Attribute_Accuracy_Report:
Collection sites, sampling dates, and taxa were all cross-checked and verified to match field data. Individual extraction numbers, genus, and pool size were verified to be correct according to laboratory notes. Sterile techniques were used throughout all molecular protocols, including positive controls for DNA extractions and negative controls included in all nested-PCR reactions. All assigned GenBank accession numbers were cross-checked for accuracy.
Attribute values fall within expected ranges and data were proofed for the presence of duplication and omission.
No data were omitted and there are no missing data. NA's in cells indicate that no Plasmodium DNA was detected in pools and thus no GenBank accession numbers were assigned.
Methodology:
Methodology_Type: Field
Methodology_Description:
Mosquitoes were collected from three locations (Campbell creek, Eagle River, and Mirror Lake) near Anchorage, Alaska, USA during the summer of 2016. Modified CDC model 512 miniature light traps were assembled at each location and opened for two 24-h periods per week from 8 May to 8 August. Traps were affixed to stands approximately 1.5 m tall and fitted with kill-jar assemblies containing 100% ethanol and supplemental CO2 systems to aid in mosquito attraction. Kill-jars were collected at the end of each 24-h period and mosquitoes were kept in ethanol until subsequent dissection and DNA extraction.
Methodology:
Methodology_Type: Lab
Methodology_Description:
Mosquitoes were identified to genus using morphological keys (Aquatic Biology Associates Inc, Corvallis, OR, USA). Further identification to species level was not possible due to discoloration from the ethanol used to preserve specimens. Identified mosquitoes were sorted by genus, sampling location, and sampling period prior to dissection. Samples were combined into pools of 1-25 individuals of the same genus and sampling period. Sterile techniques were used to separate mosquito abdomen and head/thorax segments.
Methodology:
Methodology_Type: Lab
Methodology_Description:
Genomic DNA was extracted from both abdomen and head/thorax pools of each sample using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA) and a modified method described by Plichart et al. (2006). All DNA extractions were subjected to a PCR positive control which amplified a region of the cytochrome oxidase I gene (COI) prior to Plasmodium screening. All extractions that proved positive via COI positive control were screened for Plasmodium infection in triplicate using nested-PCR methods (Hellgren et al. 2004). Amplicons were visualized on 1% agarose gels stained with Gel Red (Biotium, Hayward, CA, USA).
Methodology:
Methodology_Type: Lab
Methodology_Description:
Samples that produced amplicons when visualized on agarose gels were subsequently sequenced to verify the presence of Plasmodium DNA. Products were treated with Exo-SapIT (USB Inc., Cleveland, OH, U.S.A.) at a 3:7 dilution and sent to Functional Biosciences (MGE Innovation Center, Madison, WI, USA) for sequencing. A 479 bp fragment of Plasmodium mitochondrial DNA cytochrome b gene was bidirectionally sequenced using Big Dye Terminator v3.1 mix and analyzed on an ABI 3730xl automated DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.)
Methodology:
Methodology_Type: Lab
Methodology_Description:
A subset of thin blood smears from resident birds at our study sites were screened using light microscopy to identify Plasmodium morphospecies present in the area. Smears were fixed with absolute methanol, stained with 10% Giemsa, and examined under 1000X magnification. Plasmodium morphospecies observed were identified using taxonomic keys following Valkiūnas (2005).
Source_Information:
Source_Citation:
Citation_Information:
Originator: Plichart, C.
Originator: Sechan, Y.
Originator: Davies, N.
Originator: Legrand, A
Publication_Date: 2006
Title:
PCR and dissection as tools to monitor filarial infection of Aedes polynesiensis mosquitoes in French Polynesia
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Filaria Journal
Issue_Identification: 2
Publication_Information:
Publication_Place: online
Publisher: Biomedcentral
Other_Citation_Details:
Plichart, C., Y. Sechan, N. Davies, and A. M. Legrand. 2006. PCR and dissection as tools to monitor filarial infection of Aedes polynesiensis mosquitoes in French Polynesia. Filaria J. 5: 1–9. doi:10.1186/1475-2883-5-2
Online_Linkage: hhttps://doi.org/10.1186/1475-2883-5-2
Type_of_Source_Media: journal article
Source_Time_Period_of_Content:
Time_Period_Information:
Single_Date/Time:
Calendar_Date: 2006
Source_Currentness_Reference: publication date
Source_Citation_Abbreviation: Plichart et al. 2006
Source_Contribution: Reference for extracting genomic DNA from mosquitoes
Source_Information:
Source_Citation:
Citation_Information:
Originator: Hellgren, O.
