. 2011), and a behaviorally more efficient posture with respect to the substrate when bottom feeding (Schemmel 1980).

Despite the plethora of studies, it remains unclear what the food sources are for adult Astyanax in their natural cave environment. Wilkens and Burns (1972) studied the gut content of 17 specimens, 3–10 cm total length from Río Subterraneo Cave in the Micos area. Their stomachs and intestines were filled with “gunk” with insect sclerites and moths’ scales, which was interpreted as guano from insectivorous bats. Some large specimens of the Micos fish also contained partly digested fish bodies, suggesting carrion feeding on their conspecifics. Espinasa et al. (2017) examined the gut content of five Pachón adults, 3.6–4.6 cm long. Their gut contents also primarily contained gunk and pigmented chitin fragments originating from the exoskeleton of arthropods as part of the guano of bats. They concluded that stomach contents of adults were consistent with a diet primarily

2019 Speleobiology Notes 10: 28–37 28 Coppola & Espinasa comprised of partially decomposed material, guano, or detritus from the mud. To our knowledge, no one has found the remains of other stygobionts or troglobites in the stomach contents of adult Astyanax that could hint they are active hunters of the diverse cave-adapted fauna with whom they cohabitate the caves of the El Abra region. Some authors have even suggested that adult Astyanax diet consists almost completely of bat guano rather than live and mobile organisms (Kasumyan and Marusov 2015).

A recent study has shown that contrary to adults, juveniles (fry) are efficient predators in their natural cave environment (Espinasa et al. 2017). Their primary food items are live, aquatic crustaceans. The guts of post-larval, pre-juvenile stage individuals (1–2 cm) contained an average of 17.9 microscopic crustaceans (water fleas-Cladocera, copepods, ostracods, and isopods) per individual. At least 60.6% by volume of their food items are the likely product of active hunting of microscopic crustaceans. Espinasa et al. (2017) concluded that food regimes change between post-larval and adult stages to become more dependent on partially decomposed material, guano, or detritus from the mud.

Many stygobites are generalists and opportunistic, adapted to eating all sort of organic matter carried into the underground passages. Espinasa et al. (2017) found that one troglomorphic adult Astyanax from Pachón cave, apart from gunk and guano, had eaten a fly. Another specimen ate a pigmented, eyed beetle, both insects of obvious surface origin. With adult Astyanax being the largest aquatic organism in the El Abra karstic area, is it possible they sometimes occupy the position of top predator in the underground food chain or are they simply scavengers eating guano, carcasses, detritus, and gunk?

A reason to doubt that adult cave Astyanax are nothing more than opportunistic scavengers that only rely on their senses of smell and taste to find food is the presence of other non-smell detecting adaptations that are fine-tuned for locating live prey. Many crustaceans in the water column produce 30–40 Hz water fluctuations while swimming (Lang 1980). Most Pachón fish have Vibration Attraction Behavior (VAB), while most surface fish lack it. 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 2016). QTL analyses have shown overlapping genomic loci for VAB, superficial neuromast number at the eye orbit, and eye size in two regions of the Astyanax genome (Yoshizawa 2016).

However, VAB could be a feature that is adaptive in juveniles, when they actively hunt for microscopic crustaceans, and vestigial in their adult life as they change their feedings habits towards a scavenger diet. Indeed, Yoshizawa et al. (2014) has shown that adult, mature cavefish (>6 cm long) have reduced sensitivities of orbit and infraorbital superficial neuromasts when compared with smaller, younger adults (<4 cm long). Correspondingly, VAB is significantly attenuated in adult, mature cavefish. However, as pointed out by Yoshisawa (2016), in the field, adult cavefish are still attracted to 35 Hz vibration stimulus.

The purpose of this study is to report on the gut contents of an eyeless Astyanax mexicanus (De Filippi, 1853) from a different cave, Caballo Moro (Figure 1), in Sierra de

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Guatemala, Tamaulipas, Mexico. Description of the cave can be found in Elliott (2018). This cave is inhabited by both the eyed and the eyeless morph (Espinasa and Borowsky 2000), but this study is restricted to a specimen that was fully eyeless (Figure 1). As part of a larger study that will be published elsewhere, a specimen of 4.8 cm standard length was collected on 1/9/17 and deposited while still in the cave in 100% ethanol. Collecting permit no. SGPA/DGVS/02438/16 from Secretaría del Medio Ambiente y Recursos Naturales, México, was issued to Patricia Ornelas García. Gut content analyses were performed as in Espinasa et al. (2017): The specimen fixed in the field was brought to the laboratory and dissected with the aid of a Motic-K series stereomicroscope, scalpel, scissors, and dissection needles. Stomach and intestines were dissected and analyzed separately to differentiate recently ingested food from the older, more degraded and digested food. All gut contents were examined in detail with 4× to 50× magnification on a Motic-K series stereomicroscope. To obtain images of the gut contents, multiple pictures focused in different depth planes were photographed under an optic microscope. The Zerene Stacker focus stacking software was then used to obtain single images where the entire subject is in focus.

Figure 1. Caballo Moro Cave has a 90-m long lake inhabited by eyed and eyeless Astyanax. Water comes out from a sump on the left of the photo and sinks again on the right. Eyeless fish come from areas beyond these sumps where they have to sustain themselves with food other that bat guano. Inset shows the eyeless fish used for this study.

