Monitoring and Marking Techniques for the Comal Springs riffle beetle Heterelmis comalensis

I continue my self promotion:

Huston, D.C., J.R. Gibson, K.G. Ostrand, C.W. Norris and P.H. Diaz.  2015. Monitoring and marking techniques for the endangered Comal Springs riffle beetle, Heterelmis comalensis Bosse, Tuff, and Brown, 1988 (Coleoptera: Elmidae). The Coleopterists Bulletin, 69: 793-798.  PDF

This was the second paper that I worked on that involved the endangered Comal Springs riffle beetle Heterelmis comalensis.  I discussed the natural history of the beetle and its endangered species status, as well as our attempts to culture them in the laboratory in my previous post.

The goal of this particular project was to evaluate various methods for monitoring the population size of H. comalensis at Comal Springs in New Braunfels, Texas.  Two methods were tested: (1) the use of paint for a mark recapture pilot study and (2) using the "cotton cloth lure" technique for trapping.

As you are likely aware, mark-recapture methods are relatively straightforward.  You capture a group of animals, give them some sort of recognizable mark or tag, release these animals, and then recapture these animals at a later time.  The information obtained can help estimate population size and dispersal of the animals you are studying.  Well, in the end it turns out that marking tiny aquatic beetles isn't really that easy.

While there is a good amount of published literature available about marking terrestrial insects, very little work has been done in terms of marking aquatic invertebrates.  Most of the difficulties inherent in marking an aquatic invertebrate are obvious, I.E. water soluble paints won't work, paper tags will be destroyed, glue will dissolve, etc. etc.  This becomes even more complicated when you consider individuals which live in riffle environments, as these abrasive habitats can damage and obscure even water resistant tags (Freilich, 1989).  Whatever to do?  Well Wineriter and Walker (1984) did an evaluation of various marking methods for insects, and they recommended the use of water insoluble paints.  That seemed the best option, so oil-based paint pens were acquired for the job.

Now of course we couldn't just go about slapping paint willy-nilly on our endangered beetles.  I mean what if we poisoned a bunch of the little guys to death?  Killing a bunch of endangered species on accident means paperwork, so we needed a surrogate for the test.  Fortunately, a related species of riffle beetle, Heterelmis vulnerata, is relatively common and widespread in Texas and is around the same size, thus H. vulnerata seemed a good match for the surrogate.

In order to give these tiny (~2mm) aquatic beetles a mark, the following procedure was devised.  Beetles were placed in a water filled tray, with a nylon mesh placed on the bottom.  The beetles like to cling to things, so they always grab onto the nylon mesh.  Then a small drop of paint was liberated from the paint marker into a little blotter.  The mesh was then pulled from the tray and the elytra of beetles were carefully dried with a cotton swab.  Then, using an insect pin placed in a pin vice, a tiny dab of paint was placed on the beetle.  The beetle was then kept out of the water for a minute or so as the paint dried, before being returned to the water. Clearly this wasn't the easiest thing in the world.  You needed to have these beetles dry long enough for the paint to dry, but not so long as for the beetles to die.

Marking Heterelmis vulnerata with oil-based paint using an insect pin in a pin vice. Photo courtesy of J.R. Gibson

After some 20 days, none of the marked or unmarked H. vulnerata had died.  Thus it was thought that the procedure would be safe for H. comalensis in the field.  

So in the field, 100 beetles were collected and separated into 10 groups of 10 with each group being marked with a different color.  After one month only a single marked beetle was re-captured 1.7 M from its original release site.  The second month only a single beetle was re-captured (though a different individual) at its original site of recapture.  I'll cut through the long-winded part of the discussion and say that what this really told us was that the marking techniques would be difficult in the field, but they do have the potential for showing that dispersal is very low for individual H. comalensis.

The other part of this project was the temporal evaluation of the "cotton cloth lure" method.  So what is the cotton cloth lure (CCL) method?

