Monthly Archives: March 2017

Journal Club: Chemical Camouflage and George Harrison

Welcome to the Roughly Annual Solar Sea Slug Journal Club.

Today’s paper came from the Proceeding of the National Academy of Sciences a few years ago (Rasher et al., 2015, Proc Natl Acad Sci 112:12110).  I came across it again when I was updating records for this site, and, because it is germane to one of my pet theories, it seemed perfectly suited for an extended discussion. You’ll see how George Harrison fits into the story later.  Yes, this post will meander a bit, but the fact that you are reading the Solar Sea Slug Blog suggests you may have some time on your hands.

The paper is a very nice exploration of the interactions between herbivores and their food plants.  Up here on dry land, insects tend to specialize on particular food plants, and bugs and plants have evolved together in something of an arms race.  Insects use volatile chemicals produced by the plants to locate them, plants produce defensive chemicals to keep from being eaten and from being infected by insect-borne pathogens, insects develop resistance to the plant chemicals, and sometimes use them for their own defense, and so on.  The authors wondered if they could identify a similar web of interactions in the marine environment.

Elysia tuca in Box of Slugs 2.

The algae Halimeda incrassata would seem to be rather unpalatable.  It produces a collection of defensive chemicals, and is highly calcified, making it a crunchy, bad tasting mouthful.  Despite the defenses, Elysia tuca, a tiny and distinctively-patterned species, is commonly found on Halimeda.  The interaction between E. tuca and H. incrassata allowed the authors to ask how similar the relationship between a mollusc and a marine alga is to those of insects and terrestrial plants.

Halimeda incrassata, in Box of Slugs 2. Note the segmented appearance, which will be important in understanding Figures 3 and 4 of the paper. 3/12/17.

In order to compare the relationship between E. tuca and Halimeda to terrestrial plant-insect interactions, the study focused on five specific questions:
1)  Is E. tuca really a specialist? This is important for the development of an intimate plant-herbivore relationship.
2)  Does E. tuca find Halimeda based on chemical cues?
3)  What are the cues that E. tuca uses?
4)  What are the ecological consequences of E. tuca feeding on H. incrassata?
5)  Does Halimeda use counter-defenses to limit the damage inflicted by E. tuca.

With regard to E. tuca being a specialist, the answer was a pretty resounding “yes.”  They collected specimens of about 10 species of algae and marine plants at two sites, took them to the lab, and counted the numbers of E. tuca on each.  With a few exceptions (also in the genus Halimeda), E. tuca were only found on H. incrassata.  Further, when given the choice between many different algae species in the lab (Fig 1A, below), or three different species of Halimeda in the lab (Fig 1B), or in the field (Fig 1C) the slugs greatly preferred H. incrassata.  To test whether the slugs were following chemical cues, the experimenters soaked cotton balls in water that had held H. incrassata (Fig 1D), and found that E. tuca much preferred these to cotton balls soaked in plain seawater.  With regard to the questions posed by the paper, the results indicate that 1) E. tuca is a specialist, and 2) they find their host based on chemical cues.

Fig. 1. Elysia host preference. Number of trials in which an Elysia colonized one of 14 common seaweeds and seagrasses (n = 20) (A), three co-occurring seaweeds in the genus Halimeda (n = 20) (B and C), or a cotton ball laced with H. incrassata-conditioned seawater vs. seawater only (n = 40) (D), when offered in a still water arena (A, B, and D) or in the field (C). Choice was assessed after 2 h (A–C) or within a 5-min period (D). Results were analyzed by a Cochran’s Q (A–C) or Fisher’s exact (D) test. In A–C, different letters above bars indicate significant differences among seaweeds in terms of Elysia colonization frequency, as determined by Wilcoxon sign tests (corrected for multiple comparisons). AL, A. longicaulis; CC, Caulerpa cupressoides; CP, Caulerpa prolifera; CS, Caulerpa sertularioides; DC, Dictyosphaeria cavernosa; HI, H. incrassata; HM, H. monile; HO, H. opuntia; PC, Penicillus capitatus; PD, Penicillus dumetosus; RP, Rhipocephalus phoenix; SF, S. filiforme; TT, T. testudinum; US, Udotea sp

