Espinasa, Sloat, Parker & Robinson

A new cave population of catfish from Mexico. What’s on their menu? Frog legs

Luis Espinasa1, Solomon A. Sloat1, Katherine Parker1, and Jenna Robinson1

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

Key Words: Ictalurus balsanus, catfish, frog, troglophile, troglobiont, stomach contents, Papagayo River, Balsas River, Guerrero, México.

Catfishes (order Siluriformes) are a diverse group of ray-finned fishes that also have the second most cave-restricted fish species in the world behind Cypriniformes (Bockmann and Castro 2010; Soares and Niemiller 2013). The abundance of catfishes in subterranean habitats has been explained by “their morphological/biological ‘preadaptations’, viz. their predominantly nocturnal activity (and related chemo- orientation), cryptobiotic habits, and generalized carnivorous (or detritivorous) diet” (Bockmann and Castro 2010). Within Siluriformes, the family Ictaluridae has three genera (Prietella, Satan, and Trogloglanis) with at least four troglobitic species that lack externally visible eyes and are depigmented (Romero and Paulson 2001; Proudlove 2010). Multiple presumed troglophiles with a mosaic reduction of eyes and pigmentation have also been described (e.g., Bichuette and Trajano 2008; Reis et al. 2006; Secutti et al. 2011). We report here a new ictalurid catfish inhabiting a cave in southern Mexico, which, despite having eyes and pigmentation, can be classified as a troglophile or possibly even a troglobiont.

In the spring of 2001, members of the Sociedad Mexicana de Exploraciones Subterraneas (SMES) explored a virgin cave Sotano del Garbanzal #2 (Chickpea-Field Pit #2), located 7 km south of the town of Acahuizotla, near the city of Chilpancingo, in the state of Guerrero, Mexico (17o18’34” N, 99o25’55” W, 670 m above sea level). The total length of Sotano del Garbanzal is 471 m and its depth is 37.4 m. There are two pits in this cave: the 11-m entrance pit and a second 15-m pit near the end of the cave. A small stream runs throughout the length of the cave. After the second pit, the nature of the cave stream changes from fast-flowing with many small waterfalls to gently and slowly meandering through pools where it is evident that a hydrological base level has been reached. At the end of this gallery a sump is encountered, which has prevented further exploration. The nearest surface stream is Rio Acahuizotla, 1.35 km away. At 530 m above sea level, Rio Acahuizotla is about 100 m below the final sump. Rio Acahuizotla joins the Rio Azul, which itself is part of the Papagayo River drainage that empties near the city of Acapulco. The Papagayo River drainage contains no known catfish species

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(see below), so this may be the first confirmed report of a siluriform from this drainage. At the time of exploration, the cave stream’s flow was estimated to be about two liters per second. During the rainy season, this cave undoubtedly experiences major floods as the entrance doline has a catchment area of at least 7 km2.

Although bats are not abundant, the stream carries organic debris into the cave to support the subterranean ecosystem. Several accidental species and possible trogloxenes, such as beetles and other winged insects, are found throughout the cave. A possible troglobiotic harvestman (opilionid) was observed but remains to be studied. Some of the most interesting inhabitants of Sotano del Garbanzal are catfish. The objectives of this study are to diagnose and describe the morphology of the catfish population in Sotano del Garbanzal, examine eye histology, document diet, and highlight the ambiguities regarding how this cave population is related to surface catfish of the region.

Figure 1. Three catfish collected from Sotano del Garbanzal, Guerrero, Mexico. Specimens exhibited variability in pigmentation, with some being quite pale. Notice also that the fish in the background has proportionally longer fins than the fish in the foreground. Photograph by Luis Espinasa.

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Figure 2. Surface Ictalurus balsanus from Jalisco in the northern part of the Balsas basin (top) and a specimen from Sotano del Garbanzal (bottom). In addition to the apparent differences in pigmentation and proportionally longer fins, note that the buccal whiskers (arrow) are much longer in the cave specimen. Photo of surface specimen by John Lyons and cave specimen by Luis Espinasa.

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The catfish at Sotano del Garbanzal are found only at the base level of the cave after the second pit. They are particularly abundant in the sump where they can reach densities of at least an estimated one fish per square meter. It is likely that a large population exists beyond the sump. An exploration of the cave in 2005, four years after the initial collection showed that the population was still present and with similar densities. Specimens are rather large, with most observed individuals ca. 20 cm total length (Figure 1); although no attempt was made in the field to record size distribution of the population. Catfish from Sotano del Garbanzal apparently have normal sized eyes when compared to surface- dwelling catfishes, but exhibit great variability in body pigmentation (Figure 1). Some individuals are almost homogenously pigmented, but others are quite pale with just an assortment of blotched patches of pigmentation. No measurements of body structures were performed in the field, but it is evident that pectoral fin length is also variable based on three specimens collected. The most depigmented individual has proportionally longer pectoral fins when compared to the most pigmented individual (Figure 1). A mosaic nature of pigmentation and eye variation, as observed in the catfish population at Sotano del Garbanzal, has been reported previously from caves, in particular in several Brazilian siluriforms (Bichuette and Trajano 2008; Reis et al. 2006; Secutti et al. 2011). Their whiskers are very long, reaching the base of their pectoral fins in all three collected specimens. By comparison, whiskers of surface Ictalurus balsanus barely reach the base of the head (Figure 2).

