Thermal shock treatment for controlling exotic Caulerpa brachypus

Thermal shock treatment for controlling exotic Caulerpa brachypus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thermal shock treatment for controlling exotic Caulerpa brachypus Michele Rogalin-Henderson, Jessica McLay, Andrew G. Jeffs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8713916/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A number of macroalgal species in the genus Caulerpa are characterised as some of the most invasive marine species in the world. There are a limited range of interventions for dealing with incursions of exotic Caulerpa species that have acceptable ecological impacts. This initial study assessed the potential to use heated seawater to kill fragments of C. brachypus , a recent invader of concern in Aotearoa - New Zealand. In the laboratory, Caulerpa fragments were exposed to heated seawater treatments of 30 to 80°C in 5°C increments for each of four experimental treatment durations of either 5, 10, 30 or 60 seconds and then cultured for one week in stable conditions. At 50°C or higher for 5 seconds or more, and at 45°C or higher for 30 seconds or longer, all fragments died, whereas below these treatment combinations most fragments remained alive, with 100% survival in the control with only a sham treatment handling. In the field, Caulerpa sod segments were submerged for 30 seconds in 40 to 70°C in 10°C increments and returned to the sea for three weeks. Segments of C. brachypus exposed to thermal shock treatments of 50, 60, and 70°C resulted in a significant loss of C. brachypus cover ( p < 0.05), but not complete elimination. These results indicate the potential to use thermal shock for controlling invasive C. brachypus that supports the management and protection of Aotearoa - New Zealand’s unique marine ecosystems. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction A number of species in the macroalgal genus Caulerpa are common as invasive exotic marine organisms, with ecologically impactful incursions in a number of locations throughout the world. The locations for these incursions include the Mediterranean Sea, the Americas, and Oceania (Anderson, 2005; Creese, et.al., 2004 ; Meinesz, et.al., 2001 ). As with many other marine invasive species, invasive Caulerpa species rapidly outcompete and replace native benthos, resulting in a loss of biodiversity and habitat for native fishes, sessile invertebrates, and in faunal species, such as shellfish (Parreira, et.al., 2021 ). For example, vegetative growth in invasive C. taxifolia is reported at up to 13 mm per day in the summer months (Creese, et.al., 2004 )d racemosa at up to 20 mm per day (Piazzi, et.al., 2001 ), enabling these species to form a dense carpet that smothers the benthos (Meinesz, et.al., 2001 ). Global efforts to control invasions of exotic Caulerpa spp. have mostly had limited success (Creese, et.al., 2004 ). Furthermore, most of the control methods have significant drawbacks, such as an adverse ecological impact on native species, excessive expense, difficulty scaling up, and increasing the likelihood of spread through increased fragmentation (Creese, et.al., 2004 ). Early discovery and prompt treatment appear to be key factors for the successful eradication of invasive Caulerpa spp. (Giakoumi, et.al., 2019 ). Once an incursion of an exotic Caulerpa spp. has become well established in an ecosystem it is nearly impossible to eradicate (Bax, et.al., 2003 ). A number of control methods use various chemicals to kill invasive Caulerpa spp. These include algaecides, salt/osmotic shock, chlorine, and other various chemicals (Creese, et.al., 2004 ). Of these, chlorine and salt treatment have been shown to be effective in two instances (Creese, et.al., 2004 ; Woodfield & Merkel, 2004 ). Chlorine, infused under a benthic mat, was successful in treating and eliminating an invasion of C. taxifolia in California (Woodfield & Merkel, 2004 ). Unfortunately, the ecological impact of using this method is significant, as it also eradicates associated native flora and fauna (Williams & Schroeder, 2004 ). Chemical treatments that have been tried, without success, in the United States and the Mediterranean Sea include copper sulphate, algaecides/herbicides, hydrogen peroxide, and acetic acid (Aquatic Biosecurity, 2009 ; Walters, 2009 ). These methods also adversely impact native species. Osmotic shock through the application of fresh water was unsuccessful for controlling C. taxifolia in NSW, Australia, and it was also found to be logistically challenging to access sufficient fresh water to achieve sufficiently low salinity to be effective (Aquatic Biosecurity, 2009 ). Salt treatment has been used effectively on C. taxifolia in an estuarine invasion in NSW (Creese, et.al., 2004 ). However, this treatment adversely impacted native seagrasses and is quite labour intensive. This may be an effective method applied in small or initial incursions (Aquatic Biosecurity, 2009 ; Glasby, 2013 ). Smothering methods of control, such as the use of benthic matting (e.g., tarpaulins or jute mats) or covering of the invasive Caulerpa spp. with sediment, have been applied in the Mediterranean, the United States, and Australia, but without successful eradication (Walters, 2009 ). Collateral damage on native fauna and flora is significant with smothering methods (Williams & Shroeder, 2004). Smothering is thought to be likely to be more effective for eliminating small patches of C. taxifolia in new areas provided the covering can remain in place for up to a month to be fully effective (Creese, et.al., 2004 ; Walters, 2009 ). The removal of exotic Caulerpa spp. through suction dredging of the seafloor has been used in Australia and the Mediterranean Sea (Walters, 2009 ). However, dredging is responsible for significant fragmentation of the Caulerpa spp., which increases the risk of spread, and has not been demonstrated to eradicate Caulerpa spp. successfully, making it generally a poor choice for control (Walters, 2009 ). There has been some success with suction dredging, with close monitoring and repeat treatments in one small incursion of C. racemosa var. cylindracea in the Adriatic Sea (Zuljevic & Antolic, 2002). Suction-assisted hand removal of Caulerpa spp. by divers appears to reduce the amount of fragmentation, but this method is labour intensive and relies on repeated application (Creese, et.al., 2004 ). However, this method is more ecologically friendly than chemical treatments, such as chlorine, and in one location in California this method was specifically selected for the invasive C. prolifera to avoid killing co-occurring endangered species (Merkel, 2021; Parreira, et.al., 2021 ). Invasive exotic C. brachypus was first discovered in Aotearoa - New Zealand in June of 2021 at Aotea - Great Barrier Island in the Hauraki Gulf near Auckland, along with an invasive exotic congener C . parvifolia (Scott, 2024 ). Since its initial discovery, C. brachypus has spread to numerous locations in the Hauraki Gulf, Coromandel Peninsula, and the Bay of Islands, and ongoing monitoring is continuing to identify new locations (Biosecurity NZ, 2024b). The potential adverse impact for the Hauraki Gulf and the Bay of Islands from this incursion indicates a need for effective methods for management to arrest the spread of C. brachypus and potentially eradicate it. Methods for controlling exotic Caulerpa species that are being applied in Aotearoa - New Zealand include UV light exposure, benthic matting with salt or chlorine, suction dredging, and hand removal by divers (Biosecurity, NZ, 2024a). The use of UV light was found to be effective for controlling both C. parvifolia and C. brachypus (Rogalin-Henderson, et.al., 2025 ; Thorburn, 2025 ; Zhuang, et.al., 2024 ). However, this method requires repeated treatments, and the regrowth rate and ecological impact is yet to be studied. Benthic matting, particularly combined with the application of chlorine beneath the matting, has been demonstrated to be effective at controlling the invasive Caulerpa species in New Zealand (Ammon et al., 2019 ; McRae, 2025 ). This method also kills all other species in the treated area, which potentially leaves the area prone to future invasion (Uya, et.al., 2018 ). Suction dredging has been applied in Omakiwi Cove in the Bay of Islands of Aotearoa - New Zealand to attempt to remove invasive C . brachypus (Davidson, et.al., 2024 ). While the use of suction dredging for removing large areas of biomass was found to be effective, it also generates fragments of the targeted macroalgae during the process and does not remove all of the targeted Caulerpa species (Davidson, et.al., 2024 ). Floating fragments released through any handling of most Caulerpa species are thought to have the potential to establish in new locations (Davis, et.al, 2009 ). Thermal shock treatment involves using a temperature shock to kill invasive aquatic organisms which has been demonstrated to be an effective means of killing some invasive pest seaweeds, and has also been used for the treatment of unwanted microalgae and seaweed in aquaculture situations and in ballast tanks (Forest & Blakemore, 2006; Quilez-Badia, et.al., 2008 ; Shannon, et.al., 2018 ; Wotton, et.al., 2004 ). For example, thermal shock treatment, in the form of heated seawater, was used to eradicate a potentially invasive seaweed, wakame, Undaria pinnatifida , from a sunken ship in the Chatham Islands, located offshore from mainland Aotearoa - New Zealand (Wotton, et.al., 2004 ). To deliver this treatment, plywood boxes with foam seals were placed over the seaweed on the hull of the vessel and heating elements inside the box increased the temperature of the enclosed seawater to 70°C for 10 minutes. The method was effective for eradicating the seaweed, with no subsequent regrowth detected. Thermal shock treatments were also used in an aquaculture setting to eradicate U. pinnatifida from seed mussels in New Zealand (Forrest & Blakemore, 2006 ). Complete mortality of the seaweed was achieved by immersing the seed mussels in 55°C seawater for 5 seconds, without causing an adverse impact on the mussels, suggesting that where the target macroalgae has a low threshold to thermal stress, it might be exploited as a thermal shock treatment whilst limiting damage to associated organisms which have greater thermal tolerance. Two seaweeds, Myriophyllum aquaticum and Crassula helmsii were killed using hot seawater at temperatures of 45 to 60°C with various exposure periods (Shannon, et.al., 2018 ). Complete mortality of both seaweeds was reliably achieved with immersion at temperatures of 60°C for 10 seconds exposure. Thermal shock treatment has also been used to treat ballast water aboard a commercial vessel, with a temperature of 55°C being found to eradicate both zooplankton and phytoplankton in the ballast tanks (Quilez-Badia, et.al., 2008 ). There is only one example of thermal shock being used to control invasive Caulerpa spp. in situ, where small rocks covered in C. webbiana were removed from the seafloor, treated with heated seawater at 68°C for 10 seconds and then put back on the seafloor (Cardigos, et. al., 2015 ). While the thermal shock treatment method successfully eradicated the seaweed from the rocks, the lack of technology to apply heat to the seaweed underwater on a larger scale prevented further practical application of thermal shock methods in this location. Thermal shock treatments have also been demonstrated to reliably kill C. taxifolia in the laboratory, where fragments were exposed to heated seawater at 72°C for either 1 or 2 hours and then placed in substrate and monitored for mortality (Williams & Shroeder, 2004). All but one of the 20 treated fragments died, with the remaining showing a small area of green (i.e., chlorophyll-a) in a buried portion of the fragment. This study is an outlier in the small body of research on thermal shock treatment of macroalgae, given the lengthy time of exposure to the heat treatment. Whether C. taxifolia is much hardier, or if burial of the fragments was relevant to the persistence observed in a small number of the