Microbial diet of endangered tree snails across phyllospheres

Snails were once numerous across the Hawaiian archipelago and filled numerous roles across  multiple habitats. However, mass extinctions have occurred due to predator introductions and habitat loss, and therefore human intervention is probably the only way that many of these species will avoid this trend of decline.

 

We used high-throughput Miseq DNA sequencing to determine variance in the diet of a federally endangered tree snail, Achatinella mustelina, which is endemic to the island of Oahu. These snails dwell on trees and graze microbes that grow on  leaf surfaces - technically referred to as the "phyllosphere". The microbiology of the diet of these animals could be crucial to understanding how they can be successfully relocated into predator-proof enclosures or ex situ culture facilities.

 

This work was done in the botany department of the University of Hawai’i at Manoa for Professor Anthony Amend. The Amend lab is devoted to the study of fungal ecology in a variety of terrestrial and aquatic contexts.

Diet across the range of Achatinella mustelina, Achatinella sowerbyana and Achatinella lila.

Beta diversity plots of microbes in the feces of snails and from leaf surfaces. Top left shows fungi from Waikanai ranges, top right shows bacteria for the same snails. The bottom image is of snails from the Ko'olau ranges.

A fundamental question about a species' feeding behavior is “how does its diet vary across its geographic range”? This is a crucial thing to understand if a threatened species is to be managed by relocation into predator free enclosures or artificial habitats. When conservationists began to rescue snails by translocating them to artificial ex situ facilities they did some of this fundamental ecological research: they isolated strains of fungi from the snails’ host plant and conducted trials into whether snails would feed and grow on particular strains. Advances in DNA sequencing technology over the last few years mean that we no longer need to use a time consuming approach of testing if a fungus is snail-food by isolating it from the snails' habitat and then conducting feeding  trials (although there are some benefits to this approach). We can instead sequence eDNA directly from the snails feces to discover what  was  in their gut, and also sequence eDNA from the snails habitat to see what food was available to them.

 

We looked at the diet of four populations of Achatinella mustelina on the Waianae mountain range (from within and outside of predator proof enclosures). Dr Melissa Price conducted a separate study where she collected feces from the snails Achatinella sowerbyana and Achatinella lila and leaf microbiomes from the windward slopes of the Ko’olau mountain range. Finding remnant populations of these snails is extremely difficult and our work relied heavily on the efforts and expertise of conservationists from the OANRP, PCSU and US Fish and Wildlife Service.

Comparisons were made between the diversity of fungal species on leaves (what the snails might eat) and the diversity of fungi in the snails feces (what they have actually eaten). Similar fungi occurred in both leaves and feces, but their abundances varied. In the Ko’olau Ranges the diversity of fungi on leaves is considerably more divergent than in their feces, suggesting that A. sowerbyana and A. lila could be selectively consuming or digesting fungi. Melissa also found that snails in the Ko’olau mountain range  tended to graze fungi off leaves of a particular tree, the ʻōhiʻa lehua (Metrosideros spp.).

Clockwise from left: Tree snails occur on the branches of 'ōhiʻa (Metrosideros) trees more often than by chance. A bee visits an  'ōhiʻa flower. An Achatinella mustelina tree snail. A light microscope image of an 'ōhiʻa leaf that has been cleared of pigment by soaking in sodium hydroxide. The same leaf section, but viewed under fluorescence after staining with calcofluor white to visualise the hyphae of epiphytic (and endophytic) fungi that pierce through the leaf stomata.

O'Rorke, R., Cobian, G.M., Holland, B.S., Price, M.R., Costello, V., Amend, A.S. (2015) Dining local: the microbial diet of a snail that grazes microbial communities is geographically structured. Environmental Microbiology 17, 1753-1764 PDF Journal Webpage

Price, M.R., O’Rorke, R., Hadfield, M.G., Amend, A.S. (2017) Diet Selection at Three Spatial Scales: Implications for Conservation of an Endangered Hawaiian Tree Snail. Biotropica. 49, 130–136. PDF Journal Webpage

Improving Snail Culture

The Cladosporium used to supplement the diet of captive snails does occur in the wild. However, it does dominate the captive diet.

Set up of the feeding choice experiment used to determine if snails have food preferences.

