Effects of a bacteria-produced algicide on non-target marine invertebrate species
Algicide preparation
Four batches of algicide were used for experiments, labeled Batch 3, Batch 4–5–6, Batch 7, and Batch 8, following methods used by Grasso27. For each batch, a single colony of Shewanella sp. IRI-160 was transferred from a modified LM medium plate to liquid LM medium for overnight growth, then inoculated into f/2 with 0.05% casamino acids and incubated for 10 days at room temperature with bubbling. Bacteria and other compounds greater than 60 kDa in size were filtered out using a HemoFlow HF80S 60 kDa dialysis cartridge (Fresenius Medical Care, Waltham, MA), creating a batch of sterile filtered exudate referred to as IRI-160AA. Samples of the algicide were diluted with ultrapure water, then total nitrogen (TN) was measured with a TOC-V total organic carbon analyzer equipped with a Total Nitrogen Measuring Unit (Shimadzu Corp., Kyoto, Japan). The algicide has approximately 5.02 mg/L TN. The 24-h EC50 for K. veneficum differed among batches but was always close to 1% (actual EC50s ranged from 0.93% in Batch 4–5–6 to 1.5% in Batch 3), thus a 1% concentration of the algicide was included in all invertebrate assays27. Animals were also exposed to a media control to ensure mortality was due to the algicide.
Statistical analyses
For all statistics, data were analyzed using Shapiro–Wilk normality tests and Brown-Forsythe equal variance tests. If they failed either, data were transformed and reanalyzed. If transformed data passed both tests, then analysis proceeded. If neither log or square-root transformed data passed both normality and equal variance tests, then a non-parametric test was run if possible. Specific details on statistical analyses are provided in each section below.
Copepod mortality
Mortality experiments followed established methods for determining acute toxicity in aquatic animals30,31,33,49. For A. tonsa adults, we collected animals in Fall of 2018 after sunset near the mouth of the Broadkill River (Delaware, USA) using a plankton net. Cod ends were diluted and maintained in field collected seawater with ambient food at room temperature (~ 20 °C) until use in experiments. Adults were filtered out of the bulk collection with a 500-μm mesh, then sorted for adult females. We transferred one adult female (n = 24 for 40%, 48 for 30%, and 72 for all other concentrations) into each well of a 12-well plate containing 5 mL of test solution; test solutions included a seawater control (0%); algicide mixtures prepared from Batch 3 of the IRI-160AA in 20 psu, 0.2 μm-filtered sea water collected from Indian River Inlet, DE, USA (FSW) (1%, 5%, 10%, 13.5%, 18%, 24%, 30%, and 40% v/v); and a 24% media solution as a media control. The plates were incubated at 25 °C in low-light (~ 2.37 × 1013 photons cm-2 s−1) on a 14:10 h day:night cycle for 48 h. Every 6 h for the first 24 h, and again at 48 h, we counted the number alive and dead.
For A. tonsa nauplii, adult females and males were placed in two 1 L beakers at room temperature with a 150-μm mesh placed several centimeters off the bottom (to prevent egg cannibalism), a slow bubbler (~ 2 small bubbles s−1), and ambient seawater diluted with 20 psu FSW until the water was mostly clear. Adults were allowed to mate in the beaker for approximately 24 h, after which we removed the mesh, thus removing the adults and leaving behind any nauplii and eggs. After another 24 h, the contents of the beakers were poured through a 20-μm mesh, and we extracted the nauplii and placed them into experimental treatments (0% seawater control, algicide at 1%, 5%, 10%, 13.5%, 18%, 24%, and 30% v/v concentrations, plus a 24% media control; n = 48 animals for all concentrations) following the procedure outlined above for the adult female copepods. This experiment was conducted three times; the first two mortality experiments used Batch 3 of the IRI-160AA, and the third mortality experiment used Batch 8.
From the data collected, we generated a Probit model50 and obtained a 24-h LC50. Another approach looks at mortality over several time points in order to generate a time series of survival (e.g., Robineau et al.51, Keller et al.52). This also allows the generation of an LC50 at several time points (e.g., 6, 12, 18, and 24 h), which can better inform how a certain animal may survive over time. We used SigmaPlot to generate graphs of survival over time, and R statistical software53 and the R package ecotoxicology54 for generating and graphing the Probit model and running a χ2 test to evaluate the model.