treated fragments, is unclear. Collectively, these previous findings suggest that thermal shock treatment may be an effective method for the control of invasive Caulerpa species in Aotearoa - New Zealand. Therefore, the current study examines the efficacy of thermal shock treatments of exotic C. brachypus as a potential method of control of this invasive seaweed. Methods Laboratory experiment Samples of healthy C. brachypus were collected by divers on 22 July 2024 from Blind Bay at Aotea-Great Barrier Island (-36.262271°, 175.432261° WGS 84) in 5 m of water where the seaweed was growing as an extensive covering on sandy substrate. Samples of the sandy substrate together with the C. brachypus were dug out of the sediment with a hand trowel and placed in 8 ×15 cm plastic nursery trays and transported to the laboratory in seawater in biosecure containers. Upon arrival at the laboratory 232 individual C. brachypus fragments, each 30–40 mm in overall length and consisting of a stolon, rhizoids, and several thalli, were excised using a scalpel and forceps and each placed in a covered 100 mm diameter glass petri dish with no substrate and 50 ml of seawater. The seawater was obtained from the Waitematā Harbour and filtered through a 1 µm activated carbon filter and sterilized with ultraviolet light. Fragments of C. brachypus were exposed to heated seawater treatments of 30 to 80°C in 5°C increments, (i.e., 30, 35, 40°C etc) for each of four experimental treatment periods of either 5, 10, 30 or 60 seconds duration, i.e., a total of 44 temperature and duration combinations. Five replicate fragments of C. brachypus were subjected to each treatment combination. Seawater was heated in a 250 ml beaker on a hotplate with a magnetic stirrer and the temperature measured with a digital thermometer. At the selected treatment temperature, each C. brachypus fragment was each submerged in the heated seawater, with timer used to determine the duration of the treatments. At the end of the treatment duration, the fragment was immediately removed from the heated water and submerged in a 250 ml beaker filled with seawater at 18.5°C for 10 seconds. In addition to the temperature duration treatments for each of the four experimental treatment periods (i.e., 5, 10, 30, and 60 seconds) a control treatment was also undertaken with three randomly selected fragments that were submerged in the beaker with the 18.5°C water for each of the four treatment durations, then resubmerged in the same beaker for 10 seconds. Following the holding period in the 18.5°C water all of the fragments were placed in clean seawater in their individually labelled petri dish at 18.5°C. The labelled petri dishes were then placed in a plant culture cabinet set at a constant 18.5°C on a light:dark cycle of 12:12 hours to approximate mean annual conditions in the Hauraki Gulf. The fragments were visually assessed for viability 7 days after treatment, and each classified as alive or dead based on their tissue colour to assess chlorophyll-a content, with brown colouration indicating dead tissue and green colouration indicating living tissue with the presence of chlorophyll-a. To undertake an observer-independent assessment of the alive versus dead state of the experimental C. brachypus fragments, digital images of each fragment were taken before and after treatment using the 48-megapixel camera on an iPhone 15 at a 200 mm distance and illuminated by a 2.5 W LED lamp with a white background. Each digital image was analysed by sampling the colour at three random locations on each of the thalli, stolon, and rhizoid of the fragment to gather corresponding RGB (i.e., red-green-blue) data using PixSpy software ( www.pixspy.com ). The RGB data sets collected through PixSpy were imported to a spreadsheet for analysis. The mean of these sets of RGB data (from three locations on each of the thalli, stolon, and rhizoids), represented as numeric values for each colour component (i.e., R, G and B) was obtained by calculating the sum of the values for each component and dividing by the number of values, providing a mean colour for each component of the fragment, i.e., RGB = (∑r/n, ∑g/n, ∑b/n). For each mean the corresponding RGB colour translation was plotted in a grid for comparison and the data used for statistical comparison. Statistical Analyses The analysis of RGB data sampled from images of seaweed and plants has been demonstrated to be an effective method for estimating chlorophyll-a content (Su, et.al., 2008 ; Riccardi, et.al., 2014 ). When compared to SPAD, hyperspectral fluorescence, chlorophyll-a meter and chemical methods, with RGB colourimetric analysis being found to correlate significantly for chlorophyll-a measures (Dey, et.al., 2016 ; Zhang, et. al., 2022 ). As a loss of chlorophyll-a is associated with cell senescence and death, it provides a method of analysis for independently assessing the human visual assessment of viability for the experimentally treated fragments of C . brachypus (Hendry, et. al., 1987 ; Hortensteiner, 2004 ). Plotting the ratio of mean R/G readings against the mean B component from C . brachypus post-treatment showed a clear divide. To find an equation for the line that divided the alive and dead fragments with minimal misclassification, support vector machines (SVMs) were applied to the data set. Within the SVM method, five times as much weight was given to correctly classifying alive fragments as to dead ones to provide greater confidence for avoiding the misclassification of alive fragments as being dead. The method was constrained to estimate a straight line defined by an intercept and slope as the plot which indicated that this was appropriate, and this restriction results in a simple equation that can be used more easily in practice. The SVM analyses were implemented using the SVM function in the e1071 package in R version 4.4.2. A ten-fold cross-validation was used to get final intercept and slope values for the SVM line. In this process, the data is spilt into 10 random parts/folds. In the first iteration, the first fold of data is set aside as test data and the classification line was estimated (or ‘trained’) on the remaining 90%. The slope and intercept from this first fold were recorded and then the misclassification rates (one for dead fragments and one for alive fragments) was calculated using the test data. This was repeated another nine times, with a different fold of data set aside as the test data each time. The final intercept and slope for the line are the mean values over the ten iterations and the final reported misclassification rates are the mean misclassification rates from the ten iterations. Field experiment A field experiment using thermal shock treatment of C. brachypus growing in 1–2 m depth in Omakiwi Bay in the Bay of Islands (35°14.281’S, 174°14.652E) was undertaken in July 2025. Segments of randomly selected sod to a depth of around 5 cm in the sediment and containing a thick sward of C. brachypus were removed from the seafloor and placed into 30 × 50 cm weighted plastic boxes (Fig. 1 ). Three replicate boxes containing C. brachypus were exposed to each of the following heated seawater treatments of 70, 60, 50, or 40°C for 30 seconds, as well as a control using a sham treatment with unheated seawater. The treatment consisted of removing the ambient temperature seawater from the sample boxes then photographing with a GoPro 13 digital camera before rapidly filling each sample box with seawater preheated to the required temperature. The sample boxes were then quickly removed at the end of the exposure period and placed in ambient seawater at 16°C. For each replicate the seawater temperature was monitored using two digital temperature measurement probes, one placed at the surface among the thalli and one at the base of the substrate to measure the treatment temperature that was reached at the rhizoids that were buried at their lowest point in the sediment. After treatment, the boxes were returned to their original location on the seafloor and marked with a buoy so they could be relocated. All samples were retrieved 3 weeks later and photographed with a GoPro 13 digital camera. The digital images taken using a GoPro 13 digital camera were analysed by randomly selecting 100 points in each image using Coral Point Count software (CPCe) (Roberts, et.al., 2025; Kohler & Gill, 2006). At each point the cover was determined to have Caulerpa cover or sediment. The 100 points from each of the digital images were used to assess changes in the biomass of C. brachypus as measured by percent surface cover. Statistical Analysis Differences in the measured points of cover of C. brachypus among treatments was assessed with a chi-square contingency table by pooling all point cover counts for all replicates within each treatment. The cover of C. brachypus for before and after were compared among treatments in two separate contingency tables to establish any differences that may have been present at the outset of the experiment, as well as 3 weeks after their experimental treatment. Where a significant overall result was identified for the before or after comparisons, pairwise post-hoc binary chi-square tests were conducted to isolate the source of the difference among the individual treatments. A Bonferroni-Holm correction was used to control for error inflation due to multiple testing of the same data set. The before treatment data set was at the limit of the assumptions for low proportions for a chi-square tests, but given the large sample size (i.e., total of 1500 binary sampling points), the test can be considered to be sufficiently robust to this analysis (Curran, et. al., 1996 ). Results Laboratory Experiment All fragments of C. brachypus in the control treatment that were only subjected to handling all remained alive. In contrast, all fragments of C. brachypus treated with seawater at 50°C or higher temperatures for 5 seconds or more were visually assessed to have died as indicated by the appearance of the complete loss of chlorophyll-a from the tissues of the Caulerpa fragment (Fig. 2 ). Exposure of fragments to 45°C or higher temperatures for 30 seconds or longer caused all treated Caulerpa fragments to die. The C. brachypus fragments treated with heated seawater at 45°C for 5 and 10 seconds all survived, as did all fragments at lower temperatures and for all durations of exposure, i.e., 45°C and lower, and for exposure times of 5, 10, 30 or 60 seconds. A visual representation of the mean RGB values shows a distinct cut off at these described thresholds, wherein the samples show a loss of chlorophyll-a (i.e., green colouration), indicating a loss of viability (Fig. 3 ). Overall, the pattern of colouration presented in the figure, with dark green representing live C . brachypus tissue versus brown representing dead, is consistent with the alive and dead categorisation from the human visual assessment alone The variation in the SVM lines estimated at each iteration of the 10 cross-validations was small, with similar slopes and intercepts produced at each iteration. The final SVM classification line (Fig. 4 ) estimated that the misclassification rate for the alive fragments as dead by human visual assessment was 3.7% (Table 1 ). The 95% confidence interval for this rate indicates that the plausible range for the true misclassification rate of alive fragments could be up to 7.4%. In contrast, the misclassification rate for the dead fragments as alive by human visual assessment was 4.4%, with a 95% confidence interval of 1.1–6.9% (Table 1 ). A closer examination of the individual misclassifications found that the alive fragments based on colour that were categorized as dead by human visual assessment were all in treatments of 45°C and lower, almost entirely for short exposures (i.e., 10 seconds or less), and were mostly rhizoids (57%), compared to stolons and thalli, 38% and 5%, respectively. The individual misclassifications of dead fragments based on colour that were categorized as alive by human visual assessment were all in treatments of 45°C and higher, and were for a mix of exposure periods, and were mostly stolons (60%), compared to rhizoids and thalli, 26% and 13%, respectively. Table 1 Estimate of misclassification rates and standard errors (SE) based on 10-fold cross-validation of SVM classification line between alive and dead fragments of Caulerpa brachypus 7 days after various thermal shock treatments. SE estimated by the standard deviations of the 10 misclassification rates and CI estimated for the 2.5 and 97.5 percentiles from the 10 misclassification rates in the cross-validation. Classification line Equation of line 590–474*(R/G) - B Misclassification rate for alive fragments Estimate of rate ± SE 3.7% ± 2.1% 95% CI (1.3%, 7.4%) Misclassification rate for dead fragments Estimate of rate ± SE 4.4% ± 1.2% 95% CI (1.1%, 6.9%) Field Experiment The temperature readings from the thermometers placed among the thalli of the C. brachypus whilst it was exposed to the various thermal shock treatments showed a steady increase in temperature toward the assigned treatment in temperature over the 30 second treatment period, except for the control treatment which remained at ambient seawater temperature, which was 16°C (Table 2 ). None of the thermal treatments reached their assigned treatment temperature within the thalli of C. brachypus , except for the 40°C treatment. The temperature readings from the substrate showed minimal increase in temperature over the 30 seconds of hot water treatment, with only an increase of a few degrees recorded in the 40°C and 50°C treatments (Table 2 ). Consequently, none of the substrates measured in the thermal shock treatments reached their assigned temperatures within the 30 second treatment period. Table 2 The highest temperature readings taken from among the thalli and from 3 cm below the surface of the substrate in plastic boxes containing sods of Caulerpa brachypus during exposure to various thermal shock treatments consisting of applying ambient seawater (control) or heated seawater for 30 seconds. Temperature Treatment Thalli Substrate - Rhizoid 70°C 62.1°C 16.9°C 60°C 51.1°C 15.9°C 50°C 43.1°C 20.2°C 40°C 40.1°C 19.6°C Control 16.0°C 16.0°C Of the 15 sample boxes treated and placed back into the substrate from where they were collected, three were displaced and the remainder were heavily silted as was the surrounding C. brachypus meadow. This was likely due to tsunami activity along the eastern coast of Aotearoa – New Zealand from 30 July to 1 August 2025, which created abrupt tidal surges including in shallow areas of the Bay of Islands (TCDC, 2025). The mean percentage cover of C . brachypus in the boxes was close to 100% in each of the five thermal shock treatments immediately before their exposure to their corresponding heat treatments (Fig. 5 ). However, there were small differences in the cover of C . brachypus among the treatments at the outset of the experiment (χ 2 = 11.50, p = 0.021). The cover in both the 40°C and 50°C treatments (i.e., both 97.0% cover) were less than for the control which was 100% cover ( p = 0.05). There were no other differences in the cover of C . brachypus among the treatments at the outset of the experiment. Three weeks after the thermal shock treatments there were marked differences in the cover of C . brachypus among the treatments (χ 2 = 627.03, p < 0.0001) (Fig. 5 ). All pairwise comparisons of treatments were different ( p = 0.05). There was no difference in the cover of C . brachypus in the control before and 3 weeks after treatment ( p = 0.08), remaining close to 100% cover. However, the treatment exposed to 70°C showed the largest reduction in C . brachypus cover, with a mean loss of 89.7% of cover (Fig. 5 ). The 40°C exposure resulted in the smallest reduction in cover, with a mean loss of 9.3% of cover. The 50°C and 60°C treatments resulted in a mean loss of cover of 72.0% and 56.2%, respectively, i.e., the loss from the 50°C treatment was higher than for the 60°C treatment. Discussion Both the laboratory and field results from this current study show the potential effectiveness of thermal shock treatments for killing C. brachypus . Immersion of C. brachypus fragments in seawater at 50°C and higher temperatures for 5 seconds, or 45°C and higher temperatures for 30 seconds and longer caused all fragments to die in the current laboratory experiment as assessed by a human observer. Independent assessment of direct measures of differences in colour for alive versus dead C. brachypus in the laboratory experiment largely confirmed the human visual assessment, but with a mean misclassification rate for live fragments visually assessed as dead estimated to be 3.7% (± 2.1% S.E.), which may somewhat exaggerate the effectiveness of thermal shock treatments on C. brachypus in the treatments of 45°C and higher. The higher stolon component of misclassification may be due to its generally darker initial colouration compared to rhizoids and thalli, which suggests that a generalised alive versus dead colour scale for all morphological components may lead to misclassifications for individual components. A human visual observer is likely to unconsciously compensate for this by comparing differences in the coloration of the individual components of the macroalgae. The mean misclassification rate for dead fragments visually assessed as alive, which was estimated to be 4.4% (± 1.2% S.E.), which may suggest that the effectiveness of thermal shock treatments at lower temperatures may be somewhat higher than reported, as these misclassifications occurred in the 45°C and lower treatment sets, in the shorter durations (i.e. 5 and 10 second sets). In these observations it was the rhizoid component most often misclassified. Rhizoids are most often buried in the substrate and their chlorophyll-a content may be typically lower that the emergent parts of the macroalgae making it harder to reliably determine its alive or dead status. The results of the field experiment show the potential effectiveness of thermal shock treatments for controlling C. brachypus in situ. Immersion of sods of C. brachypus in seawater at temperatures of 50, 60, and 70°C for 30 seconds all caused a marked reduction in cover when compared to the 40°C and Control treatments ( p < 0.05). These results suggest a similar sensitivity to elevated temperatures as determined in the laboratory experiment, i.e., a threshold of 45°C for 30 seconds. The percentage cover of C. brachypus at 3 weeks after treatment tended to decrease with increasing treatment temperature, almost reaching complete elimination in the 70°C treatment, i.e., mean 9.0% ± SE percentage cover remaining. While the 50°C treatment had less remaining C, brachypus cover than the 60°C treatment group (i.e., 25.0% versus 42.5%), the declines in cover otherwise corresponded with the increasing treatment temperature. Unlike the laboratory experiments, using isolated individual small fragments of C. brachypus , none of the treatment replicates showed a complete elimination of the macroalgae from the treated replicate sods of C. brachypus indicating that the macroalgae could have remained viable. Temperature readings from among the thalli sward of C. brachypus and from within the substrate where the rhizoids are buried indicated that during the 30 second thermal shock treatment period the increase in these temperatures was relatively slow and did not reach the intended treatment temperature, especially in the substrate. This was most likely due to the thermal mass of the C. brachypus biomass and substrate, both requiring longer exposure for the transfer of the additional heat energy to meet the temperature required to kill the macroalgal tissue. This was also the most likely cause for the persistence of the C . brachypus following even the highest temperature treatment of 70°C. Hence, attaining threshold temperatures during the treatment period appears to be critical for ensuring all parts of the C . brachypus are killed, and will most likely require longer treatment periods to ensure the required temperatures are achieved. Overall, the sensitivity of C . brachypus to thermal shock treatment was similar to the results reported from a similar field experiment using C. webbiana wherein all samples were killed when exposed to 68°C for 10 seconds (Cardigos, et. al., 2015 ). In contrast, C. taxifolia , appears more tolerant of thermal shock treatment with a laboratory experiment demonstrating that fragments exposed to hot seawater at 72°C for 60 minutes were killed, with the exception of a small spot of a potentially viable green tissue remaining on one of the fragments that was buried (Williams & Shroeder, 2004). Thermal shock treatment on other macroalgal species indicate similar temperature thresholds to the current study. Two studies on U . pinnatifida , one in situ and the other in an aquaculture setting, achieved successful eradication. The first, in situ, used seawater heated to 70°C for 10 minutes, while the aquaculture study used seawater heated to 55°C for a 5 second exposure duration (Forrest & Blakemore, 2006 ; Wotton, et.al., 2004 ). Furthermore, another similar study found successful eradication of M. aquaticum and C. helmsii at 60°C for 10 seconds (Shannon, et.al., 2018 ). The results of the field experiment, while promising, are preliminary. Replicating the experiment may offer more definitive results by addressing the limitations of the current experiment. For the laboratory study, holding the samples for several weeks beyond the single experimental week used in the current study could potentially minimise classification errors, as this would confirm that the fragments categorised as dead do not recover. Using sods of C . brachypus , rather than fragments in any further laboratory experiments, could provide a more reliable assessment of the temperature threshold needed to counter the insulating effect of substrate on buried stolons and rhizoids (Cardigos, et.al., 2015 ). Ambient temperature in relation to the thermal shock threshold was not examined in this current study but may be important in terms of ensuring the efficacy of any thermal thresholds for field treatments. There is research evidence that invasive Caulerpa species are weakened by cold temperatures in the winter months, hence there is a potential for heightened effectiveness of thermal shock treatment when compared with summer conditions (Ceccherelli & Piazzi, 2001 ; Keeler, et.al., 2025 ). Ideally, future experiments could be conducted in situ, which would control for the possibility that handling of the C . brachypus in the field experiment for this current study may have had an effect on the results. Such direct treatment would be an excellent precursor for developing methods for larger scale in situ treatment of invasive C . brachypus . A key advantage for using thermal shock treatment is that this method uses no chemicals and at the lower temperature thresholds (i.e., 55°C for 10 seconds) has the potential to leave adjacent native flora and fauna unharmed when implemented in the field (Forrest & Blakemore, 2006 ). Considerations for use as a potential tool for eradication include engineering new technology to deliver hot water to the Caulerpa spp. on the seafloor at a consistent temperature and at low pressure to avoid fragmentation, such as via a remotely operated vehicle. Conclusion The preliminary results from this study indicate that thermal shock treatment has the potential to be an effective method for controlling C . brachypus . However, further refinement of temperature and exposure periods are needed to ensure complete killing of the macroalgae in situ. The practicalities of the delivery of elevated temperatures to C . brachypus on the seafloor also warrant further consideration and development if this method of control is to have any capacity to be applied in situ at scale. Declarations Funding source: Te Moananui O Toi Trust Key words: invasive, Caulerpa brachypus , thermal shock, marine biosecurity, exotic macroalgae Acknowledgements We would like to acknowledge Te Moananui O Toi Trust for funding our research. A special thank you to David Cockell for his consultation and support. Our sincere thanks goes to Francisco Marquez and his team for the seawater