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Protein-Protein Interaction Experiment

Introduction Saccharomyces cerevisiae is a unicellular eukaryotic fungus (Clark 2010). Investigating protein-protein interactions involved in the activation of GAL4 genes in yeast that regulate the metabolic breakdown of carbon energy sources is the purpose of this project.
In the yeast two hybrid system the binding domain is supposed to bind to the UAS of the GAL4 gene in the yeast (BIOL 466). The bait is bound to the UAS and the prey is bound to the binding domain; the activation domain is attached to the prey. If the bait and the prey interact it corresponds to an interaction between the respective attached domains that will allow for the activation domain to recruit the transcriptional machinery required to transcribe the reporter gene which is HIS3 (BIOL 466). This process provides insight into why the relatively small number of eukaryotic genes of yeast, and similarly humans, can have so many functional protein manifestations (BIOL 466). This, in part, is due to protein-protein interactions resulting in the variation of function upon the interaction and modification of proteins. Interacting proteins can serve many purposes in cellular function diversity (Rapely 2008). The way products interact can be based on how they are modified after translation, which proteins interactions are taking place at a given time, and the environments and where these processes are taking place (Rapely 2008). The purpose of this experiment is to use protein-protein interactions to monitor how Gal4p domains responsible for activation and binding interact in yeast by manipulating the GAL4 gene to provide a signal that can easily be monitored.
Protein-protein interactions can serve as a way of signalling fitting proteins and to help dictate their function. This can be used in molecular biology to determine whether an aunt two parts of the gene need to interact in order for the expression of a gene to take place. In the Yeast Two Hybrid (Y2H) experiment protein-protein interactions are used to determine the functionality of the GAL4 gene responsible for transcription of the protein Gal4p (BIOL 466). Yeast two hybrid (Y2H) can be done in a forward fashion or a reverse fashion. The forward fashion investigates whether protein-protein interactions take place, resulting in a reporter gene signal, while reverse investigates disrupting protein-protein interactions resulting in a toxic reporter gene signal (BIOL 466). The process involves GAL4 that is required for galactose gene expression in the presence of glucose (Clark 2010). It is a transcription factor with two domains that are involved in either DNA binding or transcription activation (Clark 2010). It also finds to a region on the Yeast genome called the UAS. These two domains can be split from each other and still function independently, similar to the physical separation of the Alpha and omega fragments in E. coli Alpha-complementation (BIOL 466). A reporter gene for places the GAL4 wild type gene to monitor the interaction of the bait and prey (Rapley 2008). In the case of this project, the reporter gene is wild type HIS3 (BIOL 466 lab. Man.).
RT-PCR is an in vitro process that presents a solution to the problem of introns in eukaryotic DNA by converting mRNA products into cDNA (complementary DNA) which consists only of exons that commonly code for the genes of interest in this project (Rapley 2008; Clark 2010). Reverse transcriptase derived from retroviruses often have the ability to degrade RNA-DNA double stranded hybrids which is not a desirable function for the RT-PCR and is often eliminated n the reverse transcriptase enzyme used (Rapley 2008). Similar to PCR, RT-PCR requires the addition of dNTPs, primers for the amplified region of interest and buffers with the appropriate salt concentrations and temperature for the enzyme to function. The primers used in the project will be designed to amplify the mRNA of interest as opposed to other methods that use random primers with no selectivity or dT oligonucleotide primers designed to ensure amplification of mRNA over rRNA in RT-PCR (Rapley 2008). The RNA is denatured first in the process in the presence of primers. Then enzyme, buffer, dNTPs, and salts are added with the inclusion of RNase-denaturing solution like DEPC H20 (Rapley 2008). The temperature is set for ideal enzyme function for the conversion of the mRNA fragment of interest until the solution is heated to inactivate the extension cycle (Rapley 2008).
The yeast two hybrid (Y2H) process involves the isolation of yeast RNA and its conversion into a cDNA templates. Through the use of PCR the cDNA is amplified after which the prey will be ligated into the vector (Rapley, 2008). The purpose of this experiment is to monitor the interaction of the bait and prey and compare the results to four plasmid preparations on an agarose gel.
The bait and prey system is used in the Y2H system to determine Protein-protein interaction (Rapley, 2008). By connecting the bait and prey system to genes in the yeast and introducing a measurable signal that corresponds to protein-protein interaction, the experiment can be monitored (Clark 2010).
