San Marcos Salamander immune to the invasive trematode Centrocestus formosanus

Artificial exposure of adult San Marcos salamanders and larval leopard frogs to the cercariae of Centrocestus formosanus

That is the title of my first ever published paper.  Not the most beautiful title, but it gets the point across.  Here's the citation and a link to the PDF:

Huston, D.C., V. Cantu and D.G. Huffman. 2014.  Artificial exposure of adult San Marcos salamanders and larval leopard frogs to the cercariae of Centrocestus formosanus. The Journal of Parasitology, 100: 239-241. PDF

Centrocestus formosanus is a species of digenetic trematode of the family Heterophyidae.  Trematodes (AKA flukes) are parasitic worms with complex multi-host life-cycles. Sexually mature adult trematodes are found in a vertebrate host, known as the “definitive host” in life-cycle terminology. However, the complex life-cycles of trematodes almost always include a molluscan (in rare cases a polychaete) 1st intermediate host, and may include 2nd and 3rd intermediate hosts as well. Mobile trematode larvae, known as cercariae, emerge from the molluscan host and find their way to the definitive host through seemingly limitless evolutionary strategies and through the exploitation of multiple trophic levels and feeding guilds.

In order to discuss my little paper, I need to provide some extensive background on the historical and biological significance of C. formosanus in North America.

Allow me to provide a diagram of the life-cycle of C. formosanus as it generally occurs in Texas:

Generalized life-cycle of Centrocestus formosanus as it occurs in Texas

Although I'm sure you are already finding this incredibly interesting, here comes the real hook: Centrocestus formosanus is not a native species to Texas!  Thus its arrival to North America represents a biological invasion.

Described by Nishigori way back in 1924 the very name C. formosanus clues you into where the parasite is originally from.  The island of Taiwan was formerly referred to as Formosa.  So how did C. formosanus make it all the way to North America?  I'll refer you to the original narrative by Scholz and Salgado-Maldanado (2000)  who discuss the introduction and spread of the parasite throughout Mexico.

Scholz and Salgado-Maldanado (2000) review evidence that C. formosanus was first introduced to Mexico through the import of a now widespread invasive species of snail Melanoides tuberculata.  These snails were introduced to Mexico as early as 1979, likely to culture as food for some start-up black carp fish farms.  Infected snails must have made it all the way from Asia to Mexico.  These invasive snails have now spread widely across South America.  Once in Mexico, cercariae emerged from the snails, infected small local fish, and were thus transmitted to local fish eating birds, striated herons.  The infected herons were able to distributed eggs of C. formosanus widely, thus increasing the range of the parasite.

Next, how did C. formosanus arrive in Texas?  Well Texas is home to many spring systems, many of which are perennial flowing from deep limestone aquifers.  These springs also have the benefit of being highly stable in terms of water quality and temperature, ideal habitats for tropical snails like M. tuberculata.  So we all know that stuff like regulations and import/export laws weren't as big of a deal back in the day as they are now, and that's pretty much how M. tuberculata made it into Texas.  Let me throw in a bit of abbreviated prose from my master's thesis (because I'm too lazy to write new stuff):

Melanoides tuberculata is thought to have been introduced to the USA through the aquarium trade sometime prior to 1950 (Murray, 1971). Populations of the snail were first reported in Texas in 1964, when the snails were found in the San Antonio River, Bexar County (Murray, 1964). The snail was later reported in Landa Lake, New Braunfels, Texas (Murray and Woopschall, 1965). Since that time, M. tuberculata has been reported in multiple spring-fed systems throughout Texas (Karatayev et al., 2009) and the USA (Benson and Neilson, 2013).

The introduction and establishment of this snail species first became a serious concern when a species of avian eyefluke was found parasitizing waterfowl in the San Antonio zoo, subsequently identified as Philophthalmus gralli (Murray and Haines, 1969; Nollen and Murray, 1978). Later, two additional species of trematodes were discovered in Texas, the first being Centrocestus formosanus, a heterophyid trematode which parasitizes the gills of fish (Mitchell et al., 2000; 2005). The second trematode species identified was Haplorchis pumilio (also a heterophyid), whose larval stages were found parasitizing M. tuberculata and an additional invasive snail species Tarebia granifera (Tolley-Jordan and Owen, 2008).

Clearly this snail and its associated parasites could be a problem.  A paper by Mitchell et al. (2000) was the one that really raised the alarm.  They found that Centrocestus formosanus was not only infecting, but causing serious pathological reactions, in an endangered species of fish endemic to the San Marcos and Comal spring systems called the fountain darter (Etheostoma fonticola).  Around the same time, C. formosanus was found infecting another threatened species of fish in West Texas, the Devils River Minnow (McDermott et al. 2014).  So what was the problem?  Well check out this picture of uninfected fish gills compared with infected fish gills:

Gill arches from Devils River minnows (Dionda diaboli) one uninfected (left) and one infected with multiple Centrocestus formosanus metacercariae (right)

Clearly there isn't a lot of real estate left over for breathing. This was of concern as these already threatened populations of endemic fish were having to deal with a brand new parasite in which they were not adapted to resist.

I could go on and on about this, and I did already during my time in Texas, so I don't really want to copy/paste my entire thesis into this blog post.  Plus I haven't even gotten to talking about my first research paper, which this post is supposed to be about.  Well its going to be a bit anti-climatic, but here we go.