The next question regarded the identity of the chemical attractants from H. incrassata (which will be henceforth referred to as “Halimeda”).  Compounds were extracted from Halimeda with methanol, and the individual components of the extracts were separated as described in the supplementary methods.  Each fraction was tested for attractiveness to slugs using the cotton ball colonization assay described in Figure 1, above.  The first compound they described, 4-hydroxybenzoic acid (4-HBA; Figure 2A, left) is found in both “vegetative” Halimeda in the normal growing stage, and in “reproductive” Halimeda that are undergoing spawning events.   When 4-HBA was placed on cloth patches next to Halimeda, the plants were colonized by significantly more Elysia than to controls with cloth soaked in the solvent but no 4-HBA (Figure 2B, left panel).

The reproductive stage of Halimeda is significantly more attractive than the vegetative stage, in part because the reproductive cells (gametes) are a rich source of nutrients.  When patches soaked in extract from reproductive plants were placed next to Halimeda plants, they attracted more than twice as many slugs as those from vegetative plants (Figure 2B, right).  This led the authors to identify halimedatetraacetate (HTA; Figure 2A, right) a chemical compound enriched in reproductive Halimeda.  It was known that HTA deters feeding on Halimeda by other species, and that E. tuca sequesters HTA in its tissues.  The authors went on to show that an extract from E. tuca that contained HTA deterred feeding by predatory wrasse.

This brings us to question #4, what are the ecological consequences of E. tuca grazing on Halimeda?  Surprisingly, the effects of such tiny slugs are significant.  The fact that the slugs feed on reproductive structures (which have the highest HTA content) is expected to substantially reduce the plants’ fecundity.  Further, when the authors manipulated the numbers of Elysia on plants in the field, those with more slugs showed less growth (Figure 3A) and more branch loss (Figure 3B).  Placing E. tuca in enclosures on branches (Figure 3D) also caused more branch loss compared with enclosures with no slugs.  So E. tuca can cause significant damage to Halimeda.  Because H. incrassata aids the development of seagrass beds, and generates the majority of carbonate sediments (a.k.a., nice white sand) in those areas, the authors suggest that grazing by E. tuca can have ecosystem-wide consequences.

How can a small slug that sucks sap cause such dramatic loss of plant tissue?  One hypothesis is that the plant self-amputates segments that have been fed upon by Elysia.  The model Rasher et al. propose is that the plants are trying to avoid the introduction of pathogens by the slugs by sacrificing segments.  After culturing fungi from the slugs’ radullae, which they use to pierce the plants’ tissues, they tested one fungal species they referred to as Et-2.  Halimeda innoculated with the fungus dropped segments above the injection site (Figure 4A).  Injection of a fungus that is a pathogen of other species did not have the same effect (Figure 4B).  The data are therefore consistent with the hypothesis that loss of segments is a defensive strategy in response to feeding by E. tuca, suggesting that the answer to question #5 is also yes.

The authors conclude that the answer to all of the questions they posed is “yes,” and that marine plant herbivore interaction described above strongly resembles those in terrestrial ecosystems, despite more than 400 million years of separation between the participating species.

At some point, this paper got me thinking of a potential alternative function for kleptoplasty.  Shall we meander our way there?

By the end of last summer, I was finding most of the prevailing theories regarding kleptoplasty to be rather unsatisfying.  While not every aspect of biology must have a function, kleptoplasty has costs that must be offset.  It takes energy to segregate and store the chloroplasts, and they must be protected from the immune system.  Plus, the animals that are active in the sunlight are exposed to predation and damage from UV light.  Despite these costs, Elysia is a very successful genus, with species found worldwide in the shallows of tropical and temperate seas.  Therefore kelptoplasty must provide a significant benefit.

