Corinna A Pinzari Mia Takai Stephanie Mladinich Dennis A LaPointe M Renee Bellinger 20250915 tabular digital data Understanding seasonal mosquito distribution, abundance, and factors that facilitate the spread of avian malaria in Haleakalā National Park. https://doi.org/10.5066/P13QHKMG These data consist of quantitative PCR results obtained from a targeted assay designed to detect the presence of Culex quinquefasciatus DNA shed into water. Under controlled laboratory conditions, water samples in three replicate container systems were collected during a laboratory experiment that spanned the larval developmental life stages of Culex quinquefasciatus from egg rafts through adult emergence, with sampling also encompassing the pre-egg raft experimental placement timepoint and beyond post-adult emergence (n=40 samples). The data also include negative control water samples taken alongside experiment samples (n=18) and blank samples taken as controls during the DNA extraction process (n=2). These data were collected to test the potential influence of Culex quinquefasciatus larval developmental stages on the detection of Culex eDNA shed in water sources. The laboratory experiment was conducted by drawing pre-experiment sample blanks from each of three experimental water containers, then placing a single egg raft in each water container and sampling the water throughout the developmental life cycle of Culex, continuing water sample collection for up to six weeks post-adult emergence from the containers. The experiment spanned a total of approximately 92 days. Water samples were processed by extracting environmental DNA from each water sample filter followed by evaluating the environmental DNA signal of Culex using a targeted Culex assay and quantitative PCR. 20240121 20250305 ground condition Complete None planned Hawaii Volcanoes National Park -155.250614 -155.246429 19.421187 19.418334 ISO 19115 Topic Category biota environment health None mosquito larval development quantitative polymerase chain reaction mosquito monitoring USGS Thesaurus water sampling field inventory and monitoring environmental DNA USGS Metadata Identifier USGS:6881d3acd4be0232bdf2ae29 Common geographic areas Hawaii Hawaii Volcanoes National Park None Culex quinquefasciatus Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Ecdysozoa Phylum Arthropoda Subphylum Hexapoda Class Insecta Subclass Pterygota Infraclass Neoptera Superorder Holometabola Order Diptera Suborder Nematocera Infraorder Culicomorpha Family Culicidae Subfamily Culicinae Tribe Culicini Genus Culex Subgenus Culex (Culex) Species Culex quinquefasciatus TSN: 126490 None. Please see 'Distribution Info' for details. None. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. M. Renee Bellinger Pacific Island Ecosystems Research Center Research Geneticist mailing

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Hawaii National Park Hawaii 96718 United States 808-300-9713 mbellinger@usgs.gov Funding support provided by the USGS through the Natural Resources Protection Program and the Biological Threats and Invasive Species Program. Additional funding was provided by the Bipartisan Infrastructure Law (BIL). Additional administrative support from USGS Pacific Island Ecosystems Research Center and the Hawai'i Cooperative Studies Unit - University of Hawai'i at Hilo. Microsoft Windows 10 Enterprise, Version 22H2, OS Build 19045.3803: Microsoft Office 2016, Excel Entered data was checked against laboratory generated data twice, first by USGS lab manager and then by USGS project leader, before being used for analysis. Extraction blanks were utilized during DNA extraction protocols. Positive and negatives controls were performed during quantitative PCR, in addition to dilution standards. The data are logically consistent. No records appear outside of the norm. All categorical values are standard codes and all numerical values fall within expected limits. Dataset is complete. Three (A,B,C) experimental containers (volume equivalent to 6 US quarts) were each filled with 1 liter of tap water and a lid was placed on each tray to limit evaporation. A pre-experiment water sample was taken from each container prior to the addition of a single egg raft. The Culex quinquefasciatus egg rafts were collected from an invasive, wild population of Culex that breeds in the Hawai'i Volcanoes National Park, Hawaii using ovicups (n = 3) baited with "stinky water" (USGS-PIERC protocol). The