Essentially this methodology was pioneered for the Comal Springs riffle beetle by my colleague and co-author Randy Gibson.  Randy once said that he developed the method based on some techniques used by cave biologists.  Apparently these cavers would throw old mop heads down into cave pools.  As the mop heads decomposed, biofilms would grow upon mops and it would attract cave invertebrates which could be captured when the mop heads were retrieved.

In the CCL method for riffle beetles, pieces of cotton/nylon sheet are cut into squares, folded up, and buried in the interstitial gravel habitats surrounding spring openings and upwellings.  Again, biofilms will grow on the cotton as it decomposes, which seems to attract grazers like riffle beetles.  This method also attracts multiple other species including the common riffle beetle Microcylloepus pusillus and ocassionally the endangered Pecks Cave amphipod Stygobromus pecki and the endangered Comal Springs dryopoid beetle Stygoparnus comalensis.

The goal here was to watch these lures over a long period of time (17 weeks) and see if the "capture" success varied.  Presumably, if part of the lure was decomposing, eventually there would only be inert fibers left, upon which the lure should no longer be useful.  Furthermore, perhaps there would be a peak time in which the lure was the most attractive to H. comalensis (meaning capture success was highest).

Again, I'll cut through the long winded discussion and just show the graphical results:

Three cotton cloth lures (A,B,C) buried 1 M apart from one another in Comal Springs for 17 weeks and the number of Heterelmis comalensis beetles found on each lure during each weekly sampling effort.

As you can see from this graph, it looks like capture success for H. comalensis is best between 7 and 10 weeks after the deployment of lures.  My presumption is this is when the biofilm fauna has really begun to flourish, and there is lots of good food available for the beetles.  Also, it looks like the capture success drops off rather rapidly after 10 weeks, presumable after all the organic matter is gone and only the inert nylon fibers remain.

In conclusion: This project was a pretty useful pilot study into the best methodologies for evaluating Comal Springs riffle beetle population sizes.  The lure method still remains the best technique, because it is minimally damaging to the habitat and beetles are able to be returned alive at the site of capture.

References:

Freilich, J. E. 1989. A method for tagging individual benthic macroinvertebrates. Journal of the North American Benthological Society 9: 351–354.

Wineriter, S. A., and T. J. Walker. 1984. Insect marking techniques: durability of materials. Entomological News 95: 117–123.

The Comal Springs riffle beetle Heterelmis Comalensis pupates underwater

Underwater Pupation by the Comal Springs Riffle Beetle, Heterelmis Comalensis Bosse, Tuff, and Brown, 1988 (Coleoptera: Elmidae), with an Update on Culture Techniques

Ah my second beetle paper.  This one was a product of my time working at the U.S. Fish and Wildlife Service (USFWS) San Marcos Aquatic Resources Center (SMARC) with my co-author on this publication J. Randy Gibson.  As always, here is the citation for the paper and a link to a PDF for your reading pleasure:

Huston, D.C., and J.R. Gibson. 2015. Underwater Pupation by the Comal Springs Riffle Beetle, Heterelmis Comalensis Bosse, Tuff, and Brown, 1988 (Coleoptera: Elmidae), with an Update on Culture Techniques." The Coleopterists Bulletin 69: 521-524. PDF

So who is the Comal Springs riffle beetle?  The little guys look like this and are around 2mm long. 

A Heterelmis comalensis adult.  Photo by J.R. Gibson, USFWS SMARC.

Heterelmis comalensis is only known from the headwaters of Comal Springs in New Braunfels, Texas and San Marcos springs in San Marcos, Texas (USFWS, 2007; Gibson et al. 2008).  These springs originate from the Edwards Aquifer, a massive aquatic limestone subterranean system.  The subterranean parts of the aquifer support a large number of endemic aquatic cave species (called "Stygobionts").  Furthermore, the above ground spring systems fed by these waters also contain a high amount of species endemism.  The Comal Springs riffle beetle, H. comalensis, is just one of these species.  This beetle is found in very close association with the upwellings and spring openings where water exits the subterranean system (Gibson et al. 2008).  The beetle is completely aquatic throughout its life.  This is why H. comalensis is in danger of extinction; reduced spring flow because of drought and excessive groundwater extraction due to human demand could lead to the complete destruction of the beetle's habitat.  Heterelmis comalensis is listed as endangered by the USFWS, and is listed as critically imperiled by NatureServe.