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Stomach contents were determined as in Espinasa et al. (2017). The specimen’s stomach was 85 % full of gunk with pigmented insect sclerites and moth scales which can be interpreted as bat guano. The intestine was 5% full of this same guano. The most interesting gut contents were two stygobitic crustaceans. In the stomach there was a 0.3 mm long microcrustacean (Figure 2) and a macrocrustacean.

Figure 2. A microcrustacean tentatively identified as a cladoceran water flea or as a Nauplius larva was found in the stomach contents of an eyeless adult Astyanax. Notice that the specimen is well-preserved, with all structures still attached and even internal structures still visible. This suggests that the specimen was eaten while still alive and not ingested as a carcass partially decomposed.

Divided between stomach and intestine were fragments of what could be assembled into an almost fully depigmented, troglomorphic macrocrustacean. In the stomach was a 1.5 mm long head with the base of the antennae still attached (Figure 3A). In the intestine there were 1–2 mm long fragments of pereopods (Figure 3B-C), at least three segments of the abdomen spanning a length of about 3 mm (Figure 3F), plus many isolated fragments of pleopods and/or uropods (Figure 3D-E, G). Based on the dimensions of these structures, when alive, this crustacean would have been about 4–5 mm wide and 1–1.5 cm long (Figure 4). This is a considerably large food item for the 4.8 cm Astyanax that ate it.

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Figure 3. Remains of a stygobitic crustacean found in the gut contents: A. head; B. fragment of pereopods; C. dactylus; D-G. pleopods and/or uropods. Its stygobitic nature is inferred from the lack of eyes and pigment.

What is the identity of this crustacean? Despite the state of being partially digested and some missing structures, its identity can be revealed. Elliott (2018) suggested several species of cave adapted macrocrustaceans that could be preyed upon by Astyanax, including decapod shrimps of the genus Troglomexicanus, mysid shrimps of the genus Spelaeomysis, and isopods of the genera Speocirolana and Brackenridgia. The nature of the pleopods can be used to differentiate among groups and supports that this is an isopod. The aforementioned decapod and mysid shrimps have narrow pleopods divided distally into segments. Isopods of these genera have broad, paddle-like pleopods such as Figures 3D-F, 4 and 5.

While it may be difficult to establish from a partially digested food item if this was a live that was hunted and preyed upon or an already deceased organism that was ingested by the Astyanax fish, there are factors that make us suspect it was the former. The almost complete form of the isopod specimen as well as the relative state of preservation, especially noticeable in the well-preserved head, support that it was ingested while still alive and not a partially decayed carcass. This applies especially to the water flea. All of the body parts were present and perfectly articulated. While some uncertainty may remain regarding the isopod, this water flea was most likely hunted and preyed upon when alive.

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Figure 4. When all fragments are assembled, they appear to be the remains of a single isopod whose length was about 1–1.5 cm long. Perhaps too big to be swallowed whole by a 4.8-cm Astyanax. If the specimen was hunted while still alive, it may have been chewed into pieces, thus explaining the absence of portions of the thorax.

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Figure 5. Macrocrustaceans encountered in Caballo Moro Cave include pill bugs (Isopoda. left) and shrimps (right). The isopods have large blade-like pleopods in the abdomen, while in the shrimps they are small and thin. This suggests that the crustacean ingested by the Astyanax is an isopod. The isopod in this photo is 3-cm long and the shrimp is 1-cm long.

The relevance of this study is that despite many years of study and copious literature on the enhanced feeding attributes of this cave-adapted fish, this is the first report where an actual stygobitic species has been reported to be a food item for adult Astyanax. Furthermore, there is good evidence that this adult fish hunted and preyed upon live crustaceans. While this had been reported for juveniles and fry in the past (Espinasa et al. 2017), all previous reports on stomach contents for adults had failed to find convincing evidence that troglomorphic organisms were being ingested by adult Astyanax.

To date, the single most common food item reported for adult fish has been bat guano. Undoubtedly this represents a collecting bias. Humans typically can only enter caves until a sump is reached, but these sumps prevent the passage of both humans and bats. The largest portion of the hydrologic network of caves is beyond these sumps (Espinasa and Espinasa 2015). Likewise, the largest proportion of the troglomorphic populations of Astyanax inhabit these areas without bats where Astyanax must sustain itself through other means. Caballo Moro Cave has a 90-m long lake. Water comes out from a massive sump just to be lost at another sump at the end of the lake. The water current can be strong and eyeless fish have been observed swimming out of these sumps. Undoubtedly, they are feeding in the areas beyond these sumps. Up to now, it had been a conundrum as to why adult cave Astyanax presented VAB when their gut contents had items only

2019 Speleobiology Notes 10: 28–37 34 Coppola & Espinasa detected by smell and not by vibrations. We report here that adult Astyanax can also be active hunters of crustaceans, which may vibrate at such frequencies. While adults may still be generalist scavengers that eat guano, carcasses, detritus, and gunk, we provide evidence that adult Astyanax can eat other cave adapted species and be opportunistic hunters. Future studies may confirm their position as the top predator of this underground system, geographical variation and seasonal variation in the diet of the fish.

Acknowledgments

We would like to thank Patricia Ornelas-Garcia who obtained a collective collection permit. This study was supported by Marist College and its School of Science.

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