Sevilleta National Wildlife Refuge
The Sevilleta National Wildlife Refuge is a protected park in New Mexico located in the Chihuahuan desert around 20 miles north of Socorro, New Mexico. The Rio Salado flows through the refuge and is a tributary of the Rio Grande joining the Rio Grande just 15 miles north of Socorro, New Mexico. The refuge area currently hosts the Sevilleta Long Term Ecological Research (LTER) Program by the University of New Mexico. While research into parasites in mammals mainly rodents (Wilson et al., 1997) and other species that habituate in the Sevilleta National Wildlife Refuge has been done, there has not been any studies on snails and digenean parasites in the region. Understanding and mapping the physid snail and digenean parasite habitat could help
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estimate the biodiversity of the digenean parasite. Periodic inspections of this area will provide insights of freshwater snails from the Sevilleta and overnight shedding for cercarial emergence to identify snails that carry the parasites for identification. The anatomy of digenean larvae may provide clues for determination of the type of parasites (e.g., Schell, 1985), but identification to the species level is extremely difficult using morphology alone (Horak et al., 2002). Therefore, characterization of the parasites collected can be aided by recording sequence data from both cercariae and their snail hosts, followed by phylogenetic analyses of parasite-derived sequences to investigate the systematic placement of the parasites among digenea as proposed by Brant et al. (2006). Collecting this data will help in the understanding of how extent the habitat of the physid snails and digenean parasites are for ecological and biomedical purposes.
Shady lakes in an artificial group of ponds located in Albuquerque, NM near the Rio Grande River.
The ponds originally started out as a bait farm that supplied fishers with worms and carp for bait fishing. However, in 1962 the Phillips bought it and turned it into a local fishing spot. The Phillips refurbished the bait farm to a local fishing, ecosystem by cleaning up the water with both an artificial filtration system and with a natural filtration system consisting of certain plants, lilies plants and fish to keep the waters clean. Included in the this man made ecosystem are game fish, like rainbow trout and black fish this turned the ponds into a favorite local fishing spot where the whole family can have fun fishing, and enjoying this picturesque scenery. However, even though the waters are somewhat clean and well maintained with numerous natural and man-made filters, it still has a population of snails that carry digenean parasites. The digenean parasites are a large group of parasites that include both non-infectious parasites, to infectious parasites. The snail parasite population living in Shady Lakes is non-infectious to humans, but may be an important model system for similarly related parasites such as Schistosoma, which causes the disease Schistosomiasis in humans.
Schistosomiasis is a parasitic disease caused by blood flukes (trematodes) of the genus Schistosoma. After malaria and intestinal helminthiasis, schistosomiasis is the third most devastating tropical disease …show more content…
in the world, being a major source of morbidity and mortality in developing countries in Africa, South America, the Caribbean, the Middle East, and Asia (“CDC - Neglected Tropical Diseases - Resources” 2015).
More than 207 million people, 85% of whom live in Africa, are infected with schistosomiasis, and an estimated 700 million people are at risk of infection in 76 countries where the disease is considered endemic, as their agricultural work, domestic chores, and recreational activities expose them to infested water.Globally, 200,000 deaths are attributed to schistosomiasis annually.[3] Transmission is interrupted in some countries (“Medscape: Medscape Access” 2015).
Twenty-four Schistosoma species are currently recognized. These are categorized into 4 distinct groups, but only a few of these are known to be pathogenic to humans. The most prevalent human pathogens are S. mansoni and S. japonicum, primarily causing gastrointestinal disease, and S. haematobium, causing mainly genitourinary disease. S. mekongi and S. intercalatum are less prevalent but are also known to cause infections in humans (Papamatheakis et al. 2014).
To better understand the local transmission dynamics of schistosomiasis, A total of 7 pulmonate gastropods was collected from two diverse regions. The snails were morphologically identified as physid freshwater snails collected from aquatic vegetation along the banks of Shady Lake (Latitude 35°12'59.15"N, Longitude 106°35'54.52"W) within the city limits of Albuquerque, New Mexico, U.S.A., in September 2015. In addition, two morphology identified physid snails were collected in the Sevilleta National Wildlife Refuge (Latitude 34°13'51.6"N Longitude 106°40'48.0"W) , New Mexico, U.S.A along a floodplain on July 2015.
The snails were placed in 24-well plates overnight and examined for the release of cercariae. Positive snails and shed cercariae were preserved in 80% ethanol for downstream DNA extraction.