So what we did at the SMARC, was maintain little colonies of endangered species from around the local Central Texas springs, such as Texas wild rice (Zizania texana), fountain darters (Etheostoma fonticola), Devils river minnows (Dionda diaboli), and of course, Comal springs riffle beetles!

Unfortunately, there had been little success in setting up a self sustaining colony of H. comalensis.  We just knew so little about these beetles and what their culture requirements might be.  The true nature of things as simple as what they really eat still eludes us to this day.  It seems like they scrape biofilms that grow on submerged plant roots and decaying woody debris (Gibson et al. 2008), though this still has not been scientifically confirmed.  Therefore, its been quite difficult to figure out how to raise these guys through all life stages in captivity.  In fact, trying to get a colony of these beetles up and running has been going on for over 10 years (Fries, 2003)!  For a long time, it seemed that the majority of the beetle larvae would just never pupate into adults.  The real change in pupation success came when Randy and I designed a new style of culture container, which we call the EasySpring!

Easy Spring aquatic invertebrate culture container

How does it work?  Obviously this thing is built out of a big plastic tote container.  Spring water is pumped into the spray bar through the black tube.  The spray bar has a bunch of tiny holes drilled in it, thus spraying a little line of water along the side of the container at a pretty good pressure.  The water runs down the side into the rock pile.  For riffle beetles, we pile up rock, layering bits of leaf and wood in between the rocks.  The U shaped standpipe also has holes drilled along the length.  It maintains the water depth, while allowing the water to exit the container in a sort of linear fashion.  All this mimics the natural habitat of H. comalensis as best we could in the lab with the materials we had.  When we added H. comalensis larvae to the container we began finding adults within a few months.

This is when we began noticing something strange.  Every 1-3 months I would go through these containers and carefully remove all the rocks while collecting the beetle larvae and adults.  Then I'd sit down for the rest of the day and count.  And it takes forever to find all the little first and second instars, let me tell you.  Well I started to notice that sometimes when I would pick up a rock that was near the bottom of the container, pupae would pop out and float around on the surface of the water.  Why and how were the beetles forming pupae under the water?  This was especially interesting seeing as Elliott (2008) in a review of the ecology of riffle beetles, had made the rather definitive statement, "pupation always occurs above the water line".  Well it looked like H. comalensis had gone rogue and wasn't following the rules.

A Heterelmis comalensis pupa.  Photo by J.R. Gibson, USFWS SMARC.

So we reckoned that the beetles were pupating underwater.  How?  Well the pupae were super hydrophobic.  I'm still not sure if that was because they held an airspace inside the carapace, or if all the tiny hairs on the carapace were serving to trap air themselves, serving like the plastron of the adults.  Hmm, I never described the plastron did I?  Well I'll give a the quick and dirty description.  A plastron is a tight bundle of hydrophobic hairs found on the abdomen of riffle beetles.  The plastron traps air in a little film on the beetle, and the spiracles of the beetle open into the plastron.  This allows the beetle to breath underwater indefinitely. 

While pretty interesting, the hydrophobia presented a problem for us.  We couldn't see the pupae where they were found normally in our containers, they were just too small and were always between rocks.  If we collected the pupae from the container and put them in something else, they floated, so we couldn't prove that they could eclose (escape the pupal carapace) under the water line.  We needed something to get ourselves to believe these guys were pupating underwater.  The solution?  Well we tried a few things, but in the end we kept the pupae in a small re-purposed plastic air lock thing as an aquatic flow through chamber with one of those little steel mesh rings you put in your faucet in order to keep the pupae forced underwater.

Heterelmis comalensis pupa in a plastic flow through chamber kept forcefully underwater with a steel mesh ring.