Crab mortality
We conducted mortality experiments for the blue crab (Callinectes sapidus) in larval (Z1-stage zoeae) and postlarval (megalopae) stages in a similar manner to mortality experiments with Acartia tonsa. We collected ovigerous female blue crabs during the Summer of 2018 by dip net and drop net at sunset from the Delaware Bay (similar to methods used by Kernehan55) in Cape Henlopen State Park and maintained them in a recirculating water tray containing filtered ambient seawater (~ 30 psu) at room temperature. We staged egg masses every few days55, and females predicted to hatch within ~ 3 days were moved to 7-gallon buckets in a 25 °C incubator containing ~ 30 psu sea water and a bubbler. Zoea larvae (Z1-stage) hatched from these females were kept in large finger bowls with 30 psu sea water at room temperature and were fed lab-reared rotifers (Brachionus rotundiformis, Reed Mariculture). These animals became subjects for mortality and sub-lethal experiments within approximately a day of hatching. Four experiments were conducted; three mortality experiments used Batch 4–5–6 of the IRI-160AA, while the fourth experiment (24 individuals for each concentration) used Batch 7.
Megalopae were collected by plankton net set on rising tides at night during the Summer and Fall of 2018. They were maintained in large finger bowls at room temperature and fed with Artemia nauplii and went into experiments within a few days of collection. Only megalopae in intermolt based on morphology56 were used in experiments. Megalopae experiments used Batch 3 of the IRI-160AA.
Both zoeae and megalopae were exposed to 1%, 5%, 10%, 13.5%, 18%, and 24% algicide concentrations, plus a 0% seawater control and a 24% media control (n = 84 animals for the 0% concentration and 60 for all other concentrations for zoeae, and n = 24 animals for megalopae for all concentrations). Animals were incubated at 25 °C under low-light (~ 2.37 × 1013 photons cm-2 s-1) on a 14:10 light:dark cycle for the duration of experiments. We checked on zoeae and megalopae every 6 h for 24 h; megalopae were checked at an additional 48-h time point.
Oyster mortality
Oyster larvae (eyed pediveligers of Crassostrea virginica) were provided by University of Maryland’s Horn Point Laboratory. Animals were maintained on a damp coffee filter in a sealed plastic container on ice during transport, then released into room-temperature fingerbowls containing 20 psu water and fed a locally-isolated alga (Storeatula major) at room temperature. Experiments occurred in similar fashion to those conducted on Acartia tonsa and Callinectes sapidus. Larvae were assayed in 12-well plates (n = 36 animals for all concentrations). Animals were exposed to 1%, 5%, 10%, 13.5%, 18%, and 24% algicide concentrations, plus a 0% seawater control, and 24% media control. Animals were incubated at 25 °C under a 14:10 light:dark cycle for the duration of experiments. Survival was evaluated every 6 h for 24 h and again at 48 h. Larvae were additionally examined at the start of the experiment and at the 24- and 48-h time points for an activity assay. These experiments used Batch 3 of the IRI-160AA.
Wild-type adult C. virginica were collected from the Delaware Bay near the University of Delaware Lewes Campus, while Haskins-disease-resistant strain individuals were collected from aquaculture cages maintained by the Delaware Center for the Inland Bays. On the first day, individuals were cleaned with a wire brush, and divided into two buckets containing approximately 10 L of 20 psu seawater and were fed Isochrysis galbana (~ 100,000 cells L−1). On the second day the water was changed and they were again fed. On the third day, water was changed and animals were not fed. On the fourth day, individuals were removed from the buckets, dried with a paper towel, labeled with permanent marker, and placed in pairs into forty-one 1 L plastic containers containing 1 L of various algicide solutions: 0%, 1%, 5%, 10%, 13.5%, 18%, and 24% (n = 28 for 0%, 22 for 1% and 18%, and 20 for all other concentrations). Individuals were checked every 6 h for 24 h and assessed if they were alive or dead. Closed individuals were assumed to be alive. If open individuals were observed, we gently tapped on the container to see if the individual shut its shell; animals that responded to this stimulus were marked as alive. Only animals that did not respond to repeated stimuli were scored as dead. Proportion surviving was compared across algicide concentration and strain. These experiments all used Batch 8 of the IRI-160AA.