Originator: Waldenstrom, J.
Originator: Bensch, S.
Publication_Date: 2004
Title:
A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Journal of Parasitology
Issue_Identification: 90
Publication_Information:
Publication_Place: online
Publisher: American Society of Parasitologists
Other_Citation_Details:
Hellgren, O., J. Waldenstrom, S. Bensch. 2004. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J. of Parasitology, 90(4):797-802 doi:10.1645/GE-184R1
Online_Linkage: https://doi.org/10.1645/GE-184R1
Type_of_Source_Media: journal article
Source_Time_Period_of_Content:
Time_Period_Information:
Single_Date/Time:
Calendar_Date: 2004
Source_Currentness_Reference: publication date
Source_Citation_Abbreviation: Hellgren et al. 2004
Source_Contribution:
Reference for detecting haemosporidian DNA in mosquito body segments
Source_Information:
Source_Citation:
Citation_Information:
Originator: Valkiūnas, G.
Publication_Date: 2005
Title: Avian Malaria Parasites and Other Haemosporidia
Geospatial_Data_Presentation_Form: book
Publication_Information:
Publication_Place: New York
Publisher: CRC Press
Other_Citation_Details:
Valkiūnas, G. 2005. Avian Malaria Parasites and Other Haemosporidia. CRC Press, NY. 947 pp.
Type_of_Source_Media: book
Source_Time_Period_of_Content:
Time_Period_Information:
Single_Date/Time:
Calendar_Date: 2005
Source_Currentness_Reference: publication date
Source_Citation_Abbreviation: Valkiūnas 2005
Source_Contribution: Reference for identifying haemosporidia
Source_Information:
Source_Citation:
Citation_Information:
Originator: National Center for Biotechnology Information (NCBI) GenBank
Publication_Date: Unknown
Title: Reference database of known Hematozoa DNA sequences, NCBI
Geospatial_Data_Presentation_Form: application/service
Online_Linkage:
Type_of_Source_Media: digital database file
Source_Time_Period_of_Content:
Time_Period_Information:
Single_Date/Time:
Calendar_Date: 2019
Source_Currentness_Reference: observed
Source_Citation_Abbreviation: NCBI nucleotide BLAST
Source_Contribution:
DNA sequences obtained in the current study were queried against all known hematozoa sequences in National Center for Biotechnology Information (NCBI) GenBank using the Basic Local Alignment Search Tool (BLAST) and determined to be positive if there was >90% consensus with one or more entries in GenBank
Process_Step:
Process_Description: Field notes were transcribed into electronic spreadsheets.
Process_Date: Unknown
Process_Step:
Process_Description:
Sequencher version 5.1 (Gene Codes Corp., Ann Arbor, MI) was used to examine and align all sequence data. International Union of Pure and Applied Chemistry (IUPAC) ambiguity codes were assigned to base pairs with uncertain or multiple nucleic acid identity.
Process_Date: Unknown
Process_Step:
Process_Description:
Genetic sequences were assigned to genera (Plasmodium) using the nucleotide BLAST (Basic Local Alignment Search Tool) function available through the National Center for Biotechnology Information (NCBI). Assignment was based on the top NCBI BLAST result with a minimum identity score of at least 90%.
Process_Date: Unknown
Process_Step:
Process_Description:
Blood parasite infections were confirmed when sequencing resulted in a bi-directionally aligned consensus sequence of target mtDNA that was successfully assigned to genus using the nucleotide BLAST (Basic Local Alignment Search Tool) function. Sequences that were not bi-directionally aligned and sequences with <90% identity were considered negative to reduce the possible occurrence of false positives.
Process_Date: Unknown