total number of eggs per egg raft, manually counted using a digital microscope (USGS-PIERC protocol), averaged 150.1 (SD = 4.9). One egg raft was placed in each experimental container tray (Experiment Day 1). Between Day 3 (egg raft hatch day) and Day 45-55 (adult post-emergence) of the study period the containers were sampled every two to seven days, then sampled every week or second week until the water in the container evaporated, at approximately day 80-92. When water samples were filtered for environmental DNA collection, the life stage of larval development was observed and recorded for each container. Lab-reared mosquitoes were raised at approximately 28˚C, 70-80% relative humidity, a 12:12 hour light:dark cycle, and maintained on a powdered yeast supplement (50:50 by volume mix of yeast hydrolysate and albumin) fed to larvae once every three days. Each water sample consisted of collecting and filtering 500 mL of water from each container using the following equipment and methods: Water was passed through a Whatman glass microfiber filter circle 0.7 microns, 25 mm diameter, seated inside a plastic filter holder, that was attached to a luer-lock style 50 mL syringe. Components of the water filter sampling kits consisted of 1-50 mL BD Luer-Lok Syringe (#309653), Sartorius Stedim Biotech re-usable syringe filter holder (#16517 E) containing a GE Healthcare Life Sciences Whatman 25 mm diameter 0.7-micron pore size glass microfiber filter (#1825-025), sterile plastic forceps, and a sterile 2 mL conical vial with tight locking lid filled with 1 mL 100% molecular grade ethanol for filter and eDNA preservation). Between lab experiment sampling, the re-useable syringe filter holders were disassembled, thoroughly decontaminated by overnight soaking in 50% bleach:50% tap water, washed and scrubbed with Alconox laboratory-grade, residue-free soap, rinsed with deionized water, soaked overnight in 10% bleach, and subjected to a final deionized water rinse. Whatman glass microfiber filter and 50 mL syringe technique: We used a modified version of the protocol in from National Park Service (NPS) CALeDNA (https://static1.squarespace.com/static/57c7ad5bcd0f68f5c730e9e8/t/6257024f402a14216dcd4974/1649869391804/CALeDNA+Water+Sampling+Protocol.pdf) and Mladinich 2023. Water was filtered using a 50 mL luer-lock syringe, with the glass microfiber filter (25mm diameter circle, 0.7 microns) housed inside the reuseable filter holder. The 50 ml syringe was removed from the packaging and 50 ml of sampling source water was drawn up from the container to rinse and prime the syringe. This water was then disposed back in the container it was drawn up from. A new volume of 50 mL of water was then drawn up and the filter in housing was screwed onto the end of the syringe. The water sample was then flushed through the syringe for collection of eDNA on the filter contained in the filter housing. The filter housing was then removed, and the previous step was repeated until up to 500 mL of water was obtained from the container or the filtering became overly difficult. Any remaining water in the syringe was discarded back into the container and 50 mL of air was drawn in. The filter in housing was screwed back onto the syringe, the air pushed through to remove any excess water. Following filtration, the filter was removed from the housing using sterile disposable forceps, folded in half twice, and placed in a 2 mL vial with 1.5 ml of 100% (200 proof) molecular grade ethanol as the DNA preservative. References: National Park Service & CALeDNA: (https://static1.squarespace.com/static/57c7ad5bcd0f68f5c730e9e8/t/6257024f402a14216dcd4974/1649869391804/CALeDNA+Water+Sampling+Protocol.pdf) USGS-PIERC protocol: Culex quinquefasciatus Oviposition Sampling Egg Fecundity and Viability Version 3. Written by Dennis LaPointe. Modified August 2023 by Dennis LaPointe and Lauren Smith. Revised by Stephanie Mladinich and Katherine McClure November 2023. Additions by Seth Judge and Dennis LaPointe December 2023. Mladinich, Stephanie. "Pockets and Pathways to Invasion: Developing Improved Mosquito Monitoring in High Elevation Forests on Hawaiʻi Island." Master's thesis, University of Hawai'i at Hilo, 2023. 20240131 Corinna Pinzari PIERC Biologist mailing