Genomic DNA was extracted from positive snails using a hexadecyltrimethylammonium bromide (CTAB) -based method (Winnepenninckx et al., 1993). Briefly, Snail tissue was homogenized in lysis buffer (100 mM Tris/HCL, 1.4 M NaCl, 20 mM EDTA, 2% [CTAB], 0.2% (v/v) beta mercapto ethanol). To this was added 10 μl of proteinase K (10 mg/ml) and the whole mixed placed in a water bath at 60°C maximum 30 minutes. Subsequently, an equal volume of chloroform/isoamyl alcohol (24:1) was added to the digest and gently mixed. Tubes containing the digest were then spun at 13,000 rpm for 5 minutes. The upper aqueous layer was transferred to a clean tube and equal amount of isopropanol was used to induce precipitation, the DNA was centrifuged again at 13,000 rpm for 5 minutes to form a pellet. The absolute ethanol was removed and 1 ml Ammonium acetate/76% ETOH were added following 15 minutes incubation.After that spun the sample for 5 minutes at maximum rpm, and the pellet washed in 75% ethanol before a final centrifugation at 13,000 rpm. The ethanol was then discarded and the pellet air dried for 5 minutes before dissolution in 50 microliter milliQ water(Kane et al. 2008).
Genomic DNA was extracted from parasite using DNAZOL reagent. Briefly spinning the sample for a minute, then removing the supernatant and add DNAzol into it. Next mixed the reaction by vortexing, leave them on bench top for 2 minutes, then add 100% EtOH to precipitate the DNA.Later remove supernatant, and wash the pellet with wash with 75% EtOH, spin the sample at max rpm, for 5 minutes, remove EtOH, repeat the wash and dry for 5 minutes by leaving tube open. At the final step, add 50 microliter milliQ water to dissolve pellet, stored the samples at -20℃.
Following quantification and a check for purity with a Nanodrop ND-1000 and ND- 8000 Spectrophotometer (Nanodrop Technologies Inc).
Genomic DNA was copied and augmented using the polymerase chain reaction (PCR) and various combinations of 16S primers (16SAr-F = PU178 and 16SBr-R = PL1462;Palumbi et al., 1991) for a 600 base pair segment and CO1 primers (LCO1490 and HCO2198; Folmer et al.,1994) for a 700 base pair segment.Fragments of nuclear rDNA genes of the parasites were PCR amplified using primers Worm-A and Worm-B to target 18S rDNA which is about 1800 bp long and LSU-5 and 1500R for 28S rDNA is about 1400 bp in length. Primers are listed in Table 1.
Applied Biosystems GeneAmp PCR System 2400 or 2700 thermal cycler was used throughout the project. PCR was also performed on the samples using AmpliTaq Gold (Applied Biosystems). Each 50 μL PCR reaction contains, 10U AmpliTaq Gold, 1.6 mM of each dNTP, 1mM each primer and 4 mM of MgCl2, 2μL of snail and 10 μL of parasite genomic DNA and rest MilliQ water. Cycling conditions for both snail and parasite PCR reactions were as follows: one cycle of 95°C for 10 min, 30 cycles of 94°C for 30 sec, 60°C for 60 sec and 72°C for 60 sec and a final single cycle of 72°C for 7 min following storage at 4°C . PCR fragments were separated on a 1% agarose gel.
PCR inhibitors were removed by further purification with a QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA) used according to the manufacturer's instructions. Purified DNA was stored at 4°C until it was used for direct DNA sequencing.
Approximately 30 μg of the purified products was then used as templates for direct sequencing of both forward and reverse strands, using the same amplification primers in an automatic ABI 310 DNA Sequencer with BigDye v.3.1(Applied Biosystems, Foster City California, U.S.A.) sequencing Ready reaction Kit. Sequences were obtained in duplicate from both strands, edited by eye, and assembled using Sequencher v. 5.4.4(GC codes, Ann Arbor, Michigan, U.S.A.).All sequences generated in this study were deposited in the GenBank of the National Center for Biotechnology Information (NCBI).
The sequences were compared with other sequences in GenBank using BLAST for further approximate identification.
The BLAST results were used to select a subset of taxa from a previous dataset of concatenated digenean 18S and 28S sequences (Brant et al., 2006) to provide relevant ingroups and outgroups for alignment with the experimentally obtained parasite-derived sequences (alignments available on request). Phylogenetic analyses using standard methods of maximum parsimony (MP), maximum likelihood (ML), and minimum evolution (ME) were carried out using PAUP* ver. 4.0b1019 (Swofford, 2001). Modeltest was used to determine the best nucleotide substitution model based on Akaike information criteria for the combined data for use in ML and ME analyses (Posada and Crandall, 1998). The following model was selected: GTR+I+G. Gaps were treated as missing data information residues. Parsimony trees were reconstructed using heuristic searches (100 replicates), random taxon-input order, and tree-bisection and reconnection (TBR) branch swapping. Optimal ME and ML trees were determined from heuristic searches (10 replicates), random taxon-input order, and TBR. Nodal support was estimated by bootstrap (500 replicates) and was determined for MP, ME, and ML trees using heuristic searches (10 replicates for both MP and ME; 5 replicates for ML), each with random taxon-input
order.
