Final Report for the Hawaii Invasive Species Council FY2021
Investigating microbial linkages within holdfast filaments of the invasive leather
mudweed, Avrainvillea lacerata, in coastal Oahu

Project Start Date 1/1/2021- Project End Date 12/31/2021

Prepared by Scott Van De Verg scottvan7@hawaii.edu and Dr. Celia Smith celia@hawaii.edu

Deliverable 1: Map with Avrainvillea lacerata and native species microbial
study design delimited

Figure 1: Six square plots of 10*10 m2 were delineated in collaboration with Mālama Maunalua on therelevant reef region (red circle) at Kuliʻouʻou, to test the efficacy of H2O2 concentration in various
assemblages unique as compared to the previous funded project at Paikō. Site selection was limited as wewere limited in the number of people that could participate due to COVID-19. We kept the field team to a
reduced number of students to maintain physical distancing of at least six ft apart.

Deliverable 2: # plots (100 m2 areas each) treated for Avrainvillea lacerata
The delineation of six plots was made in collaboration with Mālama Maunalua, who hadapproximate estimates of average percent cover of Avrainvillea plants over time throughout theKuliʻouʻou Reef region on the east side of Maunalua Bay. The site we chose to work in wasconsiderably more distant from shore than the previous HISC funded site in 2020 at Paikō, which
was about 300 meters from the shoreline at Kuliʻouʻou Beach Park. The substrate was similar tothe substrate at Paikō with a mix of rocky and sandy substrate, albeit more sand was found at theKuliʻouʻou site. Additionally, there was higher percentage of cover of benthic native algal taxa at
this site, which made it a good candidate for this year’s study as we could see if the treatments
could be used to help in restoration sites with abundant native flora.
Twenty plants in each plot (80 plants total) were chosen out of a total area of 400 m2.
Individual plants were chosen at random points along a transect within the plots for the purposesof the scientific experiment. 30mL of 3% and 10% H2O2 were administered to ten plants in eachplot to test the efficacy of the treatment on individual plants. The plants that were selected atrandom were first identified to species using the Hawaiian Reef Plants field guide and weremarked with a small and diminutive marking system (Huisman et al., 2007). A small PVC ring
was labeled and attached to the plants using a zip tie and a lead weight was added for stability.
The lead weights were attached to the zip tie with fishing line to prevent the marks from falling
off due to tidal influxes or increased wave action.
The treatments were considerably less effective in this site as compared to the Paikō reefregion, with no significant difference being found between the treatment and control. We think
there are many reasons why this might have happened in this site. For instance, there isconsiderably higher water flow in Kuliʻouʻou as the site is in close proximity to the channel forboat traffic. This water flow causes a greater dilution of the treatment over time than in lower
motion water flow, causing the time to reach reaction equilibrium to decrease. Additionally,
there may have been error involved in the specific Diving Pulse Amplitude ModulatedFluorometer unit we were using in 2021 as we sent our two other operational units that we usedin 2020 for factory repair, calibration, and pressurization testing for the housing to insure use atdepth.
Although there were errors in our measurements of photosynthesis, we observed
discoloration in the individuals that were treated with both concentrations of hydrogen peroxide,in congruence to our observations at Paikō. The surrounding macroscopic biota was observed to
be unaffected by the targeted treatments to Avrainvillea lacerata in this site. No distinct
mortality was observed as the individuals were highly embedded in the substrate and benthic
flora and as such were more cryptic and harder to make distinct observations on.

Deliverable 3: # plots (100 m2 areas each) with seagrass and native algaetransplants