Copepod sub-lethality
Respiration
We conducted respiration experiments on A. tonsa adult females and young nauplii in a 24-well microplate respirometer (Loligo Systems). First, we sorted animals into fingerbowls containing 100 mL of their respective algicide concentrations. After 24 h of algicide exposure, we removed animals via pipette and put one animal into each well of the respirometer plate (200 μL wells for adult females and 80 μL for nauplii) filled with 0.2 μm filtered FSW, then sealed the plate with Parafilm and a weight. Each experiment also had 4 to 6 wells with only FSW to calculate background oxygen consumption. The experiment occurred in darkness within a 25 °C incubator at night and lasted several hours (n = 26–39 animals for adult females, 11–18 for nauplii). Oxygen concentrations in each well were recorded every minute. At the end of the experiment, respiration rates were calculated in R statistical software using the respR package57 over a period of time when the animals were still in independent respiration, and a one-way ANOVA on ranks in SigmaPlot (Systat Software, San Jose, CA) compared treatments. Experiments with adult females used Batch 3 of IRI-160AA, while nauplii experiments used Batch 8.
Activity
Experiments determining effects on swimming activity utilized Locomotor Activity Monitors (LAMs; TriKinetics). Three beams of infrared light cross a 3 mL test tube containing an animal and register when the animal crosses the beams. We sorted batches of adult female A. tonsa into fingerbowls containing different algicide treatments. Animals were incubated at 25 °C in low-light conditions (~ 2.37 × 1013 photons cm−2 s−1) for 24 h on a 11:13-h light:dark cycle. Animals were pipetted into plastic test tubes (one animal per tube) containing ~ 3 mL of FSW, which then went into the LAMs (n = 21–36 animals). The experiment lasted 24 h with beam breaks summed at one-minute intervals, allowing the data to be analyzed wholly for the 24-h period as well as across different light phases to account for light:dark mediated activity rhythms. Experiments started in the afternoon and ran overnight, creating an initial light phase (L1), a dark phase (D), and a second light phase (L2). Comparing treatments across the entire time period was done using a one-way ANOVA on ranks, while analyzing the data based on the different light phases was performed via a one-way repeated-measures ANOVA. Additionally, at the end of the LAM activity experiments we collected the individuals and noted mortality. This data was analyzed via a one-way ANOVA on ranks. Copepod activity experiments used Batch 3 of the IRI-160AA. Nauplii were too small to generate a reliable signal in the LAMs and were not used in these experiments.
Crab sub-lethality
Respiration
Respiration experiments followed methods described for A. tonsa above and involved zoeae and megalopae. A one-way ANOVA on ranks was calculated using the data for each life stage. The first four zoeae experiments used Batch 4–5-6 of IRI-160AA, while the last two experiments used Batch 7. Megalopae experiments all used Batch 3.
Activity
Activity level experiments followed methods described for A. tonsa above and involved zoeae and megalopae. The 24-h data were analyzed using a one-way ANOVA on square root transformed data for zoeae, and a one-way ANOVA on ranks for megalopae. The data broken down by light phase were analyzed via one-way repeated measures ANOVA on log-transformed data for both zoeae and megalopae. These experiments all used Batch 3 of IRI-160AA.
At the end of experiments we collected the individuals and noted mortality. This data was analyzed via a one-way ANOVA for zoeae and a one-way ANOVA on ranks for the megalopae.
Metamorphosis
We sorted megalopae into finger bowls containing 100 mL of filtered estuary water with different concentrations of the IRI-160AA algicide (0%, 1%, and 17% v/v). After 24-h of exposure, we sorted animals into 12-well plates containing FSW (n = 60 individuals for each treatment). Water was changed daily, and animals were fed freshly hatched Artemia daily. Every 12 h, we counted how many megalopae had molted into first crabs until most had metamorphosed (5.5 days) and used a Kaplan–Meier Survival Analysis with a Gehan-Breslow test to determine if there was a difference in time to metamorphosis (TTM) across treatments. These experiments used Batch 3 of the IRI-160AA.
Abdomen Pumping and Grooming
Crabs with egg masses were collected from the Delaware Bay near Lewes, DE and separated into numbered baskets and maintained in a flow-through sea water table. They were fed thawed squid (Loligo opalescens) every day, and eggs were photographed every two to three days under a dissecting scope until they reached ~ 6 days until hatching (i.e., late-stage sensu Tankersley et al.)36. Homogenized egg water (seawater plus homogenized eggs, designated SW + HE, ~ 20 eggs mL−1) was utilized to induce pumping and grooming behavior and made according to Tankersley et al.36.