Well, it worked.  We were able to watch the beetles pupate in these things, and always found live adults in these chambers when we put pupae in them.  Though I haven't demonstrated this yet, I think that the hydrophobic quality of the pupae comes from hairs on the outside of the carapace.  There is evidence for that in this photo.  Can you see the little air bubble attached to the pupae?  It seemed like every single pupae had these bubbles.  I think it quite possible that the adult beetles use these bubbles to establish their own plastron upon eclosion.  But only future research will tell!

REFERENCES

Elliott, J. M. 2008. The ecology of riffle beetles (Coleoptera:Elmidae). Freshwater Reviews 1: 189–203.

Gibson, J. R., S. J. Harden, and J. N. Fries. 2008. Survey and distribution of invertebrates from selected springs of the Edwards Aquifer in Comal and Hays Counties, Texas. Southwestern Naturalist 53: 74–84.

United States Fish and Wildlife Service (USFWS). 2007. Endangered and threatened wildlife and plants; Designation of critical habitat for the Peck’s cave amphipod,Comal Springs dryopid beetle, and Comal Springs riffle beetle; Final Rule. Federal Register 72: 39248–39283.

Epicauta polingi (Coleoptera: Meloidae) eats Mountain Laurel and Guajillo

Epicauta polingi (Coleoptera: Meloidae) Feeding on Mountain Laurel (Sophora
secundiflora) and Guajillo (Acacia berlandieri) in West Texas

My third research paper (using the word "paper" lightly here), and the first one where I fooled with beetles.  This was one of my favorite projects of all time.  Why?  Well it definitely wasn't the paradigm shifting new knowledge unveiled. It also wasn't the part where BioOne added a random accent mark to the second O in Coleoptera making us look silly.

I mean check this thing out, its really only two paragraphs long:

Huston, D.C., D. Araujo, J.R. Gibson, and J.T. Hutchinson. 2014. Epicauta polingi (Coleoptera: Meloidae) feeding on mountain laurel (Sophora secundiflora) and guajillo (Acacia berlandieri) in West Texas. Southwestern Entomologist, 39: 887-890. PDF

If you read the paper you will know that what we reported were two new host plants for a species of blister beetle called Epicauta polingi.

Two Epicauta polingi beetles (Coleoptera: Meloidae)

While that's not earth shattering to most, I am a firm believer in the value of scientific notes.  I think its incredibly important for us to record anything and everything we can about the natural world, no matter how small, as the natural history of most animals is completely unknown.  For most invertebrates, the only thing we know about them is their name and the morphology described by the scientist who named them.  While I beam with pride at the notion that now we know E. polingi eats two additional plants than previously known, thanks to the efforts of myself and my team of adventurers, the true fun in this project was the work that went into figuring out what species of beetle I had in the first place!

Epicauta polingi adult feeding on a Sophora secundiflora leaf.

Taxonomic work often ends up in obscure journals, or as large hard copy volumes of which there were only so many copies.  The vast majority of taxonomic work has not yet been digitized.  In this case I needed a volume called "The Taxonomy of North American Epicauta (Coleoptera: Moloidae), With a Revision of the Nominate Subgenus and Survey of Courtship Behavior" by the eminent Dr. John D. Pinto.  I reckoned this book had the taxonomic keys I needed in order to properly key my beetles to species.  Unfortunately, this book is out of print, fortunately I was currently both a student at Texas State University and an employee of the U.S. Fish and Wildlife Service, so I had great inter-library loan options.  In the end, I got a hard copy mailed to me and was able to use the keys.  If you enjoy things like examining little spines on beetle legs and/or measuring and calculating antennae length ratios, then beetle taxonomy is for you!

Measuring antennal segments in order to help determine what species of Epicauta I had.

Well to prevent general boredom, I'll cut this short and say that it took a combined effort of the team (and in the end the help of Dr. John Pinto himself) to get this guy identified properly.