The yeast two-hybrid takes advantage of proteins that bind to other proteins (Clark 2010). By using a protein with two domains, such as a binding and activation domain, these domains can be manipulated to provide a signal that corresponds to leather not they are interacting with each other (Clark 2010). For this project, the binding domain of Gal4p and also its activation domain are manipulated to use the GAL4 gene of yeast as a system to monitor Protein-Protein interactions. The protein’s binding domain customarily a binds to the UAS domain upstream from the GAL4 and the activation domain usually binds RNA polymerase for transcription (BIOL 466). In the two-hybrid system, the two domains of the protein are split apart because they can still function if they are not attached to each other; these pieces can be attached to other proteins that have a known affinity for each other which are called the “bait” and the “prey” (Clark 2010). By taking advantage of MAP kinase activity and the fact that the MAPSP1 protein readily binds very tightly to p14, an endosomal protein (BIOL 466).
The key to monitoring the activation of the gene and interest depends on the bait and gray five each other and interacting a line for the transcription of a gene that provide some sort of signal that the interaction between the activation and by the domains through the bait and prey system has occurred (Clark 2010). For those projects that the prey is MP1, a kinase, and the bait is p14, respectively (Biol 466 lab). The bait is attached to the binding domain and the parade is attached to the activation domain.
The purpose of this experiment is to explore protein-protein interactions by manipulating the molecular machinery of the yeast using the bait and prey system and screening for the signal using the Y2H parameters set up using molecular biology applications.
Methods As per BIOL466 Lab Manual 2011 except waited only 45 minutes instead of 60 minutes for incubation in week 7; also waited 35 minutes instead of 45 minutes when incubating the tubes in Week 8; in week 9 for tube 3 used more than 0.5 g of C because the volume of plasmid required was below 2 L.
Results For this experiment, in order to amplified the desired protein product, the RT-PCR process is used to produce RNA fragments from DNA that are specific to regions of the yeast genome that contain GAL elements. The specific primers used are included below (See below). The negatively charged nucleic acid migrates to the positively charged end of the agarose gel network in the presence of the buffers and other solutions (BIOL466 Oligos, 2010)
Following the insertion of the vectors into the cells, the yeast two-hybrid screening on various plates with different medium content were used to confirm the bait and prey interaction within the cell that should result in a positive signal from the reporter gene HIS3 when no histidine is present in the medium.
The expected results for the plates containing leucine, tryptophan and histidine were that only the successful ligation and interaction of the bait and prey system will give a positive signal (Figure 2B). Positive signal corresponds to the plate with abundant amount of Colonies (plate C). The other plates-A, B, and D–have no growth.
The expected results for the plates containing leucine, tryptophan and no histidine were that only the successful insert of His3 or his3 auxotrophs as well as cells with the interaction of the bait and prey system will give a positive signal (Figure 2A). Positive signal corresponds to the plate with abundant amount of Colonies (plate B, C, and). The other plate-plate A,–has no significant growth.

Discussion The oligonucleotides for the bait and prey were designed to hybridize to upper part of the 5? end of the DNA fragment of interest as well as the 3? end of the DNA fragment of interest (BIOL 466). The fragments of interest were transformed into DH5 E. coli for fast replication of the fragments to have a large sample to purify and extract in a relatively short period of time.
In this project the restriction enzyme digestion is performed simultaneously because the enzymes thrive well in similar buffer conditions (NEB double digest). The restriction enzymes recognize different sequences in the DNA and create 5? overhangs that do not have compatibility with the other enzyme’s overhang. Therefore, relegation of the fragments with itself upon the addition of ligase will not occur and the probability of the recombinant plasmid forming increases significantly; this aspect of the primer design also requires consideration of the possibility that the restriction enzymes of choice do not cut the insert within the sequence and are only found in the multiple cloning site (MCS) of the plasmid used (Rapley 2008). RNase removal with DEPC helps minimize loss of RNA product to these enzymes that have disulfide bonds that contribute to their heat resistance (Rapley 2008).
This project was carried out by first isolating yeast RNA from cell culture. The RNA was extracted and purified in several steps resulting in a purified sample. The sample purification was confirmed via spectrophotometric analysis of the RNA sample and measuring the A260/A280 Ratio (Table 2). Following the purification steps of the RNA, the RNA was converted to cDNA through the using of reverse transcriptase polymerase chain reaction (RT-PCR). The conversion of RNA to the C cDNA template using a reverse transcriptase enzyme derived from viral functions was amplified to yield the portions of DNA containing the bait and prey sequences (Oligos, 2010) (Sequence 1 and 2). The primers used for amplification are derived from oligonucleotides from the yeast genome. These oligonucleotides for the prey (MP1) are ligated into the vector (pGADT7).
