Field collections and sample processing

Coral reef fish host species Acanthurus nigricans, Stegastes aureus, and Stegastes fasciolatus were caught at islands from Northern Line Islands (NLI) and French Polynesia (FP) during expeditions in 2010, 2019, 2020, and 2021. Sampling was conducted across three archipelagos of the central equatorial Pacific, encompassing 19 islands (Jarvis, Kingman, Kiritimati, Palmyra, Tabuaeran, and Teraina in the Northern Line Islands; Flint, Malden, Millennium, Starbuck, and Vostok in the Southern Line Islands; Huahine, Moorea, Raiatea, Rangiroa, Tahiti, Takapoto, Tetiaroa, and Tikehau in French Polynesia).

Acanthurus nigricans are a type of surgeonfish belonging to the Family Acanthuridae. Stegastes aureus and Stegastes fasciolatus are damselfish of Family Pomacentridae. Reef fish were collected from the forereef of each island from depths between 8m and 18m using three-pronged spears and hand nets. Fish were euthanized humanely according to UC San Diego IACUC protocol #S09392 and frozen for parasitological assessment.

Parasites were identified and counted by the Wood Lab at the University of Washington using standard dissection methods [see appendix E in (Wood et al. 2014)]. Parasites were identified to the lowest possible taxonomic level [see appendix F in (Wood et al. 2014)] and stored in 70% ethanol. All of the adult trematodes used in this study were sampled from host species A. nigricans, while the NLI larval samples came from S. aureus, and the FP larval samples came from S. fasciolatus.

Following morphological assessment conducted by the Wood Lab, it was determined that all of the adult trematodes sampled from the NLI were of the Family Microscaphidiidae (NLI adults), and all of the adult trematodes sampled from FP were of the Family Paramphistomatidae (FP adults). Parasite vouchers were sent to the Haupt Lab at California State University, Monterey Bay for further genetic analysis in 2022 (Barton, 2024).

DNA Extraction, PCR, and Sequencing

In most cases, DNA was extracted from all collected parasites. However, when more than 30 parasites were obtained from a single island, sequencing was generally limited to the first 30 individuals encountered in the vials. In some cases, additional samples were included before this threshold was recognized.

As detailed in Barton (2024): Each voucher was emptied into a petri dish containing 70% ethanol, one at a time. The petri dish was then examined under a dissecting microscope at the lowest magnification. A disposable glass pipette was used to move the parasite to a microcentrifuge tube containing autoclaved DI water in order to rinse the parasite of residual fish tissue. Individuals were not soaked for any longer than two hours as this could lead to degradation of the specimen. After 30 minutes to an hour, the parasite was then transferred into another microcentrifuge tube containing tissue lysis buffer and proteinase K and incubated at 56℃ for at least one hour. Larger individuals were incubated for 2 or more hours. Extractions were then conducted using either the QIAGEN QIAamp DNA Micro Kit (Cat. #51304) or the QIAGEN DNeasy Blood & Tissue Kit (Cat. #69504). The DNeasy kit was used for those parasite individuals that were larger and had more tissue present. DNA yield was estimated after extraction using a Nanodrop spectrophotometer. Due to the small amount of tissue present, DNA yields were often very small. Therefore, there were no restrictions on which samples moved forward to polymerase chain reaction (PCR).

A 743 bp segment of the mtDNA cytochrome oxidase 1 gene (CO1) was amplified using three primers designed by Brant and Loker (2009) for trematodes. PCR was performed in two consecutive 50 µL reactions. The first reaction was conducted using the DNA extraction and the forward and reverse primers. The PCR product of the first run was then used in the second reaction with the forward and internal primers. Both reactions were run on thermocyclers with the following reaction conditions: Initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 40 s, 72°C for 1 min, with a final step of 72°C for 10 min. Final PCR products were run on a 1.5% agarose gel using SYBR™ Safe DNA Gel Stain (Cat. #S33102) and the GeneRuler 1 kb DNA Ladder (Cat. #SM0314). The gels were run at 120 V for 30-45 minutes. However, we discovered that even if a band could not be seen, the sample was often still able to be sequenced. Therefore, we decided to send all amplified DNA samples off for sequencing. Amplified DNA was purified using the QIAGEN QIAquick PCR Purification Kit (Cat. #28104). Samples were sent for Sanger sequencing (MCLAB, San Francisco, CA) and were sequenced in the forward and internal directions.