While I'll maintain that I think the natural history information we reported in this manuscript is important, the aspects of this project I enjoyed most were getting the battered old copy of Pinto (1991) in the mail, spending some evenings in the lab examining legs and antennae, speaking with experts, and collaborating with my friends, colleagues and co-authors.

REFERENCES

Pinto, John D. The taxonomy of North American Epicauta (Coleoptera: Meloidae), with a revision of the nominate subgenus and a survey of courtship behavior. University of California Press, 1991.

Haplorchis pumilio in Texas

In my previous post I discussed how the introduction of the invasive snail species Melanoides tuberculata led to the subsequent introduction of the trematode parasite Centrocestus formosanus into Texas Spring systems.  Well in that post, I sort of glossed over the part where an additional species of Heterophyid trematode had been inadvertently introduced as well, this one called Haplorchis pumilio.  It seems that while Haplorchis pumilio had been known to be present in Texas at least as long as C. formosanus, this particular trematode really never received the attention it deserved.  In a project that resulted in my second publication, I found out why no one had paid attention to H. pumilio and why we probably should.  Before I get started, let me throw out a little self promotion and provide you a link to the paper: 

Huston, D. C., Worsham, M. D., Huffman, D. G., & Ostrand, K. G. (2014). Infection of fishes, including threatened and endangered species by the trematode parasite Haplorchis pumilio (Looss, 1896)(Trematoda: Heterophyidae). BioInvasions Records, 3(3), 189-194. PDF

So, remember in my last post where I provided a diagram of the life-cycle for C. formosanus in Texas?  The life-cycle for H. pumilio is pretty much the same, with two important differences.  The first of these differences is the 1st intermediate host snail.  While C. formosanus only seems to exploit Melanoides tuberculata in Texas, Haplorchis pumilio is known to utilize both Melanoides tuberculata and a related snail (also invasive) Tarebia granifera (Tolley-Jordan and Owen, 2008).

The second difference is the anatomical location of where the metacercariae are encysted in the 2nd intermediate host.  While Centrocestus formosanus metacercariae are found in the gills of the fish host, Haplorchis pumilio metacercariae are found in the cartilage where the fins attach to the body (fin insertions).

Haplorchis pumilio metacercariae encysted in the caudal peduncle of a shiner, Cyprinella venusta.

The above photograph is the tail of a minnow which I made a sort of cut out in order to see the metacercariae encysted there.  See those little brownish orange ovals?  Those are Haplorchis pumilio metacercariae!  By my eyes I count around 20 of them.  They look like this under higher magnification:

Haplorchis pumilio metacercaria.

The fin insertions are a pretty unusual place to find parasites, and is not included in most general parasitology examinations, so its not surprising that these little cysts went unnoticed in Texas spring fishes for around 15 years.  I probably wouldn't have even noticed them myself if I wasn't in the habit of exposing random animals to trematode cercariae.  Truth be told, in this case I knew that Haplorchis pumilio metacercariae HAD to be present Texas fishes, or I wouldn't keep finding infections in the snails I was collecting.  However, no one had ever reported the metacercariae in North America, and not knowing what the metacercariae really looked like, besides the general descriptions and figures provided by Sommerville (1982a; b), I decided to expose some common minnows (Cyprinella venusta) to Haplorchis pumilio cercariae from infected Melanoides tuberculata.  It turned out it was pretty easy to figure out where the cercariae were encysting as metacercariae:

A Cyprinella venusta mortality due to a rapid accumulation of Haplorchis pumilio metacercariae.

So I guess I have to admit I exposed the fish to a little bit more cercariae than was probably necessary, but I definitely didn't expect this kind of result.  The day after exposure, the fish developed these massive blood blisters on the caudal peduncle, and by the third day the blisters had burst.  All of the minnows perished, presumably due to hemorrhage.  Their noble sacrifice allowed me to find all the anatomical locations where the Haplorchis pumilio metacercariae might encyst: all the fin insertions (caudal, pectoral, pelvic, anal, dorsal) as well as the cartilaginous portions of the head.  I also learned from doing little tiny fillets, that the cercariae penetrate the epidermis of the fish all over the body, and actually travel under the skin to these preferred locations before encysting.  Quite peculiar.  

