Metagenomic analysis of Achatina fulica crop

==Background==
Achatina fulica is a species of giant land snails native to sub-Saharan Africa. It's physiological adaptability in terms of climate tolerance as well as ability to digest an incredibly broad range of plant material have made this a highly proliferative snail; it is found all over the world and is listed as one of the top 100 most invasive species. In ths US The species has gained a foothold in Hawaii,  while eradication measures are being taken in Florida.
It is known that land snails have an extensive microbiotic array in their digestive tract. It is believed that these symbiotic and mutualistic relationships enable the snail to achieve extremely high levels of digestive efficiency (upwards of 80% for many species). However, the actual composition and specific functional roles the microbiome remains unexplored.
This microbiome may also be able to lead scientists to new or improved methods for the production of biofuels. With dwindling fossil fuel supplies and rapidly increasing carbon dioxide emissions contributing to global climate change, the application of biotechnology to alternative fuel sources remains one of the most promising frontiers for industry. For instance, in traditional production of ethanol a large portion of the biomass used is discarded (or burned for heating, very inefficienty). This material is known as bagasse, which has a large energy potential of 19 MJ/kg and is made up largely of cellulose and lignin; two materials that are not readily fermentable by most organisms or industrial processes. The effeciency of Achatina fulica in the digestion of lignocellulose material suggests that it's microbiome may have novel enzymes that could be used in the biofuel industry.
Despite it's widespread abundance very few studies have focused on Achatina fulica, making it a potential gold mine of information regarding symbiosis and coevolution of an organism and it's microbiota, novel genes relating to lignocellulolytic activity, and applications to next-generation biofuels. A recent metagenomic analysis of this snail took all of this into account: they focused on relating Achatina fulica's microbiome with other herbivores, and focused on genes encoding enzymes that may be relevant to the catabolism of plant biomass.
==Methods==
The researchers traced the highest net as well as specific activity for lignocellulolytic activity to the crop of the snail, which is where they chose to collect their samples for sequencing. DNA isolation was performed by cell lysis using proteinase K(a broad spectrum serine protease) and sodium dodecyl sulfate(a widely used surfactant), followed by a phenol-chloroform extraction.
For sequencing reactions, the researchers used pyrosequencing. This type of sequencing is known as "sequencing by snythesis" and relies on the detection of a pyrophosphate group after a nucleotide is incorporated into he growing DNA strand. The synthesis is limited to one nucleotide at a time: only one type (adenine, guanine cytosine or thymine) is exposed to the reaction, thus the sequence is determined when the correct nucleotide is added and light is emitted.
==Results==
===General Overview===
This metagenomic study generated 1,297,598 total reads. 188,709 were removed as replicas while 198,922 were also removed due to low quality, giving a grand total of 909,967 reads that were used for functional characterization. Of these, approximately 82% were able to be assigned to a taxon. As it turns out every domain was represented in the snail's microbiome: eukaryotes, bacteria and archaea. Viral DNA was even detected as well. The number of reads was further reduced to ensure the analysis of more complete genes by eliminating "problematic" sequences. A sequence could be problematic if it is only a partial, a repeat, an outlier or other classifications based on the GS De Novo Assembler. This brought the final read count to 423,310, with 2,623 contigs larger than 500 bps (average of 4,474 bps).
The majority of the detected eukaryotic DNA sequences were found to belong to a few species of fungi. It has been well established that different fungi as well as protozoans aid in the digestion of cellulose in termites, but the exact function of these species within the gut of Achatina fulica remains unknown.
With respect to viruses, their populations in the human digestive tract remain relatively consistent. This suggests that there exists a mutualistic relationship between them and the resident bacteria, which may also be the case for the snail. The identified viral sequences detected in this study belonged to various types of bacteriophages. It is believed that they regulate and influence the community of bacteria that reside there.
===16S rRNA Analysis===
The primary bacterial groups found in the crop based on 16S rRNA analysis were Pseudomonas (37.5%), Sulfurospirillum (8.5%), and Stenotrophomonas (7.3%). Species from these genera are found all over nature: they represent a diverse array of bacteria that inhabit waterways, soil, and plants all over the world. Based on this, the researchers hypothesize that this enables the snail to digest a variety of different plants; from the roots of rice to fruits, vegetables and grasses. It is noted that these bacteria may not only contribute to the snail's ability to catabolize lignin and cellulose, but also assist in the metabolism of amino acids, vitamins, toxins, as well as protection from oxidative stress. 
The diversity of bacterial species present in the crop was notably similar to that found in various herbivores. Many of the identified taxa, such as Epsilonproteobacteria, are known to have a symbiotic relationship with their host. This type of relationship has not yet been described in the giant land snail however.
===Novel Findings===
Interestingly, this was the first time that viruses, archaea, and fungi were detected in the crop of a snail. This highlights the gaps in our knowledge base of the snail microbiome. Furthermore it demonstrates the rich diversity also observed in other ruminants and herbivores such as the wallaby, panda, and termite.
Subsystems-based annotations were done to asses the metabolic potential of the microbiome of Achatina fulica. While there were numerous genes related to main metabolic pathways, the researchs also noted several that deal with iron acquisition, vitamins and cofactors, potassium, sulfur and phosphorus. This snail's metagenome had an abundance of the aforementioned sequences compared to that of other herbivores, while the core metabolic pathways such as carbohydrate, nucleotide and protein synthesis/breakdown were shown to be at comparable levels. The researchers maintain that vast microbiome may lend the snail some of it's characteristic properties such as invasiveness and digestive efficiency.
===Glycoside Hydrolase Domains===
To gain insight into how the microbiota influences the digestion of plant fibers, the researchers performed database searches for glycoside hydrolase (GH) domains. Enzymes that have these domains catalyze the hydrolysis of multimeric sugars into smaller subunits by breaking glycosidic bonds. This represents a large enzyme family that is found in all forms of life.
The researchers obtained over 4,000 environmental gene tags (sequences that potentially code a protein, EGTs) belonging to 93 different carbohydrate-active enzyme families (CAZy). This indicated an large potential for polysaccharide degradation and therefore potential for plant cell wall breakdown. Many of the identified GH families have been shown to be involved in peptidoglycan cleavage (long sugars residing outside cell walls/membranes), de-branching of complex sugars, and other plant-oriented degradation enzymes including B-galactosidases, xylanases, chitinases, mannosidases, and more. Not all of the GH-containing EGTs obtained were catalytic however; many were found to be carbohydrate binding modules. While they may not directly be responsible for cellulose of lignin degradation, proteins containing these modules often bind cellulose or other complex sugars and enhance an enzyme's activity towards the bound substrate, or vice-versa.
Overall, the snail showed to have a greater fraction of GH family members than the panda and human gut microbiota in every category (cellulases, endohemicellulases, cell wall elongation, debranching enzymes, and oligosaccharide-degrading enzymes).The termite only surpassed the snail's fraction in the category of cellulases.
==Sources==
Cardoso, Alexander et al. (2012) Metagenomic Analysis of the Microbiota from the Crop of an Invasive Snail Reveals a Rich Resevoir of Novel Genes. PLoS ONE 7(11): e48505. doi:10.1371/journal.pone.0048505
Wikipedia: Achatina fulica. http://en.wikipedia.org/wiki/Achatina_fulica#cite_note-3