The RNA gel of the yeast contains some smears which may be the result of proteins or denatured nucleic acids contaminating the sample and the purity of the sample can be monitored by calculating A260/A280 (Lui 2009). The spectrophotometric measurements of concentration of nucleic acids were used to assess purity as well as normalized the values of nucleic acids inserted into the wells. This was done to insure that each lane show they represent the value for the samples used.
While conducting the many steps for this project, the first steps involved PCR of the yeast genome region of interest and cloning those fragments into two different vectors where one vector contained the prey protein which was attached to the activation domain. The other vector contained the bait which was attached to the binding domain (Clark 2010). The vectors were selected so that the gene of interest would be in the correct open reading frame (ORF) and the protein of interest would be properly transcribed. After the recombination process the yeast have both vectors the reporter gene will be expressed (Clark 2010). For this project the reporter gene is HIS3 therefore expression will allow auxotrophic histidine variants to grow in mediums that don’t have histidine (Clark 2010). The protein-protein interaction is detected using the reporter gene and plating these colonies on medium that will confirm or deny the presence of the Protein-Protein interaction within the cells.
By mating yeasts containing one vector with one kind of domain, a binding domain, with yeast containing another vector with the other kind of domain, and activating domain, the resulting hybrid likely have the bait and prey interaction which will allow for the activation of the reporter gene. The reporter gene will give a visual signal when plated on histidine because the reporter gene used is HIS3. There are possibilities of false positives in this experiment so the medium for plating was checked twice: one medium contained histidine, tryptophan, and leucine and another media contained only tryptophan and leucine (Figure 3). In the agarose gel Lanes one and two corresponds to the positive and negative controls, respectively (Figure 2). Lane one contains a band at about 450 bp and lane two has no band present, both as expected. Lane three contains the ligation with the band at the same size as lane one but the band is much darker suggesting that there is more ligation product than the control plasmid found in lane one. Also, the products of lane four suggests the insert is found based on its higher location of jell which corresponds to larger sized plasmid containing an insert.
The sample found in the A sample and A plate can be confirmed based on the relatively low presence of the plasmid that has the smallest molecular weight which corresponds to the smaller sized restriction map of the four choices. This conclusion is also supported by the presence of no colonies on the A plates suggesting the lack of Histidine production capabilities for the yeast plated on the triple drop out as well as the presence of a leucine auxotrophy on the double drop out plate indicating the presence of the Mp1 plasmid and nothing to offset the auxotrophy in either medium.
The lighter band on the agarose gel for the A sample suggests this sample was difficult to synthesize or extract though the process did take place based on evidence of the cDNA presence on the gel (Figure 1A and B). The results indicating the B sample was the Human insert of p14 is initially not clear based on the results of the plates alone which had the same results as the result for plate D in both the double and triple drop out. The results are distinguished, however, upon the analysis of the restriction maps and the agarose gels. The difference between the sedlin and p14 recombinant is based on the size of the insert in each respective sample inserted into the pGBKT7 vector (Sequence 3 and 4). The sedlin insert is much larger and so has a larger bp that corresponds to a band that would be comparatively higher than that of the p14 recombinant plasmid (Vectors 3 and 4, Figure 1A and B). From the overall evaluation of the data pertaining to samples B and D, the identity of B is the mouse sedlin recombinant. The recombinant gives a red color on the double drop out because of the mutation of the ade2 gene producing a red by product (BIOL 466).
Comparing these findings to the restriction maps generated by NEB cutter, the differences between lane four and lane three suggesting one has a larger insert than the other and noting the sedlin insert in the pGBKT7 is larger than the p14 in the same plasmid and the location of the cut sites for each leads to the conclusion that sedlin must be from lane D and the plate D is the sedlin recombinant yeast sample.
In conclusion, the use of GAL4 in the yeast and manipulating its transcription factor by separating it from its activation domain provides a way to monitor Protein-Protein interactions. These interactions are monitored using the two-hybrid system involving a bait and prey attached to each separate domain on two separate vectors which are then brought together after the yeast mate. The success of the insertion of the respective bait-attached gene into a vector, or the prey protein in the same respect, into the vector is monitored by agarose gel electrophoresis and compared to various controls. Furthermore, the Protein-Protein interactions were monitored through the activation of a reporter gene of HIS3 when plated on medium containing histidine and not containing histidine to confirm the presence of colonies where the bait and preyed are interacting and the activation by the domains of Gal4p interacts to recruit RNA polymerase to make histidine. Reporter gene activation is a screening process to identify the protein interaction of interest and provides insight into the way that Protein-Protein interactions within a cell dictate how the protein functions. The yeast two-hybrid method can be applied to the human genomic studies for monitoring genetic pathways and complex protein interactions stemming from the fact that humans have 30,000 genes that behave as though there are 150,000 genes (Rapely 2008; BIOL 466).