I reckoned that we had a gill parasite that was threatening the long term stability of fountain darter and devils river minnow populations in Texas spring systems.  Well, little fish aren't the only endangered species from our springs, we have a few others, including some very charismatic species, obligate aquatic salamanders.  The one I worked on was the San Marcos salamander (Eurycea nana).

San Marcos salamander (Eurycea nana). Photo credit U.S. Fish and Wildlife Service (San Marcos Aquatic Resources Center, San Marcos, Texas).

While I was working at the San Marcos Aquatic Resources Center (USFWS), we maintained a breeding colony of these salamanders.  Eurycea nana is a small lungless salamander (plethodontidae) with external gills.  These salamanders are obligate aquatics, spending their entire lives in the water.  Eurycea nana is found only in the headwaters and the upper reaches of the San Marcos River, Texas and is listed as threatened by the U.S. Fish and Wildlife Service.  One day I thought to myself, "hey I'm working with invasive gill parasites of endangered fishes. Those threatened salamanders have gills too. I wonder if these salamanders are at risk of infection from C. formosanus".   So I went out and collected a bunch of Melanoides tuberculata which were infected with Centrocestus formosanus and exposed a little group of these salamanders to the parasite cercariae in order to determine if the salamanders could become infected or not.  The results were downright shocking, quickly shattering my dreams of fame and fortune.

The salamanders did not become infected.  I tried again with a higher concentration of cercariae per liter of water.  No result, the salamanders were just fine.  I tried again with extra water circulation.  No infections.  These little guys were just downright immune to the parasites.

How could this be?  As I fell into madness, a last ditch effort left me exposing the only common local aquatic amphibian, leopard frog tadpoles, to C. formosanus cercariae.  Miraculously the tadpoles became infected.  So why did the salamanders not become infected?  Well in my paper I several possibilities, from "Who knows?" to my favorite explanation: active vs. passive respiration.  You see, the San Marcos salamander breathes passively through its gills and skin, absorbing oxygen directly from the water.  Fish and tadpoles breath actively, but pulling water past their gills.  I found this interesting paper by Paller and Uga (2008) where they found that a closely related species of trematode, Centrocestus armatus, requires the mechanical stimulation of being pulled into the gills by active respiration in order to stimulate attachment behavior in the cercariae.  So I speculated that it may be the passive respiration of the salamanders does not stimulate the C. formosanus cercariae in a way that initiated attachment and encystment behavior.  At the very least, we now know that the San Marcos salamander is not under any threat from the invasive trematode Centrocestus formosanus.

REFERENCES

Benson, A.J. and M.E. Neilson. 2013. USGS - NAS - Nonindigenous Aquatic Species: Melanoides tuberculatus. http://nas.er.usgs.gov/queries/FactSheet.aspx?SpeciesID=1037 Revision Date: 2/28/2013.

Karatayev, A.Y., L.E. Burlakova, V.A. Karatayev and D.K. Padilla. 2009. Introduction, distribution, spread, and impacts of exotic freshwater gastropods in Texas. Hydrobiologica 619: 181-194.

McDermott, K.S., T.L. Arsuffi, T.M. Brandt, D.C. Huston and K.G. Ostrand. 2014. Exotic digenetic trematode (Centrocestus formosanus) distribution and occurrence, its exotic snail intermediate host (Melanoides tuberculatus), and fish infection rates in West Texas Springs Systems. The Southwestern Naturalist, 59: 212-220.

Mitchell, A.J., M.J. Salmon, D.G. Huffman, A.E. Goodwin and T.M. Brandt. 2000. Prevalence and pathogenicity of a heterophyid trematode infecting the gills of an endangered fish, the Fountain Darter, in two Central Texas spring-fed rivers. Journal of Aquatic Animal Health .12: 283-289.

Mitchell, A.J., R.M. Overstreet, A.E. Goodwin and T.M. Brandt. 2005. Spread of an exotic fish-gill trematode: A far-reaching and complex problem. Fisheries 30.8: 11-16.

Murray, H.D. 1964. Tarebia granifera and Melanoides tuberculata in Texas. Annual Report to the American Malacological Union 53: 15-16.

Murray, H.D. 1971. The introduction and spread of thiarids in the United States. The Biologist 53: 133-135.

Murray, H.D. and D. Haines. 1969. Philophthalmus sp.(Trematoda) in Tarebia granifera and Melanoides tuberculatus in South Texas. American Malacological Union Annual Reports 1969: 44-45.

Murray, H.D. and L.J. Woopschall. 1965. Ecology of Melanoides tuberculata (Müller) and Tarebia granifera (Lamarck) in South Texas. American Malacological Union 32: 25-26.

Nishigori, M. 1924. On a new species of fluke, Stamnosoma formosanum, and its life history. Journal of the Medical Association of Formosa 234: 181-238.

Nollen, P.M. and H.D. Murray. 1978. Philophthalmus gralli: Identification, growth characteristics, and treatment of an oriental eyefluke of birds introduced into the continental United States. The Journal of Parasitology 64.1: 178-180.

Scholz, Tomáš, and Guillermo Salgado-Maldonado. "The introduction and dispersal of Centrocestus formosanus (Nishigori, 1924)(Digenea: Heterophyidae) in Mexico: a review." The American Midland Naturalist 143.1 (2000): 185-200.

Tolley-Jordan, L.R. and J.M. Owen. 2008. Habitat influences snail community structure and trematode infection levels in a spring-fed river, Texas, USA. Hydrobiologia 600: 29-40.