So, what good is kleptoplasty?  If you buy the arguments presented by deVries et al, photosynthesis by kleptoplasts do not supply a significant portion of the animals’ energy needs.  Is the energy produced by photosynthesis used to make starch or fat for use during lean times?  Maybe.  Could the kleptoplasts be a “living larder,” being digested when food is scarce?  The animals certainly become pale when they are starved, suggesting the kleptoplasts are being broken down, but why not just digest them at the time they are eaten and turn them into fat like the rest of us do?

One idea is that the kleptoplasts are merely used as camouflage.  In the case of E. diomedea, which spends a lot of its time hidden in its food plant, this seems sensible.  Not so much for E. crispata, which is easily visible against hard bottom reefs, which are generally not very green.  Further, it seems like there are other, less complicated ways of making or storing green pigment to match one’s surroundings.  However, let’s hold that thought for a minute.

Aside from making carbohydrate from sunlight and being green, chloroplasts produce important precursors for many biochemicals (e.g., Gould et al., 2008, Ann. Rev. Plant Biol. 59:491).  These could be used by the slugs for the synthesis of fats or essential aromatic amino acids for their own nutrition, or to be used for their prodigious production of eggs.  Given that there is absolutely no data regarding the role of photosynthesis in egg production by Elysia, this remains an attractive hypothesis.

However, an insight I thought was particularly brilliant was that chloroplasts synthesize isopentyl diphosphate (IPP), a precursor to a wide range of things, such as chlorophylls and terpenes. Some of these compounds are expected to be smelly, and, in principle, make the slugs smell like their food.  Some of the chemicals may also taste bad, rendering the soft, slow animals less palatable.

Predators of Elysia are expected to include nudibranchs, which are largely blind and find their food by smell, or fish, many of which find their prey by sight.  If the chloroplasts were pumping out chemicals that gave the slugs a smell of their food, it would make it much more difficult to find them by scent.  One bonus is that the green color of the kleptoplasts will also make it more difficult for visual predators, such as fish, to find the slugs.  On top of that, any noxious taste would protect the slugs from predators, regardless of their hunting methods.  Overall, this model seemed to have fewer caveats than any of the others.

I thought I had come up with this idea on my own.  Then I rediscovered the above paper by Rasher et al. while I was updating a saved search in the Scopus database.  PNAS is my Wednesday lunchtime reading, and I am sure that I was excited to come across a paper about Elysia, so I am certain that I read it when it came out.  I am saddened by the fact that I forgot that I had read the paper, and assume that the paper got me thinking about kleptoplasty and chemical camouflage.

Have you figured out the connection to George Harrison yet?  You have to be getting on in years or love music trivia to remember, but he produced a popular song “My Sweet Lord,” during his post-Beatles solo career.  He ran into some legal trouble when the Chiffons’ record label sued him for appropriating the melody from their highly popular “He’s So Fine.”  If you go online and listen to both of them, it won’t take you long to think “dang, he stole their melody.”  Harrison admitted that he was very familiar with the melody, and the judge ruled that he had committed “subconscious plagiarism.”  In the same way, I had no memory of even having read the Rasher paper when I was formulating ideas and researching the biosynthetic capabilities of chloroplasts.  I just thought I was being terribly clever.  Nonetheless, it is highly likely that the paper was somewhere in the recesses of my mind during the process.  Are there any truly new ideas?

But, more importantly, how does one test this?  The first step is to make some predictions, and here are a few possibilities:

  1. Comparison of compounds produced by food plants, normal Elysia, and Elysia in which photosynthesis is blocked will show compounds produced by plants and normal slugs that are absent or significantly less abundant when photosynthesis is blocked.  All we need to do is make some extracts and find a potential collaborator who is willing to perform some gas chromatography-mass spectroscopy on a small budget.
  2. Elysia in which photosynthesis has been blocked will be easier to detect or more attractive to predators.  Placing a predatory mollusc or fish in a Y-maze with a choice between an arm containing a control and one containing  a photosynthesis-blocked Elysia might do the trick.  There are plenty of hungry wrasses that we could use for the fish test, either in the lab or in Bahia.  As far as predatory slugs, Navanax or Roboastra are good candidate predators for E. diomedea in Baja California.

There are likely to be more, better experiments, but the above provide a start.