Possible sources of error include the limits of using this bait system includes the requirement that the proteins need to interact with the nucleus (Clark 2010). This limitation can be avoided by using membrane systems that can interact in the cytoplasm or other variations of the two-hybrid system (Clark 2010). Also, the presence of a beta carotene other than the one uses project interacting with the prey may occur (Rapley 2008). Perhaps using to proteins in a purified setting and monitoring their interaction as a control for this part of the experiment can provide insight in to what extent there may be a rogue bait protein (Rapley 2008).
Other sources of error may include mRNA degradation resulting in low yield of intact, complete product. Also large fragments greater than 200 bp are not very easily converted and may require a different amplification strategy like gateway cloning (Rapley 2008; BIOL 466).

Nutrient Cycle of an Isolated Cave

Introduction
The caves are simple natural laboratories. The climate of the cave is very stable and easy to define. Cave environment is composed with a twilight part close to the entrance, a middle part of full darkness and unstable temperature, finally a part of full darkness and stable temperature in deeper. The twilight part is the biggest and most diverse fauna container. The middle part contains some common species which can move to the earth. The deeper dark sides, which are the unique aspect of the cave environment and contain obligate (trolobitic) fauna. Green plant can’t live in stable darkness. So, the food reserve here in other forms (Poulson and White, 1969). Animal communities in the caves look remarkable chances for the investigation of community dynamics because of their relative simplicity. A comparatively small number of species is involved in even in most complex cave community but exceptionally large numbers of colonies of bats are present here. In absence of light, primary producers are absent or at least limited to chemosynthetic autotrophs. Sulfur and iron bacteria are present in some caves but their quantitative significance as producers has not yet been established (Barr Jr, 1967). The superficial nutritive part of cave clay in the blind amphipods of the genus Niphargus show that juvenile stages burrow widely and probably eat the clay in the bottom of cave pools. Presumably the juveniles utilize the bacterial content of the clay rather than the mineral material itself; and in any case, continued survival of the adults is dependent upon the presence of additional food (Barr Jr, 1967). In addition to absence of light, the physical environment of a cave is characterized by silence, relatively constant temperature which approximates the mean annual temperature of the region where the cave is located, high relative humidity except near entrances, is accompanied by an exceptionally low rate of evaporation (Barr Jr, 1967).
Cave Habitats and Ecology
Different types of caves contain variety of habitats within them and differ in amount and types of energy level. Cave supports heterotrophic microbial populations in the presence of huge input of organic carbon, nitrogen and phosphorus due to accumulation of guano and dead bats, if a cave has substantial or modest populations of bats (Cheeptham, 2012). Guano is a organic deposit common in cave derived from mainly feces of a variety of animals specially bats that visit or live and provide habitat rich in nitrogen, carbon and phosphorus that’s are nutrients for many insects (Cheeptham, 2012; IUCNSSC, 2014). Ecological classification of cavernicoles was first prepared by (Schiner, 1853)and improved and promoted by (Racovitza, 1907).They splits them into (1) troglobites, which are obligate species to the cave; (2) troglophiles, which live and reproduce not only in caves but also in cool, dark, moist microhabitats outside of caves they termed as facultative species; (3) trogloxenes, species those use caves for shelter throughout the day but feed outdoor at night; and (4) cave accidentals, which Confused with those species that certain small troglobites are also phreatobites (Barr Jr, 1967).

Figure-Different zones of a cave
The major energy sources of cave ecosystems are (a) organic matter flounced underground by sinking streams, and (b) the feces, eggs, and dead bodies of animals those are persist in the cave for shelter but feed outside (trogloxenes). In temperate region caves flooding and the entering of cold air throughout winter and initial spring interrupt the comparatively constant physical conditions of the cave environment (Barr Jr, 1967). The security of roosting sites is a vital element of any policy for the conservation of bats. Since caves are the foremost roosts for numerous bat species (Dalquest and Walton, 1970; Kunz, 1982). There are various types of bat species and large number of bats found in different cave, Seventeen species of bats roost in the caves of Yucatan, Mexico. The conservation of these types of sites should be of principal attention for the protection of chiropteran species (Arita, 1996).