laboratory support. References Ammon U, Swift L, Brand S, Jeffs A, Swift S (2019) Review of in-water hull encapsulation and enclosure treatments for eliminating marine biofouling. MPI Technical Paper No: 2019/06. Prepared for the Ministry for Primary Industries, The University of Auckland Anderson LWJ (2007) Control of invasive seaweeds. Bot Mar 50:418–437 Aquatic Biosecurity (2009) NSW control plan for the noxious marine alga Caulerpa taxifolia. Prepared by the Aquatic Biosecurity & Risk Management Unit for Industry & Investment NSW Govt Bax N, Williamson A, Aguero M, Gonzalez E, Geeves W (2003) Marine invasive species: a threat to global biodiversity. Mar Policy 27:313–323 Biosecurity New Zealand (2024a) Summary of current known exotic Caulerpa locations and Summary of Biosecurity New Zealand funded removal trials or removal missions. Reports compiled for the Exotic Caulerpa National Advisory Group Biosecurity New Zealand (2024b) Exotic Caulerpa strategic technical advisory group report. Biosecurity New Zealand Information Paper No. 2024/XY Cardigos F, Monteiro J, Fontes J, Parretti P, Sandos RS (2015) Fighting invasions in the marine realm, a case study with Caulerpa webbiana in the Azores. Biological Invasions in Changing Ecosystems: Vectors, Ecological Impacts, Management and Predictions, João Canning-Clode. De Gruyter Open Poland, Warsaw, Poland Ceccherelli G, Piazzi L (2001) Dispersal of Caulerpa racemosa fragments in the Mediterranean: Lack of detachment time effect on establishment. Bot Mar 44:209–213 Creese RG, Davis AR, Glasby TM (2004) Eradicating and preventing the spread of the invasive alga Caulerpa taxifolia in NSW . NSW Fisheries Final Report Series, No.64 Curran PJ, West SG, Finch JF (1996) The robustness of test statistics to nonnormality and specification error in confirmatory factor analysis. Phycological Methods 1(1):16–19 Davidson I, Fletcher L, Richards D, Bennett-Jones L, Nevill-Jackson M (2024) et.al. Omakiwi Cove exotic Caulerpa removal. Phase I: method development for mechanical suction dredging. Nelson: Cawthron Institute. Cawthron Report 4040. Prepared for Northland Regional Council and Biosecurity New Zealand Davis AR, Ferguson AM, Wright JT (2009) Structural complexity facilitates accumulation and retention of fragments of the invasive alga, Caulerpa taxifolia . J Exp Mar Biol Ecol 371:163–169 Dey AK, Sharma M, Meshram MR (2016) An analysis of leaf chlorophyll measurement method using chlorophyll meter and image processing technique. Procedia Comput Sci 85:286–292 Forrest BM, Blakemore KA (2006) Evaluation of treatments to reduce the spread of a marine plant pest with aquaculture transfers. Aquaculture 257:333–345 Giakoumi S, Katsanevakis S, Albano PG, Azzurro E, Cardoso AC, et.al (2019) Management priorities for marine invasive species. Sci Total Environ 688:976–982 Glasby TM (2013) Caulerpa taxifolia in seagrass meadows: Killer or opportunistic weed? Biol Invasions 15:1017–1035 Hendry GAF, Houghton JD, Brown SB (1987) The degradation of chlorophyll – a biological enigma. New Phytol 107:255–302 Hortensteiner S (2004) The loss of green color during chlorophyll degradation – A prerequisite to prevent cell death? Planta 219:191–194 Keeler S, Barr N, Pinkerton M, Thoral F (2025) Evaluation of exotic Caulerpa biomass reduction. Prepared for Biosecurity New Zealand. NIWA Client Report No: 2025137WN McRae S (2025) Rehabitat interim report for MPI . A report prepared by Commercial Dive Specialists Limited for the Ministry for Primary Industries Meinesz A, Belsher T, Thibaut T, Antolic B, Mustapha KB, et.al (2001) The introduced green alga Caulerpa taxifolia continues to spread in the Mediterranean. Biol Invasions 3:201–210 Merkel & Associates (2021) Rapid response and control plan for the invasive green alga Caulerpa prolifera in Newport Bay. Report for the Southern California Caulerpa Action Team. Retrieved from: https://nrm.dfg.ca.gov/FileHandler.ashx? DocumentID = 233231 Parreira F, Martinez-Crego B, Afonso CML, Machado M, Oliveira F, et.al (2021) Biodiversity consequences of Caulerpa prolifera takeover of a coastal lagoon. Estuar Coast Shelf Sci 255:107344 Piazzi L, Ceccherelli G, Cinelli F (2001) Threat to macroalgal diversity: Effects of the introduced green alga Caulerpa racemosa in the Mediterranean. Mar Ecol Prog Ser 210:149–159 Quilez-Badia G, McCollin T, Josefsen KD, Vourdachas A, Gill ME, et.al (2008) On board short-time high temperature heat treatment of ballast water: A field trial under operational conditions. Mar Pollut Bull 56:127–135 Riccardi M, Mele G, Pulveno C, Lavini, d’Andria R, et.al (2014) Non-destructive evaluation of chlorophyll content in quinoa and amaranth leaves by simple and multiple regression analysis of RGB image components. Photosynth Res 120:263–272 Rogalin-Henderson M, Zhuang A, Jeffs AG, Thorburn C (2025) Laboratory assessment of the use of ultraviolet-C light for the destruction of Caulerpa brachypus. Report compiled in fulfilment of the project objectives of the Exotic Caulerpa Elimination Grant Project # C0037506 to Advanced. Aquarium Technologies Ltd and the Ministry for Primary Industries Scott B (2024) The anatomy of an invasion: Exotic Caulerpa at Aotea Great Barrier Island. Report prepared for Aotea Great Barrier Environmental Trust Shannon C, Quinn CH, Stebbing PD, Hassall C, Dunn AM (2018) The practical application of hot water to reduce the introduction and spread of aquatic invasive alien species. Manage Biol Invasions 9(4):417–423 Su CH, Fu CC, Chang YC, Nair GR, Ye JJ, et.al (2008) Simultaneous estimation of chlorophyll-a and lipid contents in microalgae by three-color analysis. Biotechnol Bioeng 99:1034–1039 Thames Coromandel District Council (TCDC) (2025) Tsunami advisory lifted, but continue to take extra care. Retrieved from TCDC: https://www.tcdc.govt.nz/Our - Council/News-Media-and-Public-Notices/Latest-News/Tsunami-Advisory- %E2%80%93-Strong-and-unusual-currents-expected-along-NZ-coast Thorburn C (2025) Applying emerging UV-C technology for the sustainable long-term, non-chemical control of invasive Caulerpa. Report prepared by Advanced Aquarium Technologies for the Ministry for Primary Industries Uya M, Bulleri F, Gribben PE (2018) Propagules are not all equal: Traits of vegetative fragments and disturbance regulate invasion success. Ecology 99(4):957–965 Walters L (2009) Ecology and management of the invasive marine macroalga Caulerpa taxifolia. In: Inderjit (eds) Management of Invasive Weeds, 287–318. Springer, Dordrecht Williams SL, Schroeder SL (2004) Control of the invasive seaweed Caulerpa taxifolia by chlorine bleach. Mar Ecol Prog Ser 272:69–76 Woodfield R, Merkel K (2004) Control and surveillance of Caulerpa taxifolia within Agua Hedionda Lagoon, Carlsbad, California: Third year status report. Prepared for the Steering Committee of the Southern California Caulerpa Action Team by Merkel & Associates, Inc. Wotton DM, O’Brien C, Stuart MD, Fergus DJ (2004) Control success down under: Heat treatment of a sunken trawler to kill the invasive seaweed Undaria pinnatifida . Mar Pollut Bull 49:844–849 Zhang H, Ge Y, Xie X, Atefi A, Wijewardane NK, et.al (2022) High throughput analysis of leaf chlorophyll content in sorghum using RGB, hyperspectral, and fluorescence imaging and sensor fusion. Plant Methods 18:60–77 Zhuang A, Thorburn C, Caiger P, Davis W, Jeffs AG (2024) Control of invasive Caulerpa seaweed species using ultraviolet-C light. CW218971, Prepared for Auckland Council, ARIBA ID Zulijevic A, Antolic B (2005) Appearance and spread of the invasive Caulerpa species in the Adriatic Sea. 47th International Symposium ELMAR, 08–10 June 2005. Zadar, Croatia Supplementary Files ThermalshocktreatmentDATA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8713916","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583137142,"identity":"0c9898eb-d4fd-45d6-84e4-b78f80f5fd2e","order_by":0,"name":"Michele Rogalin-Henderson","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4931-5906","institution":"University of Auckland - City Campus: University of Auckland","correspondingAuthor":true,"prefix":"","firstName":"Michele","middleName":"","lastName":"Rogalin-Henderson","suffix":""},{"id":583137143,"identity":"113da1bf-9162-47eb-b490-30ec890c7c0c","order_by":1,"name":"Jessica McLay","email":"","orcid":"","institution":"University of Auckland - City Campus: University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"McLay","suffix":""},{"id":583137144,"identity":"355b7fa6-66d5-4e45-af51-d1627838d729","order_by":2,"name":"Andrew G. Jeffs","email":"","orcid":"","institution":"University of Auckland - City Campus: University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"G.","lastName":"Jeffs","suffix":""}],"badges":[],"createdAt":"2026-01-27 19:42:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8713916/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8713916/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101804406,"identity":"eafae723-a8c2-4292-ab51-fb9f578606ed","added_by":"auto","created_at":"2026-02-03 19:11:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63144,"visible":true,"origin":"","legend":"\u003cp\u003eReplicate samples of \u003cem\u003eCaulerpa brachypus\u003c/em\u003e sod in weighted boxes prior to thermal shock treatment.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/7cdce9ba01fe882795f1f4ac.jpg"},{"id":101804405,"identity":"1197474c-83d5-481e-b6d5-5fdf51d7521d","added_by":"auto","created_at":"2026-02-03 19:11:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127161,"visible":true,"origin":"","legend":"\u003cp\u003eAn example of an experimental thermal shock treatment combination for \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e showing photographs of a fragment before thermal shock treatment for 30 seconds at 45 °C and 7 days afterwards.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/5e72704b0d14575b2002cdca.jpg"},{"id":101804407,"identity":"ce9db3c9-a961-465b-8525-8d7517e8e0ee","added_by":"auto","created_at":"2026-02-03 19:11:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":169905,"visible":true,"origin":"","legend":"\u003cp\u003eMean colour of \u003cem\u003eCaulerpa brachypus\u003c/em\u003e fragments as measured 7 days after exposure to different combinations of temperature (heated water and a control of 18.5 °C) and exposure time. Each colour block represents the mean colour of three points sampled from each of the thalli, stolon, and rhizoid from each of three replicate fragments for every temperature and exposure time combination. The loss of dark green colouration (i.e., chlorophyll-a) from the \u003cem\u003eCaulerpa\u003c/em\u003e \u003cem\u003ebrachypus\u003c/em\u003etissues indicating the death of the fragment based on a\u003cem\u003e \u003c/em\u003evisual assessment.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/08153ab4c3ebed937e8ca803.jpg"},{"id":101804409,"identity":"6f81a03f-ef6a-4938-827f-14c1fd95415f","added_by":"auto","created_at":"2026-02-03 19:11:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129141,"visible":true,"origin":"","legend":"\u003cp\u003ePlot of the ratio of mean Red/Green colour against Blue colour measured for three components of fragments of \u003cem\u003eCaulerpa brachypus\u003c/em\u003e 7 days after exposure to various experimental thermal shock treatments and visually assessed by human observer as alive (green circle) or dead (red circle) (N = 4176). The independently derived SVM classification line that divides the alive and dead fragments is shown in black. The equation of the line is 590 – 474*(R/G) – B.