I think that its extremely important for me to point out that the infection pressure experienced by the fish in these exposure experiments would be many many orders of magnitude higher than what any fish would experience in the wild.  It is highly unlikely that an individual fish would acquire more than a few metacercariae per day in a natural setting.  The real concern is compound effects of continuous metacercarial acquisition over a fish's lifetime, coupled with additional stressors.

From there the story and the resulting project were very straightforward.  My colleagues and I acquired a small sample of various fishes across Texas spring systems (including archived specimens that I had previously examined for C. formosanus, waste not!), and we examined them using what we had learned from the exposure experiments.

The results of our survey showed that not only was the largemouth bass (Micropterus salmoides) hosting H. pumilio metacercariae, but so were several species of conservation concern:

The Devils River minnow (Dionda diaboli) - IUCN red list status: endangered

The fountain darter (Etheostoma fonticola) - IUCN red list status: endangered

The Rio Grande darter (Etheostoma grahami) - IUCN red list status: vulnerable

The Pecos gambusia (Gambusia nobilis) - IUCN red list status: endangered

Devils River, Texas, USA. 2014. I'm still amazed that the invasive snail Melanoides tuberculata managed to get introduced somewhere as remote as the Devils River.  Some of us speculate that contaminated scientific equipment was the cause!

Now none of these fish were hosting metacercariae in densities anywhere near what I had seen in the artificial infections with Cyprinella venusta, as was expected.  So its pretty hard to make the argument that these trematodes are having serious deleterious effects on these fish.  However, I had previously worked on a project examining fish gills for Centrocestus formosanus metacercariae (McDermott et al. 2014) in the same spring systems.  I found that about 50% of all the fish from the Haplorchis pumilio study were also infected with Centrocestus formosanus.  So it seems that many Texas spring fish species are hosting two species of exotic trematodes, increasing the parasite pressure on these already threatened populations.

Our paper was the first report of H. pumilio infecting fishes in North America north of Mexico.  Centrocestus formosanus has been reported in several other states of the USA, and I reckon that where C. formosanus is found, H. pumilio will surely follow (if it isn't already there).  Furthermore, as time goes on, additional species of exotic trematode will make their way to Texas and the rest of North America.  Melanoides tuberculata is a known host for over 100 species of parasite (Pinto and Melo, 2011).  I found a fourth species of exotic exploiting M. tuberculata shortly before I left the USA for Australia.  As more and more species of exotic parasite arrive, they will put more and more pressure on the native fauna.  As drought occurs more often and becomes more severe, and as anthropogenic effects intensify, stressors experienced by these threatened and endangered fishes will begin to compound.

I'm afraid I don't have a solution to the problems we identified.  There are currently no viable control measures for M. tuberculata in the spring systems in which it has invaded, and those measures that have been suggested (and even attempted) border on the absurd.  However, the overwhelming problem facing all Texas spring species is reduced spring flow.  One of the major causes of this is excessive extraction of groundwater.  We need a paradigm shift in our mindsets.  We need to realize the value of wild places, especially our spring fed oases in the arid southwest.  We need to use less, think more, and to realize that having a green lawn in the middle of summer in the southwest is ridiculous and irresponsible.  Be proud of your dead grass.

REFERENCES

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.

Sommerville C (1982a) The life history of Haplorchis pumilio(Looss, 1896) from cultured tilapias. Journal of Fish Diseases 5: 233–241.

Sommerville C (1982b) The pathology of Haplorchis pumilio (Looss, 1896) infections in cultured tilapias. Journal of Fish Diseases 5: 243–250.

Pinto, H.A., and A.L. Melo. 2011. A checklist of trematodes (Platyhelminthes) transmitted by Melanoides tuberculata (Mollusca: Thiaridae). Zootaxa 2799: 15-28.

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.