Three plots were identified as having above typical abundances of seagrass blade density
and were southwest of the treatment plots. These plots were identified for ideal locations forseagrass propagule source and reciprocal transplantation. However, we were limited to theamount and expertise of volunteers in this year of experimentation due to COVID-19. As there
were limitations in our field team capabilities to undergo a proper transplantation effort, wedecided to take an alternative approach to assessing microbial inhibition in the field setting using
molecular tools. We submitted biological samples from our original field site for microbial
sequencing analysis at Mānoaʻs new Microbial Genomics and Analytical Lab (MGAL;
https://icemhh.pbrc.hawaii.edu/mgal), to better understand the internal microbial community.
This is important to study as there may be inhibitory effects from these microbes that may
prevent species turnover in the sediment where these invasive species grow.
Ten samples of Avrainvillea lacerata were collected from the west end of the Paikō reef
flat. Because we were interested in interactions with the substrate, we separated the holdfast(roots) from the blade and stipes (leaves) of the individuals for a paired complementary
microbial analysis. We hypothesized that due to substrate dynamics (i.e. influence by therhizosphere/phycosphere) that there would be different microbial assemblages between thebottom half and top half of the organism. The blades and stipes were separated as one unit as
they were both growing above the substrate in the water column, potentially allowing for adifferent assemblage to inhabit the interwoven, sponge-like thallus. Fungal isolation methods andmedia for standard marine samples were used for culture.
The summary of the microbial analysis was that there was a considerable difference (atleast three fungal species) that were found in the blades and stipes of the organism that were notfound in the holdfast. However, there were other fungal species in the holdfast that were not able
to be amplified or identified and may represent a new taxon. Seeing that this was the first attempt
at trying to describe the microbial community in this organism, and the likelihood of novel andundescribed species level identifications to be found, a more specialized approach may beneeded to better refine the differences the differences between the tissue types that wereanalyzed. A few of the genera that were able to be cultured and amplified in the blades and stipes
and not the holdfast of the organisms was Fusarium and Penicillium.

Deliverable 4: # of control plots (100 m2 areas each)
Two of the six plots (200 m2) were set aside for control measurements at Kuliʻouʻou andwere separated from treatment plots to test if there were plot wide effects from selected treatment
plots. The results from these control plots were comparable to previous results from controls thatwere measured at the Paikō reef region the year before. It was possible that the use of the Diving
Pulse with red pulsed light might have been measuring different tissues than compared to the one
with blue light used the year before. For instance, the red light could have had a higher affinity tomeasuring more of the internal photosynthetic symbionts such as cyanobacteria than does theone with blue light. However, our results show that the controlled organisms were comparable
regardless of the type of light source used.

Deliverable 5: Results and recommendations
Although the results from the photosynthesis measurements showed to insignificant, theresults from this project based on observations are informative in that there could be additional
treatments developed for higher water motion areas. From the work at Paikō we know thatoxidative damage can and will impact the growth and the alga if applied for long enoughduration and intensity before the reaction reaches equilibrium. If an oxidative agent using thehydrogen peroxide as an active ingredient could be developed into a substance that can be held inplace on the organism, then a treatment could be developed for areas with higher water and could
additionally help prevent the dissolution of the treatment into non target areas via water motion.
Further chemistry is required to better adhere the aqueous solution to targeted
phospholipid bilayers. The use of surfactants is common practice in the application of biocides tomany organisms. However, surfactants may not provide the stickiness that is required to create atargeted application in the aqueous environment of highly abundant reef systems. Althoughsurfactants may not be miscible in water, they do not prevent the movement and emulsification
within the water column. Further research is required in the development of a caustic paste orsemisolid that can be applied to targeted organisms in areas with consistent water flow and wave
motion to prevent the dissolution of treatment. Containment systems may be another option.
Additional research could also investigate ways to catalyze the reaction more efficiently.
In areas of the ocean with less sunlight available, catalyzers may be needed to allow the aqueous
solution of hydrogen peroxide to effectively break down to water and oxygen. Some of thesecatalysts may include inorganic constituents such as carbonates of sodium (soda ash or bakingsoda) or compounds including iron. Whether new chemical formulations are created, the careful
study, application of treatments, and monitoring of reef communities will be necessary incontinuing to control the widespread nature of this and other marine invasive algal species.

Deliverable 6: At least one peer-reviewed publication
A publication is being proposed for the algal-fungal associations that were discovered in
the process of this project with the development of more specialized approaches, however thejournal has yet to be determined for this work. Further work to substantiate this publication will
be continued to sample the sediment itself including both the surrounding sand and mud that thetarget organism is found to be growing in. These substrates and tissue types are to resampled tofurther substantiate the results from the microbial analysis and will be slated for publication if
the results are found to be impactful. From this work, there is potential to describe novel species
that have not yet been described to science.

Literature Cited
Huisman, J. M., I. A. Abbott, and C. M. Smith. 2007. Hawaiian reef plants. University of Hawaii
Sea Grant College Program.