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Hawaii National Park Hawaii 96718 United States 808-582-0468 cpinzari@usgs.gov DNA extraction from water samples: All extractions were performed with filter tips in a negative flow air clean HEPA filtered hood in area of lab dedicated to eDNA extractions, where standard DNA extractions are not performed. UV light was used to sterilize tubes and dedicated pipettes prior to any uses. Areas were cleaned with bleach before and after extractions. During extraction steps where filters were being manipulated, we sterilized forceps between each sample by decontaminating in a 50% bleach and water solution for at least a minute, then rinsing with deionized uv-treated water, dipping in ethanol and flaming until dry. We included one extraction blank as a negative control per ~20 extractions. The DNA extracts were maintained at -20˚C until further analysis. Whatman glass microfiber filter samples: We followed a modified version of the protocol from Minamoto et al. 2019 using a Qiagen DNEasy Blood and Tissue Kit.  After sample collection, filters were removed from ethanol storage using sterile forceps and scissors, cut in half, and one filter half was placed in a 1.5 mL vial (Eppendorf DNA LoBind Microcentrifuge Tubes) for drying at ambient temperature for 30 minutes. Each 1.5 mL vial was covered with a KimWipe (Kimberly-Clark Professional) and placed under a fume hood during the drying step. Once the filter was dried, 400 uL of AL buffer and 40 uL of Proteinase K from the Qiagen DNeasy kit were pipetted directly onto the filter, followed by incubation on a heat block incubator/rocker for 1 hour at 55˚C. After heating, 220 uL of TE buffer was added to the solution, left to incubate for 1 minute and centrifuged for 3 minutes at 3000 g. Remaining liquid was squeezed from the filter using forceps and the filter was discarded. Next, 400 uL of 200 proof molecular grade ethanol was added to the solution and mixed using a pipette. The solution (in 600 uL volumes) was transferred to a DNeasy extraction column and centrifuged for 1 minute at 6000 g, replacing collection tubes and centrifuging repeatedly until the entire volume of liquid was processed. After washing the column with AW1 and AW2 washing buffers, the column was transferred to a new 1.5 mL vial where the DNA was eluted with 50-100 uL of AE buffer, incubated at room temperature for 2 mins, and centrifuged for 1 min at 6000 g. Minamoto, T. 2019. DNA extraction from glass fiber filters. Pages 43–51 in Environmental DNA sampling and experimental manual version 2.1. Ed. by eDNA Methods Standardization Committee, The eDNA Society, Otsu, Japan. 20240417 Corinna Pinzari PIERC Biologist mailing

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Hawaii National Park Hawaii 96718 United States 808-582-0468 cpinzari@usgs.gov Quantitative PCR methods: All qPCR assay tests were prepared under a laminar flow UV AirClean hood with a HEPA filter in the clean laboratory for set-up and plate loading. Filter tips were used for each step, and UV light was used to sterilize tubes and dedicated eDNA pipettes prior to use. Bench areas were cleaned with bleach before and after qPCR steps. Samples were analyzed in real time qPCR reactions using the Culex quinquefasciatus assay design designed and optimized as described in Mladinich 2023. The assay consists of a forward Primer Cq64F: 5’ - CGA GCA GAA TTA AGT CAA CCA GG - 3’, reverse primer Cq326R; 5’-CTGTTCCAGATGAAAGAGGGG-3’, and TaqMan hydrolysis probe Cq292P; 5’-56-FAM/TTC AAG TAG/ ZEN/TTT AGT AGA AAA TGG AGC TGG GAC TGG/3IABkFQ/-3’), utilizing FAM as the fluorescent dye. Additionally, we used Quantabio 2X Perfecta qPCR Tough Mix UNG (#95138-012) and Thermofisher TaqMan Exogenous Internal Positive Control Reagents Kit (IPC) (#4308323) in the assay to assess and overcome challenges in target detection caused by potential PCR inhibitors in environmental water samples. The final qPCR reaction used for Culex quinquefasciatus environmental DNA detection was as follows: 20 uL reactions consisted of 10 uL 2x Perfecta qPCR Tough Mix UNG (final concentration 1X), 1 uL forward primer (10uM) (final concentration 0.5 uM), 1 uL reverse primer (10uM) (final concentration 0.5 uM), 0.5 uL Cq292 FAM probe (final concentration 0.25 uM), 0.1 uL 50X IPC DNA, 0.5 uL 10X IPC Master Mix (includes IPC probe and primers), 3.9 uL of molecular grade water, and 5 uL of eDNA sample/positive control DNA/IPC block/NTC. All eDNA samples were run in 6 replicates with 6 replicates containing the internal postive control. Three replicates with IPC blocking reagent were used as a test of the IPC. No