Cave communities
Connectivity among communities is continued by the rearrangement of biomass, frequently by mobile animals that eat resources in one habitat and then reproduce, urinate, and/or defecate in other surroundings. This transmission of organic material affects the nutrient budget of a community and effects population and food web dynamics (Emerson and Roark, 2007). Cave-roosting species spent half of their lives inside the caves (Kunz, 1982). The security of cave atmospheres is essential to guarantee their conservation. In a parallel fashion, the presence of bats might be an essential state for the existence of cave environments. In channels with no bats, biomass thickness in a typical North American cave can be as little as 1 g/ha in ponds or 20-30 g/ha in terrestrial zones (Poulson and White, 1969). In contrast, passageways covered with bat guano present an excess of nutrients and provide very diverse groups of arthropods (Barr Jr, 1968; Harris, 1970; Poulson, 1972). For endogenous primary manufacture by chemosynthetic bacteria is insignificant, cave communities depend completely on exogenous origins of nutrients for their maintenance (Culver, 1982).

Figure-Cave communities and feeding cycle
Nutrients can be occupied into a cave in the form of detritus and plant material passed by watercourses, as dissolved organic matter infiltrating through minute cracks or exuding from tree roots (Howarth, 1972; Howarth, 1983), otherwise they can be placed inside caves as feces of trogloxenes, for example cave crickets, bats, birds, and other animals (Harris, 1970; Poulson, 1972; Culver, 1982). In various tropical caves, bat guano is by far the most significant source of nutrients. By carrying tons of organic matter to the caves, bats act as transferable links concerning cave environments with the external world (Arita, 1996). Any animal existing in a cave can be said as a cavernicole. Troglobites, which are obligate cavernicoles, are the emphasis of this appraisal. Many troglobites are offspring of troglophiles. Facultative cave populations are able to alive in or outside caves. Trogloxenes are consistent cave inhabitants that return intermittently to the exterior for food; bats and cave-crickets are examples. Main taxonomic collections of animals with various troglobitic species comprise collembolans, turbellarians, millipedes, spiders, pseudoscorpions, gastropods opilionidsisopods, amphipods, diplurans, decapods, beetles (Pselaphidae, Carabidae, Leiodidae), salamanders and fishes.(Barr and Holsinger, 1985)
Cave Nutrient Cycle
Food contribution into a cave ecosystem is attributable to two chief sources- sinking watercourses, which wash twigs, logs, bacteria, leaves and epigean animals (including zooplankton) into caves; and trogloxenes, which deposit their eggs and feces in caves and frequently die there and donate their bodies to the ecosystem (Barr Jr, 1967). Species from exterior sources include the bulk of the plankton in the Cave (Scott, 1909) and rivers inside Cave (Kofoid, 1899). Smaller individuals of the blind cavefish, Amblyopsis spelaea, feed mainly on copepods in this plankton (Poulson, 1963). Plant fragments are placed along the banks of subterranean streams, where they are gradually decomposed by bacteria and fungi. The decomposers provide food for detritus-feeding animals (e.g., diplurans, milli-pedes, and collembolans) which are then eaten by predators (e.g., opilionids, spiders, carabid beetles, pseudoscorpions). Bats and the eastern cave crickets of the genus Hadenoecus (Park and Barr, 1961) are important guano manufacturers in caves of the United States. Few troglobites are able to use the guano directly, while guano is usually populated by a characteristic assemblage of troglophiles which may be eaten by predatory troglobites (Jeannel, 1949). Seasonal differences in the physical atmosphere and food supply of temperate zone caves are often unexpectedly drastic. During late winter and spring overflowing of rivers Cave, typically raises the water level 5 or 6 m, and a maximum rise of nearly 15 m has been recorded. Additionally the flood is a drop in temperature of the water and small increases in pH, entire alkalinity, and dissolved oxygen (Barr Jr, 1967). A much longer existence time in a riparian species of cave beetle when the riparian species and another species usually found in drier, higher cave galleries were immersed in water. Many species of Pseudanophthalmus and Ameroduvalius (troglobitic Carabidae) normally feed on little tubificid annelids in the damp silt along cave streams (Barr Jr and Peck, 1965). The effects of flooding on aquatic cavernicoles, suggesting that spring floods may trigger their reproductive cycles (Poulson, 1964). Winter poses additional hazards for terrestrial troglobites. Food supplies vary seasonally in caves. Guano deposition by bats is limited to summer months, and Hadenoecus spp. feed outside the caves less often throughout winter than in summer, so there is minimum guano supply in winter. Conversely, deposition of organic detritus by watercourses is improved in winter because of flooding, but decomposition of the fragments takes place gradually over the time of several months or years. A great plankton count in Echo River of Mammoth Cave occurs only throughout late spring or summer floods, when plankton manufacture in Green River, which provides the flood waters, is great (Barr Jr, 1967). The genus Pseudanophthalmus covers about 175 species (many of them not yet described) and is known from Indiana, Kentucky, Illinois and Tennessee, Alabama, Georgia Virginia, West Virginia, Pennsylvania, and Ohio (Barr Jr and Peck, 1965). Ameroduvalius, limited to south- east Kentucky, has only three species; Nelsonites, from the Cumberland Plateau of Tennessee and Kentucky, has two; and Neaphaenops and Darlingtonea, from many parts of Kentucky, are monobasic. All of these beetles are predatory troglobites and are supposed to be remnants of a well-known soil-and-moss-dwelling periglacial fauna (Barr Jr, 1965).