Since the catfish at Sotano del Garbanzal have eyes but did not appear to respond readily to our lights while in the field, a second objective of this study was to describe the histology of their eyes. In other eyed and pigmented species of cavefish, histological examinations of eyes have found cases in which, despite exhibiting externally large eyes, individuals lack well-formed retinas (Espinasa et al. 2001; Espinasa and Jeffery 2003). In essence, cave-dwelling fish can possess eyes but be effectively blind. For this study, three fish were collected and fixed in 10% formaldehyde. Histological preparations of the eye of two fish were done. Eyes were extracted with a scalpel, embedded in paraffin, and cut into 5-µm sections using a microtome. Staining was done with hematoxilin-eosin. For comparison, histology of the eyes of Ictalurus balsanus from Jalisco in the northern part of the Balsas basin was also performed. Specimens had been stored at the University of Wisconsin Zoological Museum.

Eyes of Sotano del Garbanzal catfish possessed a cornea, lens, and anterior and posterior chambers typical of a teleost eye (Figure 3A, C, E), but the composition of the retina appeared to be variable among individuals. In one individual all the layers of the retina were present, but the neuronal layers appear particularly thin, especially when they are compared against their own pigmented epithelium (Figure 3D). In another cave specimen the retina appeared to be highly degenerated, with some layers apparently missing, including perhaps the photoreceptors, and with tissues of unclear identity (Figure 3F).

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Figure 3. Histology of the eye of surface Ictalurus balsanus (A–B) and two different individuals from Sotano del Garbanzal catfish (C–D and E–F). 1= Ganglion cell layer; 2=Inner plexiform layer; 3= Inner nuclear layer; 4= Outer plexiform layer; 5= Outer nuclear layer; 6= Photoreceptors; 7= Pigmented epithelium. Note that while the cornea, lens, and posterior chambers in the cavefish are similar to the surface fish, there is variability in the retina. Some cavefish had an apparent normal retina with all the typical layers (C–D), while in other fish some of the layers appear to be missing and degenerated (E–F). Photographs by Luis Espinasa.

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Our studies also attempted to address what catfishes of Sotano del Garbanzal are eating and what food resources sustain a putatively large and dense population. Population density of catfishes at Sotano del Garbanzal appears to be high and stable throughout multiple years based on our observations throughout a four-year period (2001–2005). To answer these questions, stomach contents were studied. As a preliminary assessment of diet, dissection of the digestive tract was performed on a single specimen out of the three specimens collected so as not to destroy the integrity of the few and valuable specimens available. Dissection was performed under a Motic K series stereomicroscope with the aid of a scalpel, dissection needles, and tweezers. Each section of the digestive tract was carefully opened and its contents were examined under high and low magnification to discern and extract both small and large identifiable food items. To photograph small food items, multiple photographs focused in different depth planes were shot under an optic microscope. Then the images were processed with the use of Zerene Stacker software to stack all of them into a single photo where all parts are in focus.

Stomach contents included body parts of arthropods, such as heads, segments of the thorax/abdomen, and legs (Figure 4A). Many of them appeared to belong to aquatic larvae of beetles and other insects. All of the arthropod sclerites were pigmented, suggesting that these food items are not from troglobiotic food sources in the cave and instead derive from surface arthropods that are flushed into the cave.

Nine bones up to 2 cm long were also found inside the digestive tract (Figure 4B). Soft tissue was already digested. They were from a right arm and shoulder girdle (one finger bone, a fused ulna and radius, humerus, and scapula), some sternal bones (two clavicles and two coracoids), and a left lower jaw. The bones were found aligned and topologically in proper order, suggesting that they belonged to a single organism from which the catfish had taken a bite and swallowed the limb and adjoining areas all together. To identify the type of organism these bones belonged to, comparisons with the skeletons found in the Marist College Biology Department collection and against photographs available online. An almost identical morphology was found when compared against a 25 cm long American Bullfrog (Rana catesbeiana) of the Marist collection (Figure 5). Support that these remains are indeed from an anuran include that the ulna and radius are fused into a single bone and that the lower jaw lacks teeth (Figure 5C), both diagnostic characteristics of anurans.