The Achatinella tree snails have been reduced from approximately 41 species to just 10 species. There is only one lonely individual remaining of the species Achatinella apexfulva and there are less than ten known individuals of A. fulgens in the wild. To minimize the risk of extinction of surviving species an ex situ breeding facility, the Hawaiian Tree Snail Conservation Laboratory, has maintained subpopulations of the snails since the late nineteen-eighties. However, despite the absence of predators these ex situ populations have not flourished. Because wild stocks of these unique animals are quickly declining, managers are anxious to improve lab conservation strategies. We used non-invasive methods and surrogate species to explore how the ex situ diet of these critically endangered species differs from their wild diet and could be enhanced.

 

Staff at the ex situ culture facility replicate the wild diet of snails by making frequent expeditions into the forest to collect bags of native foliage from which the captive snails can graze epiphytic fungi. The diet of captive snails is also supplemented with a monoculture of the fungus Cladosporium, which is provided to snails on disks of nutrient agar. We used DNA sequencing of feces to determine if the Cladosporium fungus that is used to supplement the diet of captive snails is a large component of their diet. It turned out that this isn’t really a “supplemental” diet, but instead dominates the diet (~38%). Although Cladosporium is a large component of the snail’s wild diet, but by “large” I mean it makes up ~1.5% of the fungus detected in the guts of the snail, so considerably less than 38%.

 

Studies had been conducted with snails where they were given the option of eating a fungal species or not, i.e. “single choice” experiments. These experiments determined that snails eat almost any fungus. Our eDNA studies also indicated that snails tended to indiscriminately consume any fungus available on their host tree. We wanted to use a controlled experiment to test if snails have food preferences. We didn’t use the federally endangered Achatinella snails for this, but substituted a species of tree snail in the same subfamily as them, an Auriculella. We were reluctant to use any critically endangered species because the controlled experiment involved putting multiple snails in little glass enclosures and filming them over 24 hours.

Our controlled experiment offered the snails a choice of eleven fungal and bacterial species that Kapono Gaughen and I had isolated from the snails’ habitat and grown on lawns of potato dextrose agar (typically called PDA). PDA is commonly used in fungal culture, and it is simply agar mixed with a sugar (dextrose) and potato starch. The Cladosporium fed to snails in the captive breeding program is given to them on PDA. We included a plug of PDA in our experiment as a control. My thinking was that snails would probably choose any microbial food at random, the PDA would be a control just to make sure that the snails were visiting the food choices for the food and not because, for example, the soft damp PDA creates a more attractive environment for the snails than the cold hard glass they must travel across to get to any food. To our surprise the PDA, which was meant to be a negative control, was by far the most preferred item in the experiment! Further observations confirmed that the snails are actually eating the PDA. This is a bit of a concern, as the natural habitat of the snails is not rich in nutrients and calories, so the PDA is by comparison a western-styled “junk” food of sugar and potato carbohydrate. As an alternative to provisioning captive snails with fungi grown on PDA, Kapono and I confirmed that Cladosporium can be grown in a liquid broth, collected in a sieve, and the excess of carbohydrate rinsed off, which might be a better way of delivering supplemental microbial cultures to tree snails.

 

We also found that, despite appearing to randomly graze  food, tree-snails do have preferences. In our study they tended to graze the darker pigmented species of fungi and avoid other food groups.

One of the 24 hour day/night videos used to collect data about snail feeding choice. The color of the ring indicates  that the snail spends with a particular food item. The most popular food item is the potato dextrose agar (turquoise), which was added as a negative control. Next in popularity are fungi that form dark tufts of hyphae (yellow bars in the chart).

O'Rorke, R., Holland, B.S., Cobian, G.M., Gaughen, K., Amend, A.S. (2016) Dietary preferences of Hawaiian tree snails to inform culture for conservation Biological Conservation, 198, 177–182.  PDF Journal Webpage

Mesocosm study - testing snail modification the phyllosphere

Blocked mesocosm experiment. One of each pair of mesocosms had ten snails added to it to see if the fungal community would change relative to the control mesocosm. Change in fungal community composition was measured by eDNA swabbed from leaves and change in fungal abundance by scanning electron microscope transects of leaves.