Figure- The cave food pyramid
Guano
Bat guano supports an accumulation of organisms that differs depending on the species of bat manufacturing it. Alterations in guano composition propose that guano from bats in unlike feeding guilds can affect ecosystem configuration and dynamics differently (Emerson and Roark, 2007). Allochthonous effort of nutrients such as nitrogen and phosphorus, which are found in comparatively high concentrations in bird guano, increases primary productivity in terrestrial ecosystems by improving the quality and quantity of vegetation (Polis et al., 1997). Nutrient input through guano deposition by seabirds has also been shown to increase the abundance of organisms such as detritivorous beetles on islands used by roosting seabirds (Sánchez-Piñero and Polis, 2000). In addition to its effects on primary and secondary productivity, allochthonous nutrient input can also influence community structure the presence of birds and nutrient-rich guano significantly alters the structure of intertidal communities by enhancing algal growth and settlement of invertebrates in dense algalmats (Bosman and Hockey, 1986). Such consumer-driven nutrient recycling via fecal deposition by bats also affects community structure in guano-based ecosystems. Bat guano forms the basis of a food web consisting of bacteria, fungi, protozoans, nematodes, and arthropods (Harris, 1970). Cave salamanders consume guano of grey bats (Myotis grisescens) and incorporate the nutrients they obtain through coprophagy into body tissues (Fenolio et al., 2006). The diversity of organisms associated with guano has been shown to vary depending on the diet of the bat producing it, with guano of sanguivorous, insectivorous, and frugivorous bats supporting different assemblages of invertebrates (Ferreira and Martins, 1998). Differences in guano composition (C, N,P, and mass ratios) most likely resulted from dissimilarities in nutrient composition of the diets of each bat species (Studier et al., 1994). Variation in nutrients and stoichiometric nutrient ratios of guano from bats in different feeding guilds could have considerable effects on producers, consumers, and decomposers living on or in guano.

Figure- Collection of guano from cave
As highlighted by (Sterner and Elser, 2002) and subsequently in reviews by (Vrede et al., 2004) and (Moe et al., 2005), relationships among elemental nutrients have the potential to regulate processes at many ecological levels, including production, individual and population growth, coexistence of species, rates of decomposition of organic matter, and nutrient cycling. Primary production in terrestrial ecosystems (as in marine systems) is thought to be limited by the availability of N and P (Vitousek and Howarth, 1991), and the input of these nutrients by fecal deposition can have considerable bottom-up influences in detritus-based ecosystems. Ecosystem-level effects of different nutrient contents could also result from differences in rates of conversion of nutrients in guano from biologically unavailable to available forms (Vitousek et al., 1988). Differences in guano nutrient profiles could have considerable ecological consequences ranging from effects on the growth or productivity of individual residents of guano piles to effects on ecosystem-level processes like decomposition and nutrient cycling (Emerson and Roark, 2007).
REFERENCE
ARITA, H. T. 1996. The conservation of cave-roosting bats in Yucatan, Mexico. Biological Conservation, 76, 177-185.
BARR JR, T. C. 1965. The Pseudanophthalmus of the Appalachian Valley (Coleoptera: Carabidae). American Midland Naturalist, 41-72.
BARR JR, T. C. 1967. Observations on the ecology of caves. American Naturalist, 475-491.
BARR JR, T. C. 1968. Cave ecology and the evolution of troglobites. Evolutionary biology. Springer.
BARR JR, T. C.

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