estimate the biodiversity of the digenean parasite. Periodic inspections of this area will provide insights of freshwater snails from the Sevilleta and overnight shedding for cercarial emergence to identify snails that carry the parasites for identification. The anatomy of digenean larvae may provide clues for determination of the type of parasites (e.g., Schell, 1985), but identification to the species level is extremely difficult using morphology alone (Horak et al., 2002). Therefore, characterization of the parasites collected can be aided by recording sequence data from both cercariae and their snail hosts, followed by phylogenetic analyses of parasite-derived sequences to investigate the systematic placement of the parasites among digenea as proposed by Brant et al. (2006). Collecting this data will help in the understanding of how extent the habitat of the physid snails and digenean parasites are for ecological and biomedical purposes.
Shady lakes in an artificial group of ponds located in Albuquerque, NM near the Rio Grande River.
The ponds originally started out as a bait farm that supplied fishers with worms and carp for bait fishing. However, in 1962 the Phillips bought it and turned it into a local fishing spot. The Phillips refurbished the bait farm to a local fishing, ecosystem by cleaning up the water with both an artificial filtration system and with a natural filtration system consisting of certain plants, lilies plants and fish to keep the waters clean. Included in the this man made ecosystem are game fish, like rainbow trout and black fish this turned the ponds into a favorite local fishing spot where the whole family can have fun fishing, and enjoying this picturesque scenery. However, even though the waters are somewhat clean and well maintained with numerous natural and man-made filters, it still has a population of snails that carry digenean parasites. The digenean parasites are a large group of parasites that include both non-infectious parasites, to infectious parasites. The snail parasite population living in Shady Lakes is non-infectious to humans, but may be an important model system for similarly related parasites such as Schistosoma, which causes the disease Schistosomiasis in humans.
Schistosomiasis is a parasitic disease caused by blood flukes (trematodes) of the genus Schistosoma. After malaria and intestinal helminthiasis, schistosomiasis is the third most devastating tropical disease …show more content…
in the world, being a major source of morbidity and mortality in developing countries in Africa, South America, the Caribbean, the Middle East, and Asia (“CDC - Neglected Tropical Diseases - Resources” 2015).
More than 207 million people, 85% of whom live in Africa, are infected with schistosomiasis, and an estimated 700 million people are at risk of infection in 76 countries where the disease is considered endemic, as their agricultural work, domestic chores, and recreational activities expose them to infested water.Globally, 200,000 deaths are attributed to schistosomiasis annually.[3] Transmission is interrupted in some countries (“Medscape: Medscape Access” 2015).
Twenty-four Schistosoma species are currently recognized. These are categorized into 4 distinct groups, but only a few of these are known to be pathogenic to humans. The most prevalent human pathogens are S. mansoni and S. japonicum, primarily causing gastrointestinal disease, and S. haematobium, causing mainly genitourinary disease. S. mekongi and S. intercalatum are less prevalent but are also known to cause infections in humans (Papamatheakis et al. 2014).
To better understand the local transmission dynamics of schistosomiasis, A total of 7 pulmonate gastropods was collected from two diverse regions. The snails were morphologically identified as physid freshwater snails collected from aquatic vegetation along the banks of Shady Lake (Latitude 35°12'59.15"N, Longitude 106°35'54.52"W) within the city limits of Albuquerque, New Mexico, U.S.A., in September 2015. In addition, two morphology identified physid snails were collected in the Sevilleta National Wildlife Refuge (Latitude 34°13'51.6"N Longitude 106°40'48.0"W) , New Mexico, U.S.A along a floodplain on July 2015.
The snails were placed in 24-well plates overnight and examined for the release of cercariae. Positive snails and shed cercariae were preserved in 80% ethanol for downstream DNA extraction.
