Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Parameters
• Instruments
• Project Information
• Funding

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.

DNA Extraction, PCR, and Sequencing

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 uL 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.

The consensus sequences were trimmed and assembled from the forward and reverse sequences using the Geneious assembler in Geneious Prime® ver 2023.2.1, and visually examined for accuracy and polymorphisms. All analyses were conducted from multiple sequence alignments created using the Clustal Omega 1.2.2 alignment in Geneious Prime® ver 2023.2.1. Alignments were visually examined for accuracy and excess ends were manually trimmed.

NSF Award Abstract:

Nontechnical explanation of the project's broader significance and importance

As Earth's ecosystems experience rapid biodiversity change, disease ecologists have turned to an urgent question: how might reductions in biodiversity affect the transmission of parasites? In other words, does biodiversity loss increase the abundance of parasites by eroding natural checks and balances on transmission? Alternatively, does it decrease parasite abundance by removing the free-living biodiversity on which parasites depend? This study will constitute the first comprehensive test of these questions in any ecosystem. It will evaluate the relationship between fish biodiversity and parasite abundance across 18 replicate coral reef ecosystems. Not only will the work explore whether reductions in fish biodiversity are associated with increases or decreases in parasite burdens, it will also assess whether parasite and host traits or geographical distance influence the direction and strength of this relationship. The theories that are tested are among the most important and controversial in the rapidly growing field of disease ecology and our work represents a novel, creative approach to a long standing, but unresolved research question. The work will yield transformative insights into the nature of parasite transmission in a changing world. Furthermore, the project will intimately intermingle education with research by launching the Research Internship in Molecular Ecology at California State Monterey Bay, which will place a group of underrepresented undergraduates in a central research role, and by developing and disseminating quality educational tools for teaching about parasite biodiversity through collaboration with the Network of Conservation Educators and Practitioners at the American Museum of Natural History. Parasites are often hidden and can be easy to overlook, but they are ecologically important and affect every population of marine animals.

Technical description of the project

The field of disease ecology is plagued by uncertainty and disagreement over whether biodiversity loss exacerbates parasite transmission, because it lacks the comprehensive, multi-host, multi-parasite, broad-spatial-scale dataset needed to formulate a convincing empirical test.

This project will answer this recalcitrant question, using a dataset of unprecedented replication and taxonomic and spatial resolution, by exploiting the advantages of a marine model system. The project is centered on a natural experiment in which the abundance of parasites across a highly resolved gradient of host biodiversity, for more than 77 parasite species and 18 replicate coral reef ecosystems will be quantified. Dataset will critically test hypotheses for the biodiversity-parasite abundance relationship, revealing how the direction, shape, and scale-dependence of this relationship vary across a diverse array of parasite taxa, and resolving questions of burning interest in the disease ecology literature - and of vital importance to marine conservation. This project will address the following questions: (Q1) For each parasite species detected, what is the direction and shape of the relationship between biodiversity and parasite abundance? (Q2) What factors (e.g., parasite traits like transmission strategy and host specificity, host traits like body size) determine the direction and shape of the relationship between biodiversity and parasite abundance? (Q3) How does spatial scale interact with parasite dispersal capacity to moderate the effects of biodiversity on parasite abundance? The work will integrate an existing dataset on fish biodiversity and abundance of coral reef fish parasites from six equatorial Pacific islands (the Northern Line Islands) with new sampling from 12 additional islands (the Southern Line Islands and French Polynesia). The resulting dataset will reflect the burden of >77 metazoan parasite taxa for seven species of coral reef fishes across18 islands. The work will provide the world's first data on the direction, magnitude, and shape of the biodiversity-disease relationship across a diversity of parasite taxa, host taxa, and spatial scales, and will comprehensively identify conditions under which biodiversity is likely to be important in determining the abundance of parasites - a fundamental contribution to ecology and to biological oceanography. The project will intimately integrate education with research by placing a group of underrepresented minority undergraduates in a central research role: performing the molecular analyses required to estimate parasite dispersal distance. A summer Research Internship in Molecular Ecology will be established at California State University Monterey Bay, a Hispanic-Serving Institution. The project will also underwrite the development of a peer-reviewed learning module on parasite biodiversity, to be developed and disseminated in collaboration with the American Museum of Natural History, and will support the training of two graduate students, one postdoctoral scholar, and several undergraduates.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.