Animals that feed on fungi can shape the fungal community in a several ways. For example, by grazing away biomass they release minor members of the community from competitors. They can also influence the community by dispersing spores and vegetative fragments of their food after it passes through them.

 

There is a marsh snail (named Littoraria irrorata) that “farms” the fungus that it prefers to eat. This marsh snail poops on wounded leaves, and the fungal spores or propagules in their poop grow in the wound and can be eaten later. Because this is a well-known example, and because Hawaiian Achatinella snails poop on the leaves they feed from, many biologists would ask me if I thought the Hawaiian snails were farming the fungus that they ate. We conducted an experiment to test this. The idea seemed plausible, especially because Kapono and I, and also Brenden Holland and his team, found that diverse fungal species could be isolated from poop and cultured on different agar plates.

 

Our experiment consisted of pairs of mesh bags ("mesocosms") that we placed over tree branches. At the start of the experiment we took some snails from the wild and looked at what food species were in their poop using eDNA. We then put snails into one of the mesocosms (but not the matched pair, which was a control). We then visited the study site over six weeks to see if the bags with snails would become colonized with the species of fungus that the snails fed on and pooped out. They didn’t. However, the fungi growing in enclosures with snails did differ from the control enclosures without snails; the snails created more variability in the biodiversity of the fungus. We thought that this was consistent with grazing facilitating the invasion of environmental fungus into the leaf community. With help from Tina Carvalho from the University of Hawai’i Biological Electron Microscopy Facility we compared microbe abundances between leaves from grazed and control enclosures. Our measurements were consistent with the hypothesis that snails were clearing large regions of fungi and therefore facilitating invasion.

On a tangent, I did get interested in why viable fungi in the snails' poop weren’t growing in the mesocosms. This seemed odd because leaf surfaces are very nutrient poor, so I thought it was intuitive that a poop should be one of the best fertilised places for fungi to grow – shouldn’t it? Snail feces are compacted with mucus, and although pedal mucus and fecal mucus differ a little, I thought it worth looking at pedal mucus as a proxy (apologies if too much mention of feces and mucus is making your stomach turn). I found  that Auriculella pedal mucus is very hydrophobic (repels water), and a lack of water can be a strong factor in preventing microbial growth. I thought that this was pretty interesting, especially because I could only find one other reference to snail mucus – and for that species it was hydrophilic, the complete opposite! So I started a project, that I am yet to complete, to see how snail mucus hydrophobicity varies with snail phylogeny and ecology.

Snails were left to roam over the surface of a (A-i) glass slide and (B-i) an 'ōhiʻa leaf. The mucus covered surfaces were much more hydrophobic than the no-snail controls A-ii and B-ii.

Take home message: These snails certainly modify fungal communities by grazing, which allows new fungal species to invade the leaf surface and give it a local “flavor”.  However, they do not seem to disperse the fungus after they eat it.

Leaves from mesocosms treated with snails had less abundant fungal hyphae - consistent with grazing. The leaves grazed by snails displayed a greater variance in the diversity of fungal species than either ungrazed control leaves and leaves at the beginning of the experiment (T0).

O'Rorke, R,. Tooman, L., Gaughen, K., Holland, B.S. & Amend, A.S. (2017) Not just browsing: an animal that grazes phyllosphere microbes facilitates community heterogeneity. The ISME Journal, 11, 1788–1798 PDF Journal Webpage

phyllosphere and snail diet
Microbial diet of endangered tree snails across phyllospheres Diet across the range of Achatinella mustelina, Achatinella sowerbyana and Achatinella lila.

Beta diversity plots of microbes in the feces of snails and from leaf surfaces. Top left shows fungi from Waikanai ranges, top right shows bacteria for the same snails. The bottom image is of snails from the Ko'olau ranges.

Improving Snail Culture
Mesocosm study - testing snail modification the phyllosphere

Snails were left to roam over the surface of a (A-i) glass slide and (B-i) an 'ōhiʻa leaf. The mucus covered surfaces were much more hydrophobic than the no-snail controls A-ii and B-ii.

phyllosphere and snail diet
Microbial diet of endangered tree snails across phyllospheres
Diet across the range of Achatinella mustelina, Achatinella sowerbyana and Achatinella lila.

Improving Snail Culture
Mesocosm study - testing snail modification the phyllosphere