Since the availability of frogs may be limited in this cave, we doubt that frogs are the main food source of Sotano del Garbanzal catfish. Instead, we hypothesize that a frog accidentally fell into the cave, died, and while it was in the process of decomposing, our specimen happened to take a bite of it. It is our belief that these catfish are both generalist and opportunistic and that they must be eating all sort of organic matter that the cave stream abundantly carries into the lower passages. Arthropods and plants are likely to be the primary sources of food, but undoubtedly when a carcass of a bat, frog, or any available animal happens to be carried inside the cave, catfishes will opportunistically feed.

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Figure 4. Stomach contents of a catfish from Sotano del Garbanzal. The single specimen analyzed had ingested several arthropods, particularly what appeared to be beetle larvae (A). In its stomach, there were also nine bones from an arm, pectoral girdle, sternum, and mandible (B) of a vertebrate. Specimens in A are 0.5 mm long. For scale of bones, see Figure 5.

Apart from their apparent reduced reaction to light, variable degeneration of the retina, reduced pigmentation, and longer sensory organs, several other factors hint that these catfish may be troglophiles or cave-adapted and not just accidentals that were flushed into the cave. The first is that the population is somewhat isolated from any surface source populations. The entrance doline has no flowing stream and the nearest stream is Rio Acahuizotla, 1.35 km away and 100 m below the final sump. The linear distance combined with altitudinal difference poses a challenge for surface catfish (if they even exist in Acahuizotla River), to swim upstream through underground passages to reach Sotano del Garbanzal. A second reason for assuming they are a permanent population is the

2017 Speleobiology Notes 9: 1–10 7 Espinasa, Sloat, Parker & Robinson apparent stability of this population. As mentioned above, an exploration of the cave four years after the initial collection showed that the population was still present and with similar densities.

Figure 5. Skeleton of an American Bullfrog from the Marist College collection (A and B). C. Bones found inside the stomach of the catfish. Scale for B and C is the same. Notice that the bones from the stomach content share with the frog’s bones their general shape and size, as well as the diagnostic characteristics of frogs, which include a lower jaw lacking teeth and a fused ulna and radius.

The final objective of this study was to establish how catfish at Sotano del Garbanzal are related to surface catfish of the region. Based on photographs, Sotano del Garbanzal catfish have been identified as belonging to the Balsas Catfish, Ictalurus balsanus (Jordan & Snyder, 1989), by Hector Espinosa, a renowned ichthyologist and specialist of Mexican fishes. Nonetheless, he recognized that the cave specimens have some distinctive characters that may differentiate them from surface Balsas Catfish populations. There is a further perplexing factor—Ictalurus balsanus is reported as endemic to the Balsas River

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drainage in central Mexico (Rosas-Valdez 2007), which drains into the Pacific Ocean at Lazaro Cardenas. The Papagayo River drainage, to which Sotano del Garbanzal is located, although near the Balsas, is an independent drainage that discharges at a different locality in the Pacific Ocean, near Acapulco. The ichthyological collection at the National Autonomous University of Mexico (UNAM) has no records of Ictalurus having ever been collected from the Papagayo drainage (Espinosa pers. comm.). It remains to be determined if there is an undocumented surface Ictalurus species from the Rio Acahuizotla/Papagayo River drainage or if the Sotano del Garbanzal population belongs to a different population or subspecies from I. balsanus. This should be the subject of future molecular studies. Our specimens were preserved in formaldehyde and collected so many years ago that DNA has degraded. New tissue samples would need to be collected. A hindrance is that social and political unrest in the State of Guerrero may prevent collecting samples in the near future.

In conclusion, we report herein a new population of catfish that inhabits a cave in Guerrero, Mexico. These fish have large eyes, but, nonetheless, their apparent reduced reaction to light, variable degeneration of the retina, reduced pigmentation, enlarged sensory organs, and biogeography hint that they could be a stable underground population, possibly cave adapted, and not just accidentals. We also report on the feeding habits of one individual, which included arthropods and, most interestingly, a frog.

Acknowledgments

We would like to thank the members of the SMES, in particular, Ramon Espinasa, and the students of the Universidad Autonoma del Estado de Morelos who helped in the exploration of Garbanzal cave and the collection of specimens. Héctor Salvador Espinosa Pérez, curator of the Colección Nacional de Peces (National Fish Collection) at UNAM determined the species identity of the catfish. John Lyons, Adjunct Curator of Fishes at the University of Wisconsin Zoological Museum provided the photo and eyes of a surface Balsas Catfish. Histology of the eyes were conducted at the laboratory of Nicholas Rohner, at the Stowers Institute for Medical Research. LE received partial support to conduct this study from a VPAA Summer Research Grant from Marist College. SS, KP, and JR received a Marist College Student Grant.

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