Ovigerous females were exposed to several sub-lethal concentrations of algicide combined with the homogenized egg solution and monitored for pumping and grooming behavior. Test solutions were diluted to 1.5 L with filtered 30 psu seawater, and 3.75 mL aliquot of a pre-prepared homogenized egg solution was added to achieve a final concentration of ~ 20 eggs/mL. These experiments used Batch 4–5–6, Batch 7, and Batch 3 of the IRI-160AA.
Between three and six crabs were tested at a time, and all crabs were staged the day of the experiment to verify that their eggs were no more than six days from hatching. All experiments were performed under dim red light to reduce disturbance. Each crab was tested in every treatment. A crab was placed into a translucent container (20.1 × 16.5 × 11.4 cm) with a given treatment condition and acclimated for 2.5 min. Then, for the following 2.5 min, the number of times the crab pumped its abdomen was recorded. Immediately following the end of the first crab’s measurement period, another crab was placed into the same treatment to begin its acclimation period. Each crab was returned to a flowing water table between treatments and remained there for at least twenty minutes before beginning the acclimation period of its next treatment. The treatment series began and ended with 30 psu seawater (SW), and proceeded through an increasing gradient of 0, 7, 11, and 17% IRI-160AA in SW + HE.
Each measurement period of the pumping experiments was filmed. The videos were reviewed later, and the time the crabs spent grooming their egg masses was recorded.
A χ2 test was performed for the 24 crabs tested to assess if the proportion of crabs performing the behaviors differed among treatments. A one-way repeated-measures ANOVA (Friedman Repeated Measures Analysis of Variance on Ranks) was used to assess trends in the number of pumps and the time spent grooming. Only crabs that performed the behavior were included in each analysis.
Oyster sub-lethality
Respiration
Respiration on oyster pediveligers following methods described for A. tonsa nauplii above. Two individuals were placed in each 80 µl well, with rates calculated per individual. Data were analyzed via a one-way ANOVA on Ranks. These experiments all used Batch 3 of IRI-160AA.
Activity
Activity experiments on pediveliger larvae were conducted in LAMs and followed similar methods to Acartia tonsa and Callinectes sapidus. The 24-h data was tested via a one-way ANOVA on ranks, while the data broken down by light phase was analyzed via a one-way repeated measures ANOVA. These experiments used Batch 3 of IRI-160AA.
An additional analysis of pediveliger activity occurred during the mortality experiment by ranking how active each animal appeared to be on a scale of 1 (High Activity, HA, animal was actively swimming), 2 (Medium Activity, MA, animal had its velum extended and cilia active, sometimes scooting across the bottom), 3 (Low Activity, LA, animal was enclosed in its shell but viscera moved when the shell was touched), and 4 (Dead/No Activity, D, animal was completely unresponsive even to repeated stimulation). Ranking occurred at the start of the experiment (where all animals scored as HA), at the 24-h mark, and at the 48-h mark. This assessment was analyzed via a χ2 test for both the 24-h and 48-h data sets. At the end of the LAM experiments, animals were analyzed in the same manner.
Activity experiments on the wild-type adult C. virginica occurred during the mortality experiments. At each 6-h time point, animals in the containers (0%, 1%, 5%, 10%, 13.5%, 18%, and 24% v/v IRI-160AA treatments) were scored as either Open (O) or Closed (C), and analyzed via a two-way repeated measures ANOVA on the proportion of animals that opened at each time point in each concentration.
Feeding
Feeding experiments occurred only on adult C. virginica. Animals and containers from the mortality experiments were rinsed to remove algicide residue, then filled with 1 L of 20 psu seawater and Isochrysis galbana at ~ 100,000 cells L−1, and one animal from each container was returned to it. Five milliliters from each container were removed immediately and in vivo chlorophyll a florescence was measured using a fluorometer (Turner Systems). Air stones were added to the containers to keep the algae in suspension, and lids were added to prevent liquid from bubbling out. After 6 h, another fluorescence reading was taken. Animals were given another 6 h to feed, and a final fluorescence reading was taken at the 12-h time point. Clearance rates (CR) were calculated according to Thessen et al.58 from time zero to six hours (initial rate, 0–6), and from six to twelve hours (end rate, 6–12), and compared across time ranges and treatments and strains using a three-way ANOVA. More