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/07a93f0e1e183fea57ae6319.jpg"},{"id":101804410,"identity":"743b819a-c42e-451e-ad27-ac260a261007","added_by":"auto","created_at":"2026-02-03 19:11:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159775,"visible":true,"origin":"","legend":"\u003cp\u003eThe mean percentage cover (±SE) of \u003cem\u003eCaulerpa brachypus \u003c/em\u003ecover before thermal shock treatments on 22 July and 3 weeks later. The cover in both the 40 °C and 50 °C treatments were less than the control for the before treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), and after treatments all treatments were different from one another (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/c9c186edcb963450e1f68fc2.jpg"},{"id":103049270,"identity":"134bae86-f07f-47ee-92f8-59f9a4d5ffd4","added_by":"auto","created_at":"2026-02-20 07:39:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1221036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/58e9775b-3e73-43cd-b0e1-6cc1bf57ba0e.pdf"},{"id":101804408,"identity":"09618317-9ddd-47b9-99b1-1e4419355de5","added_by":"auto","created_at":"2026-02-03 19:11:11","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15000,"visible":true,"origin":"","legend":"","description":"","filename":"ThermalshocktreatmentDATA.docx","url":"https://assets-eu.researchsquare.com/files/rs-8713916/v1/44ce3d1355297cba70f9c6b8.docx"}],"financialInterests":"","formattedTitle":"Thermal shock treatment for controlling exotic Caulerpa brachypus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA number of species in the macroalgal genus \u003cem\u003eCaulerpa\u003c/em\u003e are common as invasive exotic marine organisms, with ecologically impactful incursions in a number of locations throughout the world. The locations for these incursions include the Mediterranean Sea, the Americas, and Oceania (Anderson, 2005; Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Meinesz, et.al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As with many other marine invasive species, invasive \u003cem\u003eCaulerpa\u003c/em\u003e species rapidly outcompete and replace native benthos, resulting in a loss of biodiversity and habitat for native fishes, sessile invertebrates, and in faunal species, such as shellfish (Parreira, et.al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, vegetative growth in invasive \u003cem\u003eC. taxifolia\u003c/em\u003e is reported at up to 13 mm per day in the summer months (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)d \u003cem\u003eracemosa\u003c/em\u003e at up to 20 mm per day (Piazzi, et.al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), enabling these species to form a dense carpet that smothers the benthos (Meinesz, et.al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlobal efforts to control invasions of exotic \u003cem\u003eCaulerpa\u003c/em\u003e spp. have mostly had limited success (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Furthermore, most of the control methods have significant drawbacks, such as an adverse ecological impact on native species, excessive expense, difficulty scaling up, and increasing the likelihood of spread through increased fragmentation (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Early discovery and prompt treatment appear to be key factors for the successful eradication of invasive \u003cem\u003eCaulerpa\u003c/em\u003e spp. (Giakoumi, et.al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Once an incursion of an exotic \u003cem\u003eCaulerpa\u003c/em\u003e spp. has become well established in an ecosystem it is nearly impossible to eradicate (Bax, et.al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA number of control methods use various chemicals to kill invasive \u003cem\u003eCaulerpa\u003c/em\u003e spp. These include algaecides, salt/osmotic shock, chlorine, and other various chemicals (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Of these, chlorine and salt treatment have been shown to be effective in two instances (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Woodfield \u0026amp; Merkel, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Chlorine, infused under a benthic mat, was successful in treating and eliminating an invasion of \u003cem\u003eC. taxifolia\u003c/em\u003e in California (Woodfield \u0026amp; Merkel, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Unfortunately, the ecological impact of using this method is significant, as it also eradicates associated native flora and fauna (Williams \u0026amp; Schroeder, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Chemical treatments that have been tried, without success, in the United States and the Mediterranean Sea include copper sulphate, algaecides/herbicides, hydrogen peroxide, and acetic acid (Aquatic Biosecurity, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Walters, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These methods also adversely impact native species. Osmotic shock through the application of fresh water was unsuccessful for controlling \u003cem\u003eC. taxifolia\u003c/em\u003e in NSW, Australia, and it was also found to be logistically challenging to access sufficient fresh water to achieve sufficiently low salinity to be effective (Aquatic Biosecurity, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Salt treatment has been used effectively on \u003cem\u003eC. taxifolia\u003c/em\u003e in an estuarine invasion in NSW (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, this treatment adversely impacted native seagrasses and is quite labour intensive. This may be an effective method applied in small or initial incursions (Aquatic Biosecurity, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Glasby, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSmothering methods of control, such as the use of benthic matting (e.g., tarpaulins or jute mats) or covering of the invasive \u003cem\u003eCaulerpa\u003c/em\u003e spp. with sediment, have been applied in the Mediterranean, the United States, and Australia, but without successful eradication (Walters, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Collateral damage on native fauna and flora is significant with smothering methods (Williams \u0026amp; Shroeder, 2004). Smothering is thought to be likely to be more effective for eliminating small patches of \u003cem\u003eC. taxifolia\u003c/em\u003e in new areas provided the covering can remain in place for up to a month to be fully effective (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Walters, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe removal of exotic \u003cem\u003eCaulerpa\u003c/em\u003e spp. through suction dredging of the seafloor has been used in Australia and the Mediterranean Sea (Walters, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, dredging is responsible for significant fragmentation of the \u003cem\u003eCaulerpa\u003c/em\u003e spp., which increases the risk of spread, and has not been demonstrated to eradicate \u003cem\u003eCaulerpa\u003c/em\u003e spp. successfully, making it generally a poor choice for control (Walters, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). There has been some success with suction dredging, with close monitoring and repeat treatments in one small incursion of \u003cem\u003eC. racemosa\u003c/em\u003e var. \u003cem\u003ecylindracea\u003c/em\u003e in the Adriatic Sea (Zuljevic \u0026amp; Antolic, 2002). Suction-assisted hand removal of \u003cem\u003eCaulerpa\u003c/em\u003e spp. by divers appears to reduce the amount of fragmentation, but this method is labour intensive and relies on repeated application (Creese, et.al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, this method is more ecologically friendly than chemical treatments, such as chlorine, and in one location in California this method was specifically selected for the invasive \u003cem\u003eC. prolifera\u003c/em\u003e to avoid killing co-occurring endangered species (Merkel, 2021; Parreira, et.al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInvasive exotic \u003cem\u003eC. brachypus\u003c/em\u003e was first discovered in Aotearoa - New Zealand in June of 2021 at Aotea - Great Barrier Island in the Hauraki Gulf near Auckland, along with an invasive exotic congener \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eparvifolia\u003c/em\u003e (Scott, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Since its initial discovery, \u003cem\u003eC. brachypus\u003c/em\u003e has spread to numerous locations in the Hauraki Gulf, Coromandel Peninsula, and the Bay of Islands, and ongoing monitoring is continuing to identify new locations (Biosecurity NZ, 2024b). The potential adverse impact for the Hauraki Gulf and the Bay of Islands from this incursion indicates a need for effective methods for management to arrest the spread of \u003cem\u003eC. brachypus\u003c/em\u003e and potentially eradicate it.\u003c/p\u003e \u003cp\u003eMethods for controlling exotic \u003cem\u003eCaulerpa\u003c/em\u003e species that are being applied in Aotearoa - New Zealand include UV light exposure, benthic matting with salt or chlorine, suction dredging, and hand removal by divers (Biosecurity, NZ, 2024a). The use of UV light was found to be effective for controlling both \u003cem\u003eC. parvifolia\u003c/em\u003e and \u003cem\u003eC. brachypus\u003c/em\u003e (Rogalin-Henderson, et.al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Thorburn, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhuang, et.al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, this method requires repeated treatments, and the regrowth rate and ecological impact is yet to be studied. Benthic matting, particularly combined with the application of chlorine beneath the matting, has been demonstrated to be effective at controlling the invasive \u003cem\u003eCaulerpa\u003c/em\u003e species in New Zealand (Ammon et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; McRae, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This method also kills all other species in the treated area, which potentially leaves the area prone to future invasion (Uya, et.al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Suction dredging has been applied in Omakiwi Cove in the Bay of Islands of Aotearoa - New Zealand to attempt to remove invasive \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e (Davidson, et.al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While the use of suction dredging for removing large areas of biomass was found to be effective, it also generates fragments of the targeted macroalgae during the process and does not remove all of the targeted \u003cem\u003eCaulerpa\u003c/em\u003e species (Davidson, et.al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Floating fragments released through any handling of most \u003cem\u003eCaulerpa\u003c/em\u003e species are thought to have the potential to establish in new locations (Davis, et.al, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThermal shock treatment involves using a temperature shock to kill invasive aquatic organisms which has been demonstrated to be an effective means of killing some invasive pest seaweeds, and has also been used for the treatment of unwanted microalgae and seaweed in aquaculture situations and in ballast tanks (Forest \u0026amp; Blakemore, 2006; Quilez-Badia, et.al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shannon, et.al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wotton, et.al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For example, thermal shock treatment, in the form of heated seawater, was used to eradicate a potentially invasive seaweed, wakame, \u003cem\u003eUndaria pinnatifida\u003c/em\u003e, from a sunken ship in the Chatham Islands, located offshore from mainland Aotearoa - New Zealand (Wotton, et.al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). To deliver this treatment, plywood boxes with foam seals were placed over the seaweed on the hull of the vessel and heating elements inside the box increased the temperature of the enclosed seawater to 70\u0026deg;C for 10 minutes. The method was effective for eradicating the seaweed, with no subsequent regrowth detected. Thermal shock treatments were also used in an aquaculture setting to eradicate \u003cem\u003eU. pinnatifida\u003c/em\u003e from seed mussels in New Zealand (Forrest \u0026amp; Blakemore, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Complete mortality of the seaweed was achieved by immersing the seed mussels in 55\u0026deg;C seawater for 5 seconds, without causing an adverse impact on the mussels, suggesting that where the target macroalgae has a low threshold to thermal stress, it might be exploited as a thermal shock treatment whilst limiting damage to associated organisms which have greater thermal tolerance. Two seaweeds, \u003cem\u003eMyriophyllum aquaticum\u003c/em\u003e and \u003cem\u003eCrassula helmsii\u003c/em\u003e were killed using hot seawater at temperatures of 45 to 60\u0026deg;C with various exposure periods (Shannon, et.al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Complete mortality of both seaweeds was reliably achieved with immersion at temperatures of 60\u0026deg;C for 10 seconds exposure. Thermal shock treatment has also been used to treat ballast water aboard a commercial vessel, with a temperature of 55\u0026deg;C being found to eradicate both zooplankton and phytoplankton in the ballast tanks (Quilez-Badia, et.al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is only one example of thermal shock being used to control invasive \u003cem\u003eCaulerpa\u003c/em\u003e spp. in situ, where small rocks covered in \u003cem\u003eC. webbiana\u003c/em\u003e were removed from the seafloor, treated with heated seawater at 68\u0026deg;C for 10 seconds and then put back on the seafloor (Cardigos, et. al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). While the thermal shock treatment method successfully eradicated the seaweed from the rocks, the lack of technology to apply heat to the seaweed underwater on a larger scale prevented further practical application of thermal shock methods in this location.