Genomic DNA was extracted from positive snails using a hexadecyltrimethylammonium bromide (CTAB) -based method (Winnepenninckx et al., 1993). Briefly, Snail tissue was homogenized in lysis buffer (100 mM Tris/HCL, 1.4 M NaCl, 20 mM EDTA, 2% [CTAB], 0.2% (v/v) beta mercapto ethanol). To this was added 10 μl of proteinase K (10 mg/ml) and the whole mixed placed in a water bath at 60°C maximum 30 minutes. Subsequently, an equal volume of chloroform/isoamyl alcohol (24:1) was added to the digest and gently mixed. Tubes containing the digest were then spun at 13,000 rpm for 5 minutes. The upper aqueous layer was transferred to a clean tube and equal amount of isopropanol was used to induce precipitation, the DNA was centrifuged again at 13,000 rpm for 5 minutes to form a pellet. The absolute ethanol was removed and 1 ml Ammonium acetate/76% ETOH were added following 15 minutes incubation.After that spun the sample for 5 minutes at maximum rpm, and the pellet washed in 75% ethanol before a final centrifugation at 13,000 rpm. The ethanol was then discarded and the pellet air dried for 5 minutes before dissolution in 50 microliter milliQ water(Kane et al. 2008).
Genomic DNA was extracted from parasite using DNAZOL reagent. Briefly spinning the sample for a minute, then removing the supernatant and add DNAzol into it. Next mixed the reaction by vortexing, leave them on bench top for 2 minutes, then add 100% EtOH to precipitate the DNA.Later remove supernatant, and wash the pellet with wash with 75% EtOH, spin the sample at max rpm, for 5 minutes, remove EtOH, repeat the wash and dry for 5 minutes by leaving tube open. At the final step, add 50 microliter milliQ water to dissolve pellet, stored the samples at -20℃.
Following quantification and a check for purity with a Nanodrop ND-1000 and ND- 8000 Spectrophotometer (Nanodrop Technologies Inc).
Genomic DNA was copied and augmented using the polymerase chain reaction (PCR) and various combinations of 16S primers (16SAr-F = PU178 and 16SBr-R = PL1462;Palumbi et al., 1991) for a 600 base pair segment and CO1 primers (LCO1490 and HCO2198; Folmer et al.,1994) for a 700 base pair segment.Fragments of nuclear rDNA genes of the parasites were PCR amplified using primers Worm-A and Worm-B to target 18S rDNA which is about 1800 bp long and LSU-5 and 1500R for 28S rDNA is about 1400 bp in length. Primers are listed in Table 1.
Applied Biosystems GeneAmp PCR System 2400 or 2700 thermal cycler was used throughout the project. PCR was also performed on the samples using AmpliTaq Gold (Applied Biosystems). Each 50 μL PCR reaction contains, 10U AmpliTaq Gold, 1.6 mM of each dNTP, 1mM each primer and 4 mM of MgCl2, 2μL of snail and 10 μL of parasite genomic DNA and rest MilliQ water. Cycling conditions for both snail and parasite PCR reactions were as follows: one cycle of 95°C for 10 min, 30 cycles of 94°C for 30 sec, 60°C for 60 sec and 72°C for 60 sec and a final single cycle of 72°C for 7 min following storage at 4°C . PCR fragments were separated on a 1% agarose gel.
PCR inhibitors were removed by further purification with a QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA) used according to the manufacturer's instructions. Purified DNA was stored at 4°C until it was used for direct DNA sequencing.
Approximately 30 μg of the purified products was then used as templates for direct sequencing of both forward and reverse strands, using the same amplification primers in an automatic ABI 310 DNA Sequencer with BigDye v.3.1(Applied Biosystems, Foster City California, U.S.A.) sequencing Ready reaction Kit. Sequences were obtained in duplicate from both strands, edited by eye, and assembled using Sequencher v. 5.4.4(GC codes, Ann Arbor, Michigan, U.S.A.).All sequences generated in this study were deposited in the GenBank of the National Center for Biotechnology Information (NCBI).
The sequences were compared with other sequences in GenBank using BLAST for further approximate identification.
The BLAST results were used to select a subset of taxa from a previous dataset of concatenated digenean 18S and 28S sequences (Brant et al., 2006) to provide relevant ingroups and outgroups for alignment with the experimentally obtained parasite-derived sequences (alignments available on request). Phylogenetic analyses using standard methods of maximum parsimony (MP), maximum likelihood (ML), and minimum evolution (ME) were carried out using PAUP* ver. 4.0b1019 (Swofford, 2001). Modeltest was used to determine the best nucleotide substitution model based on Akaike information criteria for the combined data for use in ML and ME analyses (Posada and Crandall, 1998). The following model was selected: GTR+I+G. Gaps were treated as missing data information residues. Parsimony trees were reconstructed using heuristic searches (100 replicates), random taxon-input order, and tree-bisection and reconnection (TBR) branch swapping. Optimal ME and ML trees were determined from heuristic searches (10 replicates), random taxon-input order, and TBR. Nodal support was estimated by bootstrap (500 replicates) and was determined for MP, ME, and ML trees using heuristic searches (10 replicates for both MP and ME; 5 replicates for ML), each with random taxon-input
order.