template controls were used on each plate in replicates of 3 as negative controls to detect potential contamination. A positive control serial dilution series (1:1, 1:1,000, 1:10,000, and 1:1,000,000) was included as a reference standard on all qPCR plates using gDNA from C. quinquefasciatus (starting DNA concentation of 4 ng/uL) also using 3 replicates. Negative control water samples and extraction blank negative controls were included in the qPCR assay. Amplification of eDNA samples were performed on a CFX96 Real-Time PCR Detection System (BioRad). The thermocycling protocol consisted of 5 mins at 95˚C followed by 50 cycles of 10 seconds at 95˚C and 30 seconds at 53-60˚C. A cycle threshold (Ct) value is a data point in quantitative polymerase chain reaction (qPCR) that indicates the number of amplification cycles required to detect a target nucleic acid in a sample. Ct values for Culex quinquefasciatus and internal positive control amplification cycles were analyzed using BioRad CFX Manager software (version 3.1) and reported for each replicate sample as well as the mean across replicates, calculating means for positive detections only. Reference: Mladinich, Stephanie. "Pockets and Pathways to Invasion: Developing Improved Mosquito Monitoring in High Elevation Forests on Hawaiʻi Island." Master's thesis, University of Hawai'i at Hilo, 2023. 20241111 Corinna Pinzari PIERC Biologist mailing

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Hawaii National Park Hawaii 96718 United States 808-582-0468 cpinzari@usgs.gov 1_Culex Lab Experiment qPCR Results 2024 2025.csv Comma Separated Value (CSV) file containing data. Producer Defined Lab_Sample_Number Unique identification number assigned to a lab water filter sample during the extraction process. Producer Defined Unique identification number assigned to a lab water filter sample during the extraction process. Life_Stage_Observed The developmental stage of the mosquito larvae were observed during the lab water sampling event. Producer Defined pre_experiment water sample taken and filtered before a mosquito egg raft was added to the container. Producer defined egg_raft_and_hatched water sample taken and filtered when a mosquito egg raft and hatched larvae were present in the container. Producer defined negative_control water sample taken and filtered simultaneously with container derived water samples, negative control water samples consisted of UV treated deionized lab water. Producer defined first_instar water sample taken and filtered when first instar mosquito larvae were present in the container. Producer defined extraction_blank Sample was an extraction blank prepared during the DNA extraction process. Producer defined second_instar water sample taken and filtered when second instar mosquito larvae were present in the container. Producer defined later_instars_and_adult mosquitoes water sample taken and filtered when third and fourth instar mosquito larvae were present in the container. Producer defined first_and second_instars water sample taken and filtered when both first and second instar mosquito larvae were present in the container. Producer defined pupae_and_adult_mosquitoes water sample taken and filtered when both puape and adult mosquitos were present in the container. Producer defined post_emergence water sample taken and filtered after all pupae had emerged as adults in the water container. Producer defined Water_Sampling_Date Date a water sample was collected from the lab experiment and filtered. Producer Defined NAN No value recorded for DNA extraction blank control samples. Producer defined 1/31/2024 5/01/2024 Experiment_Day_Number Day of the experimental study period a water sample was taken and filtered. Producer Defined NAN No value recorded for DNA extraction blank control samples. Producer defined 1 92 day Container_Name Name assigned to the experimental container or negative control, in which water samples were derived from. Producer Defined NAN No value recorded for DNA extraction blank control samples. Producer defined A Container A Producer defined B Container B Producer defined C Container C Producer defined Neg No container, negative control water. Producer defined Extraction_Date Date that the lab water filter sample was processed for DNA extraction. Producer Defined 04/2/2024 5/10/2024 qPCR_Date Date that lab water filter sample was tested with the qPCR assay. Producer Defined 11/11/2024 3/3/2025 