\u003c/p\u003e \u003cp\u003eThermal shock treatments have also been demonstrated to reliably kill \u003cem\u003eC. taxifolia\u003c/em\u003e in the laboratory, where fragments were exposed to heated seawater at 72\u0026deg;C for either 1 or 2 hours and then placed in substrate and monitored for mortality (Williams \u0026amp; Shroeder, 2004). All but one of the 20 treated fragments died, with the remaining showing a small area of green (i.e., chlorophyll-a) in a buried portion of the fragment. This study is an outlier in the small body of research on thermal shock treatment of macroalgae, given the lengthy time of exposure to the heat treatment. Whether \u003cem\u003eC. taxifolia\u003c/em\u003e is much hardier, or if burial of the fragments was relevant to the persistence observed in a small number of the treated fragments, is unclear.\u003c/p\u003e \u003cp\u003eCollectively, these previous findings suggest that thermal shock treatment may be an effective method for the control of invasive \u003cem\u003eCaulerpa\u003c/em\u003e species in Aotearoa - New Zealand. Therefore, the current study examines the efficacy of thermal shock treatments of exotic \u003cem\u003eC. brachypus\u003c/em\u003e as a potential method of control of this invasive seaweed.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory experiment\u003c/h2\u003e \u003cp\u003eSamples of healthy \u003cem\u003eC. brachypus\u003c/em\u003e were collected by divers on 22 July 2024 from Blind Bay at Aotea-Great Barrier Island (-36.262271\u0026deg;, 175.432261\u0026deg; WGS 84) in 5 m of water where the seaweed was growing as an extensive covering on sandy substrate. Samples of the sandy substrate together with the \u003cem\u003eC. brachypus\u003c/em\u003e were dug out of the sediment with a hand trowel and placed in 8 \u0026times;15 cm plastic nursery trays and transported to the laboratory in seawater in biosecure containers.\u003c/p\u003e \u003cp\u003eUpon arrival at the laboratory 232 individual \u003cem\u003eC. brachypus\u003c/em\u003e fragments, each 30\u0026ndash;40 mm in overall length and consisting of a stolon, rhizoids, and several thalli, were excised using a scalpel and forceps and each placed in a covered 100 mm diameter glass petri dish with no substrate and 50 ml of seawater. The seawater was obtained from the Waitematā Harbour and filtered through a 1 \u0026micro;m activated carbon filter and sterilized with ultraviolet light.\u003c/p\u003e \u003cp\u003eFragments of \u003cem\u003eC. brachypus\u003c/em\u003e were exposed to heated seawater treatments of 30 to 80\u0026deg;C in 5\u0026deg;C increments, (i.e., 30, 35, 40\u0026deg;C etc) for each of four experimental treatment periods of either 5, 10, 30 or 60 seconds duration, i.e., a total of 44 temperature and duration combinations. Five replicate fragments of \u003cem\u003eC. brachypus\u003c/em\u003e were subjected to each treatment combination.\u003c/p\u003e \u003cp\u003eSeawater was heated in a 250 ml beaker on a hotplate with a magnetic stirrer and the temperature measured with a digital thermometer. At the selected treatment temperature, each \u003cem\u003eC. brachypus\u003c/em\u003e fragment was each submerged in the heated seawater, with timer used to determine the duration of the treatments. At the end of the treatment duration, the fragment was immediately removed from the heated water and submerged in a 250 ml beaker filled with seawater at 18.5\u0026deg;C for 10 seconds. In addition to the temperature duration treatments for each of the four experimental treatment periods (i.e., 5, 10, 30, and 60 seconds) a control treatment was also undertaken with three randomly selected fragments that were submerged in the beaker with the 18.5\u0026deg;C water for each of the four treatment durations, then resubmerged in the same beaker for 10 seconds. Following the holding period in the 18.5\u0026deg;C water all of the fragments were placed in clean seawater in their individually labelled petri dish at 18.5\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe labelled petri dishes were then placed in a plant culture cabinet set at a constant 18.5\u0026deg;C on a light:dark cycle of 12:12 hours to approximate mean annual conditions in the Hauraki Gulf. The fragments were visually assessed for viability 7 days after treatment, and each classified as alive or dead based on their tissue colour to assess chlorophyll-a content, with brown colouration indicating dead tissue and green colouration indicating living tissue with the presence of chlorophyll-a.\u003c/p\u003e \u003cp\u003eTo undertake an observer-independent assessment of the alive versus dead state of the experimental \u003cem\u003eC. brachypus\u003c/em\u003e fragments, digital images of each fragment were taken before and after treatment using the 48-megapixel camera on an iPhone 15 at a 200 mm distance and illuminated by a 2.5 W LED lamp with a white background. Each digital image was analysed by sampling the colour at three random locations on each of the thalli, stolon, and rhizoid of the fragment to gather corresponding RGB (i.e., red-green-blue) data using PixSpy software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.pixspy.com\" target=\"_blank\"\u003ewww.pixspy.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.pixspy.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The RGB data sets collected through PixSpy were imported to a spreadsheet for analysis. The mean of these sets of RGB data (from three locations on each of the thalli, stolon, and rhizoids), represented as numeric values for each colour component (i.e., R, G and B) was obtained by calculating the sum of the values for each component and dividing by the number of values, providing a mean colour for each component of the fragment, i.e., RGB = (\u0026sum;r/n, \u0026sum;g/n, \u0026sum;b/n). For each mean the corresponding RGB colour translation was plotted in a grid for comparison and the data used for statistical comparison.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003eThe analysis of RGB data sampled from images of seaweed and plants has been demonstrated to be an effective method for estimating chlorophyll-a content (Su, et.al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Riccardi, et.al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). When compared to SPAD, hyperspectral fluorescence, chlorophyll-a meter and chemical methods, with RGB colourimetric analysis being found to correlate significantly for chlorophyll-a measures (Dey, et.al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang, et. al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a loss of chlorophyll-a is associated with cell senescence and death, it provides a method of analysis for independently assessing the human visual assessment of viability for the experimentally treated fragments of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e (Hendry, et. al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hortensteiner, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlotting the ratio of mean R/G readings against the mean B component from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e post-treatment showed a clear divide. To find an equation for the line that divided the alive and dead fragments with minimal misclassification, support vector machines (SVMs) were applied to the data set. Within the SVM method, five times as much weight was given to correctly classifying alive fragments as to dead ones to provide greater confidence for avoiding the misclassification of alive fragments as being dead. The method was constrained to estimate a straight line defined by an intercept and slope as the plot which indicated that this was appropriate, and this restriction results in a simple equation that can be used more easily in practice. The SVM analyses were implemented using the SVM function in the e1071 package in R version 4.4.2.\u003c/p\u003e \u003cp\u003eA ten-fold cross-validation was used to get final intercept and slope values for the SVM line. In this process, the data is spilt into 10 random parts/folds. In the first iteration, the first fold of data is set aside as test data and the classification line was estimated (or \u0026lsquo;trained\u0026rsquo;) on the remaining 90%. The slope and intercept from this first fold were recorded and then the misclassification rates (one for dead fragments and one for alive fragments) was calculated using the test data. This was repeated another nine times, with a different fold of data set aside as the test data each time. The final intercept and slope for the line are the mean values over the ten iterations and the final reported misclassification rates are the mean misclassification rates from the ten iterations.\u003c/p\u003e\n\u003ch3\u003eField experiment\u003c/h3\u003e\n\u003cp\u003eA field experiment using thermal shock treatment of \u003cem\u003eC. brachypus\u003c/em\u003e growing in 1\u0026ndash;2 m depth in Omakiwi Bay in the Bay of Islands (35\u0026deg;14.281\u0026rsquo;S, 174\u0026deg;14.652E) was undertaken in July 2025.\u003c/p\u003e \u003cp\u003eSegments of randomly selected sod to a depth of around 5 cm in the sediment and containing a thick sward of \u003cem\u003eC. brachypus\u003c/em\u003e were removed from the seafloor and placed into 30 \u0026times; 50 cm weighted plastic boxes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThree replicate boxes containing \u003cem\u003eC. brachypus\u003c/em\u003e were exposed to each of the following heated seawater treatments of 70, 60, 50, or 40\u0026deg;C for 30 seconds, as well as a control using a sham treatment with unheated seawater. The treatment consisted of removing the ambient temperature seawater from the sample boxes then photographing with a GoPro 13 digital camera before rapidly filling each sample box with seawater preheated to the required temperature. The sample boxes were then quickly removed at the end of the exposure period and placed in ambient seawater at 16\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor each replicate the seawater temperature was monitored using two digital temperature measurement probes, one placed at the surface among the thalli and one at the base of the substrate to measure the treatment temperature that was reached at the rhizoids that were buried at their lowest point in the sediment.\u003c/p\u003e \u003cp\u003eAfter treatment, the boxes were returned to their original location on the seafloor and marked with a buoy so they could be relocated. All samples were retrieved 3 weeks later and photographed with a GoPro 13 digital camera.\u003c/p\u003e \u003cp\u003eThe digital images taken using a GoPro 13 digital camera were analysed by randomly selecting 100 points in each image using Coral Point Count software (CPCe) (Roberts, et.al., 2025; Kohler \u0026amp; Gill, 2006). At each point the cover was determined to have \u003cem\u003eCaulerpa\u003c/em\u003e cover or sediment. The 100 points from each of the digital images were used to assess changes in the biomass of \u003cem\u003eC. brachypus\u003c/em\u003e as measured by percent surface cover.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eDifferences in the measured points of cover of \u003cem\u003eC. brachypus\u003c/em\u003e among treatments was assessed with a chi-square contingency table by pooling all point cover counts for all replicates within each treatment. The cover of \u003cem\u003eC. brachypus\u003c/em\u003e for before and after were compared among treatments in two separate contingency tables to establish any differences that may have been present at the outset of the experiment, as well as 3 weeks after their experimental treatment. Where a significant overall result was identified for the before or after comparisons, pairwise post-hoc binary chi-square tests were conducted to isolate the source of the difference among the individual treatments. A Bonferroni-Holm correction was used to control for error inflation due to multiple testing of the same data set. The before treatment data set was at the limit of the assumptions for low proportions for a chi-square tests, but given the large sample size (i.e., total of 1500 binary sampling points), the test can be considered to be sufficiently robust to this analysis (Curran, et. al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory Experiment\u003c/h2\u003e \u003cp\u003eAll fragments of \u003cem\u003eC. brachypus\u003c/em\u003e in the control treatment that were only subjected to handling all remained alive. In contrast, all fragments of \u003cem\u003eC. brachypus\u003c/em\u003e treated with seawater at 50\u0026deg;C or higher temperatures for 5 seconds or more were visually assessed to have died as indicated by the appearance of the complete loss of chlorophyll-a from the tissues of the \u003cem\u003eCaulerpa\u003c/em\u003e fragment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Exposure of fragments to 45\u0026deg;C or higher temperatures for 30 seconds or longer caused all treated \u003cem\u003eCaulerpa\u003c/em\u003e fragments to die. The \u003cem\u003eC. brachypus\u003c/em\u003e fragments treated with heated seawater at 45\u0026deg;C for 5 and 10 seconds all survived, as did all fragments at lower temperatures and for all durations of exposure, i.e., 45\u0026deg;C and lower, and for exposure times of 5, 10, 30 or 60 seconds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA visual representation of the mean RGB values shows a distinct cut off at these described thresholds, wherein the samples show a loss of chlorophyll-a (i.e., green colouration), indicating a loss of viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, the pattern of colouration presented in the figure, with dark green representing live \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e tissue versus brown representing dead, is consistent with the alive and dead categorisation from the human visual assessment alone\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe variation in the SVM lines estimated at each iteration of the 10 cross-validations was small, with similar slopes and intercepts produced at each iteration. The final SVM classification line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) estimated that the misclassification rate for the alive fragments as dead by human visual assessment was 3.7% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 95% confidence interval for this rate indicates that the plausible range for the true misclassification rate of alive fragments could be up to 7.4%. In contrast, the misclassification rate for the dead fragments as alive by human visual assessment was 4.4%, with a 95% confidence interval of 1.1\u0026ndash;6.9% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA closer examination of the individual misclassifications found that the alive fragments based on colour that were categorized as dead by human visual assessment were all in treatments of 45\u0026deg;C and lower, almost entirely for short exposures (i.e., 10 seconds or less), and were mostly rhizoids (57%), compared to stolons and thalli, 38% and 5%, respectively. The individual misclassifications of dead fragments based on colour that were categorized as alive by human visual assessment were all in treatments of 45\u0026deg;C and higher, and were for a mix of exposure periods, and were mostly stolons (60%), compared to rhizoids and thalli, 26% and 13%, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimate of misclassification rates and standard errors (SE) based on 10-fold cross-validation of SVM classification line between alive and dead fragments of \u003cem\u003eCaulerpa brachypus\u003c/em\u003e 7 days after various thermal shock treatments. SE estimated by the standard deviations of the 10 misclassification rates and CI estimated for the 2.5 and 97.5 percentiles from the 10 misclassification rates in the cross-validation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClassification line\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEquation of line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e590\u0026ndash;474*(R/G) - B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMisclassification rate for alive fragments\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstimate of rate\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7% \u0026plusmn; 2.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1.3%, 7.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMisclassification rate for dead fragments\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstimate of rate\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.4% \u0026plusmn; 1.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1.1%, 6.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eField Experiment\u003c/h3\u003e\n\u003cp\u003eThe temperature readings from the thermometers placed among the thalli of the \u003cem\u003eC. brachypus\u003c/em\u003e whilst it was exposed to the various thermal shock treatments showed a steady increase in temperature toward the assigned treatment in temperature over the 30 second treatment period, except for the control treatment which remained at ambient seawater temperature, which was 16\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). None of the thermal treatments reached their assigned treatment temperature within the thalli of \u003cem\u003eC. brachypus\u003c/em\u003e, except for the 40\u0026deg;C treatment. The temperature readings from the substrate showed minimal increase in temperature over the 30 seconds of hot water treatment, with only an increase of a few degrees recorded in the 40\u0026deg;C and 50\u0026deg;C treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consequently, none of the substrates measured in the thermal shock treatments reached their assigned temperatures within the 30 second treatment period.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe highest temperature readings taken from among the thalli and from 3 cm below the surface of the substrate in plastic boxes containing sods of \u003cem\u003eCaulerpa brachypus\u003c/em\u003e during exposure to various thermal shock treatments consisting of applying ambient seawater (control) or heated seawater for 30 seconds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThalli\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate - Rhizoid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e70\u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.1\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.9\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e60\u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.1\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.9\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e50\u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.1\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.2\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e40\u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.1\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.6\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.0\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.0\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOf the 15 sample boxes treated and placed back into the substrate from where they were collected, three were displaced and the remainder were heavily silted as was the surrounding \u003cem\u003eC. brachypus\u003c/em\u003e meadow. This was likely due to tsunami activity along the eastern coast of Aotearoa \u0026ndash; New Zealand from 30 July to 1 August 2025, which created abrupt tidal surges including in shallow areas of the Bay of Islands (TCDC, 2025).\u003c/p\u003e \u003cp\u003eThe mean percentage cover of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e in the boxes was close to 100% in each of the five thermal shock treatments immediately before their exposure to their corresponding heat treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, there were small differences in the cover of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e among the treatments at the outset of the experiment (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021). The cover in both the 40\u0026deg;C and 50\u0026deg;C treatments (i.e., both 97.0% cover) were less than for the control which was 100% cover (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05). There were no other differences in the cover of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e among the treatments at the outset of the experiment.\u003c/p\u003e \u003cp\u003eThree weeks after the thermal shock treatments there were marked differences in the cover of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e among the treatments (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;627.03, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All pairwise comparisons of treatments were different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05). There was no difference in the cover of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e in the control before and 3 weeks after treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08), remaining close to 100% cover. However, the treatment exposed to 70\u0026deg;C showed the largest reduction in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e cover, with a mean loss of 89.7% of cover (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The 40\u0026deg;C exposure resulted in the smallest reduction in cover, with a mean loss of 9.3% of cover. The 50\u0026deg;C and 60\u0026deg;C treatments resulted in a mean loss of cover of 72.0% and 56.2%, respectively, i.e., the loss from the 50\u0026deg;C treatment was higher than for the 60\u0026deg;C treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBoth the laboratory and field results from this current study show the potential effectiveness of thermal shock treatments for killing \u003cem\u003eC. brachypus\u003c/em\u003e. Immersion of \u003cem\u003eC. brachypus\u003c/em\u003e fragments in seawater at 50\u0026deg;C and higher temperatures for 5 seconds, or 45\u0026deg;C and higher temperatures for 30 seconds and longer caused all fragments to die in the current laboratory experiment as assessed by a human observer. Independent assessment of direct measures of differences in colour for alive versus dead \u003cem\u003eC. brachypus\u003c/em\u003e in the laboratory experiment largely confirmed the human visual assessment, but with a mean misclassification rate for live fragments visually assessed as dead estimated to be 3.7% (\u0026plusmn;\u0026thinsp;2.1% S.E.), which may somewhat exaggerate the effectiveness of thermal shock treatments on \u003cem\u003eC. brachypus\u003c/em\u003e in the treatments of 45\u0026deg;C and higher. The higher stolon component of misclassification may be due to its generally darker initial colouration compared to rhizoids and thalli, which suggests that a generalised alive versus dead colour scale for all morphological components may lead to misclassifications for individual components. A human visual observer is likely to unconsciously compensate for this by comparing differences in the coloration of the individual components of the macroalgae.