Number_Replicates_Attempted_Culex_Target Number of replicates attempted for the Culex DNA target during the qPCR assay test. Producer Defined 6 6 Number_Replicates_Detected_Culex_Target Number of replicates that showed amplification for the Culex DNA target during the qPCR assay test. Producer Defined 0 6 Culex_Ct_Value_Replicate_1 The cycle threshold value result in replicate 1 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No amplification of the Culex DNA target was observed in the replicate. Producer defined 23.37 39.45 cycles Culex_Ct_Value_Replicate_2 The cycle threshold value result in replicate 2 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No amplification of the Culex DNA target was observed in the replicate. Producer defined 23.4 38.63 cycles Culex_Ct_Value_Replicate_3 The cycle threshold value result in replicate 3 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No detected amplification of the Culex DNA target observed in the replicate. Producer defined 23.31 38.64 cycles Culex_Ct_Value_Replicate_4 The cycle threshold value result in replicate 4 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No detected amplification of the Culex DNA target observed in the replicate. Producer defined 0.0 38.68 cycles Culex_Ct_Value_Replicate_5 The cycle threshold value result in replicate 5 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No detected amplification of the Culex DNA target observed in the replicate. Producer defined 0.0 38.54 cycles Culex_Ct_Value_Replicate_6 The cycle threshold value result in replicate 6 if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No detected amplification of the Culex DNA target observed in the replicate. Producer defined 23.38 40.32 cycles Mean_Ct_Value_Culex_Target The mean cycle threshold value across all attempted replicates if the Culex DNA target was amplified during the qPCR assay. Producer Defined ND No detected amplification of the Culex DNA target observed in the replicate. Producer defined 14.96 40.32 cycles Number_Replicates_Attempted_IPC_Target Number of replicates attempted for the internal positive control DNA target during the qPCR assay test. Producer Defined 6 6 Number_Replicates_Detected_IPC_Target Number of replicates that showed amplification for the internal positive control DNA target during the qPCR assay test. Producer Defined 6 6 IPC_Ct_Value_Replicate_1 The cycle threshold value result in replicate 1 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 20.77 38.56 cycles IPC_Ct_Value_Replicate_2 The cycle threshold value result in replicate 2 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 27.94 38.29 cycles IPC_Ct_Value_Replicate_3 The cycle threshold value result in replicate 3 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 28.23 38.61 cycles IPC_Ct_Value_Replicate_4 The cycle threshold value result in replicate 4 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 29.03 38.68 cycles IPC_Ct_Value_Replicate_5 The cycle threshold value result in replicate 5 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 28.29 39.17 cycles IPC_Ct_Value_Replicate_6 The cycle threshold value result in replicate 6 if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 28.53 38.46 cycles Mean_Ct_Value_IPC_Target The mean cycle threshold value across all attempted replicates if the internal positive control DNA target was amplified during the qPCR assay. Producer Defined 28.31 38.48 cycles These data were collected to test the potential influence of Culex quinquefasciatus larval development on the detection of Culex eDNA shed in water sources. A laboratory experiment was conducted by placing egg rafts in containers of water, then collecting eDNA samples from the water for approximately 92 days, including the pre-and post post-adult emergence time-period. Water sample filters were processed to extract environmental DNA, and the genetic signal of Culex within the water filter sample was tested using quantitative PCR and a targeted Culex assay. None. GS ScienceBase U.S. Geological Survey mailing address

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Denver CO 80225 United States 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Digital Data https://doi.org/10.5066/P13QHKMG None 20260408 U.S. Geological Survey – PIERC Data Steward physical

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Hawaii National Park HI 96718 US 808-210-6141 pierc-datasteward@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999