\u003c/p\u003e \u003cp\u003eThe mean misclassification rate for dead fragments visually assessed as alive, which was estimated to be 4.4% (\u0026plusmn;\u0026thinsp;1.2% S.E.), which may suggest that the effectiveness of thermal shock treatments at lower temperatures may be somewhat higher than reported, as these misclassifications occurred in the 45\u0026deg;C and lower treatment sets, in the shorter durations (i.e. 5 and 10 second sets). In these observations it was the rhizoid component most often misclassified. Rhizoids are most often buried in the substrate and their chlorophyll-a content may be typically lower that the emergent parts of the macroalgae making it harder to reliably determine its alive or dead status.\u003c/p\u003e \u003cp\u003eThe results of the field experiment show the potential effectiveness of thermal shock treatments for controlling \u003cem\u003eC. brachypus\u003c/em\u003e in situ. Immersion of sods of \u003cem\u003eC. brachypus\u003c/em\u003e in seawater at temperatures of 50, 60, and 70\u0026deg;C for 30 seconds all caused a marked reduction in cover when compared to the 40\u0026deg;C and Control treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggest a similar sensitivity to elevated temperatures as determined in the laboratory experiment, i.e., a threshold of 45\u0026deg;C for 30 seconds.\u003c/p\u003e \u003cp\u003eThe percentage cover of \u003cem\u003eC. brachypus\u003c/em\u003e at 3 weeks after treatment tended to decrease with increasing treatment temperature, almost reaching complete elimination in the 70\u0026deg;C treatment, i.e., mean 9.0% \u0026plusmn; SE percentage cover remaining. While the 50\u0026deg;C treatment had less remaining \u003cem\u003eC, brachypus\u003c/em\u003e cover than the 60\u0026deg;C treatment group (i.e., 25.0% versus 42.5%), the declines in cover otherwise corresponded with the increasing treatment temperature.\u003c/p\u003e \u003cp\u003eUnlike the laboratory experiments, using isolated individual small fragments of \u003cem\u003eC. brachypus\u003c/em\u003e, none of the treatment replicates showed a complete elimination of the macroalgae from the treated replicate sods of \u003cem\u003eC. brachypus\u003c/em\u003e indicating that the macroalgae could have remained viable. Temperature readings from among the thalli sward of \u003cem\u003eC. brachypus\u003c/em\u003e and from within the substrate where the rhizoids are buried indicated that during the 30 second thermal shock treatment period the increase in these temperatures was relatively slow and did not reach the intended treatment temperature, especially in the substrate. This was most likely due to the thermal mass of the \u003cem\u003eC. brachypus\u003c/em\u003e biomass and substrate, both requiring longer exposure for the transfer of the additional heat energy to meet the temperature required to kill the macroalgal tissue. This was also the most likely cause for the persistence of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e following even the highest temperature treatment of 70\u0026deg;C. Hence, attaining threshold temperatures during the treatment period appears to be critical for ensuring all parts of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e are killed, and will most likely require longer treatment periods to ensure the required temperatures are achieved.\u003c/p\u003e \u003cp\u003eOverall, the sensitivity of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e to thermal shock treatment was similar to the results reported from a similar field experiment using \u003cem\u003eC. webbiana\u003c/em\u003e wherein all samples were killed when exposed to 68\u0026deg;C for 10 seconds (Cardigos, et. al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, \u003cem\u003eC. taxifolia\u003c/em\u003e, appears more tolerant of thermal shock treatment with a laboratory experiment demonstrating that fragments exposed to hot seawater at 72\u0026deg;C for 60 minutes were killed, with the exception of a small spot of a potentially viable green tissue remaining on one of the fragments that was buried (Williams \u0026amp; Shroeder, 2004).\u003c/p\u003e \u003cp\u003eThermal shock treatment on other macroalgal species indicate similar temperature thresholds to the current study. Two studies on \u003cem\u003eU\u003c/em\u003e. \u003cem\u003epinnatifida\u003c/em\u003e, one in situ and the other in an aquaculture setting, achieved successful eradication. The first, in situ, used seawater heated to 70\u0026deg;C for 10 minutes, while the aquaculture study used seawater heated to 55\u0026deg;C for a 5 second exposure duration (Forrest \u0026amp; Blakemore, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Wotton, et.al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Furthermore, another similar study found successful eradication of \u003cem\u003eM. aquaticum\u003c/em\u003e and \u003cem\u003eC. helmsii\u003c/em\u003e at 60\u0026deg;C for 10 seconds (Shannon, et.al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of the field experiment, while promising, are preliminary. Replicating the experiment may offer more definitive results by addressing the limitations of the current experiment. For the laboratory study, holding the samples for several weeks beyond the single experimental week used in the current study could potentially minimise classification errors, as this would confirm that the fragments categorised as dead do not recover. Using sods of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e, rather than fragments in any further laboratory experiments, could provide a more reliable assessment of the temperature threshold needed to counter the insulating effect of substrate on buried stolons and rhizoids (Cardigos, et.al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Ambient temperature in relation to the thermal shock threshold was not examined in this current study but may be important in terms of ensuring the efficacy of any thermal thresholds for field treatments. There is research evidence that invasive \u003cem\u003eCaulerpa\u003c/em\u003e species are weakened by cold temperatures in the winter months, hence there is a potential for heightened effectiveness of thermal shock treatment when compared with summer conditions (Ceccherelli \u0026amp; Piazzi, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Keeler, et.al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Ideally, future experiments could be conducted in situ, which would control for the possibility that handling of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e in the field experiment for this current study may have had an effect on the results. Such direct treatment would be an excellent precursor for developing methods for larger scale in situ treatment of invasive \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA key advantage for using thermal shock treatment is that this method uses no chemicals and at the lower temperature thresholds (i.e., 55\u0026deg;C for 10 seconds) has the potential to leave adjacent native flora and fauna unharmed when implemented in the field (Forrest \u0026amp; Blakemore, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Considerations for use as a potential tool for eradication include engineering new technology to deliver hot water to the \u003cem\u003eCaulerpa\u003c/em\u003e spp. on the seafloor at a consistent temperature and at low pressure to avoid fragmentation, such as via a remotely operated vehicle.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe preliminary results from this study indicate that thermal shock treatment has the potential to be an effective method for controlling \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e. However, further refinement of temperature and exposure periods are needed to ensure complete killing of the macroalgae in situ. The practicalities of the delivery of elevated temperatures to \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ebrachypus\u003c/em\u003e on the seafloor also warrant further consideration and development if this method of control is to have any capacity to be applied in situ at scale.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding source:\u003c/h2\u003e \u003cp\u003eTe Moananui O Toi Trust\u003c/p\u003e \u003cp\u003eKey words: invasive, \u003cem\u003eCaulerpa brachypus\u003c/em\u003e, thermal shock, marine biosecurity, exotic macroalgae\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to acknowledge Te Moananui O Toi Trust for funding our research. A special thank you to David Cockell for his consultation and support. Our sincere thanks goes to Francisco Marquez and his team for the seawater laboratory support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmmon U, Swift L, Brand S, Jeffs A, Swift S (2019) \u003cem\u003eReview of in-water hull encapsulation and enclosure treatments for eliminating marine biofouling.\u003c/em\u003e MPI Technical Paper No: 2019/06. Prepared for the Ministry for Primary Industries, The University of Auckland\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson LWJ (2007) Control of invasive seaweeds. Bot Mar 50:418\u0026ndash;437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAquatic Biosecurity (2009) NSW control plan for the noxious marine alga Caulerpa taxifolia. 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Zadar, Croatia\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8713916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8713916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA number of macroalgal species in the genus \u003cem\u003eCaulerpa\u003c/em\u003e are characterised as some of the most invasive marine species in the world. There are a limited range of interventions for dealing with incursions of exotic \u003cem\u003eCaulerpa\u003c/em\u003e species that have acceptable ecological impacts. This initial study assessed the potential to use heated seawater to kill fragments of \u003cem\u003eC. brachypus\u003c/em\u003e, a recent invader of concern in Aotearoa - New Zealand. In the laboratory, \u003cem\u003eCaulerpa\u003c/em\u003e fragments were exposed to heated seawater treatments of 30 to 80\u0026deg;C in 5\u0026deg;C increments for each of four experimental treatment durations of either 5, 10, 30 or 60 seconds and then cultured for one week in stable conditions. At 50\u0026deg;C or higher for 5 seconds or more, and at 45\u0026deg;C or higher for 30 seconds or longer, all fragments died, whereas below these treatment combinations most fragments remained alive, with 100% survival in the control with only a sham treatment handling. In the field, \u003cem\u003eCaulerpa\u003c/em\u003e sod segments were submerged for 30 seconds in 40 to 70\u0026deg;C in 10\u0026deg;C increments and returned to the sea for three weeks. Segments of \u003cem\u003eC. brachypus\u003c/em\u003e exposed to thermal shock treatments of 50, 60, and 70\u0026deg;C resulted in a significant loss of \u003cem\u003eC. brachypus\u003c/em\u003e cover (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not complete elimination. These results indicate the potential to use thermal shock for controlling invasive \u003cem\u003eC. brachypus\u003c/em\u003e that supports the management and protection of Aotearoa - New Zealand\u0026rsquo;s unique marine ecosystems.\u003c/p\u003e","manuscriptTitle":"Thermal shock treatment for controlling exotic Caulerpa brachypus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 19:11:00","doi":"10.21203/rs.3.rs-8713916/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7655b3b0-857f-4fb5-954f-570df76444cc","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T01:14:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 19:11:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8713916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8713916","identity":"rs-8713916","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}