Experimental design:
To understand how the mode of exposure of sublethal levels of microcystin-LR (MCLR) impacts a keystone and ecological engineer species, crayfish were exposed to environmentally relevant concentrations of MCLR via water or sediment. The effects of exposure were assessed using crayfish foraging behavior, bioturbation behavior, and a food consumption (using fish gelatin as a food source) in a mesocosm assay. To mimic an acute exposure, crayfish were exposed for four days with a sublethal concentration of MCLR (10 μg/L) in small-scale dosing tanks under one of five conditions: MCLR and EtOH in the water column, MCLR and EtOH in the sediment, EtOH in the water column, EtOH in the sediment, and a control with no MCLR or EtOH. After the dosing period, crayfish were moved to an experimental mesocosm filled with flowing river water. A total of 79 trials were run and were distributed among the five treatment conditions: control (N = 14), EtOH in the sediment (N = 17), EtOH in the water column (N = 16), MCLR and EtOH in the sediment (N = 15), MCLR and EtOH in the water column (N = 17).
Collection and Housing of Organisms:
Seventy-Nine form II male rusty crayfish (Faxonius rusticus) were collected from Maple Bay in Burt Lake (45.4802 ºN, 84.6974 ºW) using hand nets and from Carp Lake River in Emmet County, Michigan, USA (45.7497 ºN, 84.8292 ºW) using minnow traps baited with Beach Cliff® sardines in soybean oil sardines. After collection, crayfish were transported to the University of Michigan Stream Research Facility for housing. Crayfish were placed in a flow through cattle trough (237.5 86.4 60.1 cm, l x w x h) which were fed from the east branch of the Maple River. Unfiltered river water flowed into the tank from a 7.6 cm PVC delivery pipe and exited the tank via a standpipe which kept the water depth at approximately 60 cm. Terracotta pots were placed in the cattle through to be used as crayfish shelters. Crayfish fed on the detritus that arrives via the flow of water. The cattle trough was placed outside and exposed to natural light:dark cycles and natural temperature fluctuations May 15th to July 25th. Only form II males were used within the trials. Post-orbital carapace length (measuring from the orbital socket to the posterior of the carapace), carapace width, and the right chelae of each crayfish were measured to the nearest 0.05 cm before use in a trial.
Microcystin-LR Exposure:
Microcystin-LR (MCLR) with a purity of 95% was purchased from Focus Biomolecules for a total quantity of 800 μg. Dosing regimens occurred in plastic aquaria (27.9 x 16.8 x 13.7 cm, l x w x h) which were filled with 1.75 L of unfiltered water and 1.35 L of sand taken from the Maple River. For water column-exposed studies, 17.5 μg MCLR was suspended in 35 μL 98% EtOH and added to 1.75 L of water to produce a final concentration of 10 μg/L of MCLR and 20 μg/L EtOH. For sediment-exposed studies, 1.35 L of water dosed with 13.5 μg MCLR suspended in 27 μL EtOH to a concentration of 10 μg/L of MCLR and 20 μg/L EtOH was mixed with 1.35 L of sediment and was allowed to sit for 24 hours. For control studies, no MCLR or EtOH was added to the system. For water column-exposed vehicle control studies, 35 μL EtOH was added to 1.75 L water to produce a final concentration 20 μL EtOH/L water. For sediment-exposed vehicle control studies, 1.35 L water dosed with 27 μL EtOH for a concentration of 20 μL EtOH/L water was mixed with the sediment and was allowed to sit for 24 hours. Exposure tanks were dosed once before each trial. Each tank was located indoors and was aerated with an air bubbler. Crayfish remained in the exposure tanks for four days.
Experimental mesocosm:
The experimental mesocosm (81.3 x 30.5 x 40.6 cm, l x w x h) consisted of concrete cinder blocks covered with plastic sheeting and were used to make eight flow-through stream mesocosms. Two 208 L plastic drums served as constant head tanks for the eight mesocosms with a single drum feeding four mesocosms. Constant head tanks were filled with water from the Maple River. Water entered the constant head tanks through 7.6 cm PVC pipes. Each plastic drum fed two 1.9 cm diameter hoses into each of the eight mesocosms with a flow rate of 0.1 ± 0.05 L/s per hose. Nylon stockings (0.01 cm2 mesh size) covered the ends of the supply pipes to filter macroinvertebrates and organic matter. The bottom of the experimental mesocosms were filled to a depth of approximately 5 cm with sand collected from the Maple River to mimic natural conditions. Approximately 5 cm of sand taken from differing locations along the Maple River to mimic natural conditions. Red light bulbs were used to illuminate the system. A wooden frame held Swann PRO-530 Cameras approximately 1.3 m above the surface of the water to record crayfish behavior.
Food Production:
Fish gelatin was prepared by blending 0.175 L boiled water, 13 g sardines, and 7 g Knox unflavored gelatin mix. After blending, the gelatin was placed into plastic scintillation vial caps, covered with plastic wrap, and refrigerated overnight. Each cap was filled with approximately 2.5 g of food .
Experimental protocol:
At the beginning of each trial, one crayfish was placed into each exposure tank between 1008 and 1130. After approximately four and a half days in the exposure tanks, crayfish were relocated into the experimental mesocosms at 2345, allowing the crayfish 15 minutes to acclimate to their new environment. Fish gelatin was weighed and then placed in the experimental mesocosm at 0000 and was promptly removed the following morning at 0800. After removal, the fish gelatin was tamped dry and weighed. Crayfish were removed from the experimental mesocosms at approximately 0800 the next morning. Crayfish behavior was recorded from 0000-0400.
Behavioral analysis:
The videos captured on the Swann PRO-530 Cameras were subsequently analyzed by two viewers blind to treatment. These video recordings were analyzed and the duration of three types of behaviors that contribute to bioturbation were recorded: foraging, working sediment, and walking. Crayfish were classified as exhibiting foraging behavior when directly on top of the food source consuming food. Crayfish were classified as working the sediment when using their chelae, their two front walking legs, or their entire body move sand. Crayfish were classified as walking when changing the location of their bodies at 1 cm/s or greater. Behaviors from video-recordings were categorized by the second.
Consumption analysis:
Crayfish consumption of food (fish gelatin) during an eight-hour period was calculated by the the difference between the weight of the fish gelatin before and after each trial. Fish gelatin was weighted before each trial. After each trial, the fish gelatin was tamped dry and weighed again.
Animal housing and tissue collection for physiological study:
Nineteen form I male rusty crayfish (mean ± standard error of carapace length; 20.73 ± 1.54 cm) were collected from the North Branch Portage River (41.2142 ºN, 83.5328 ºW) using hand nets and minnow traps baited with Beach Cliff® sardines in soybean oil sardines. Crayfish werewere were housed in (21.5 x 14.2 x 13.5 cm, l x w x h) dosage tanks for approximately 108 hours. Dosage tanks contained 1.75 L artificial pond water and 1.35 L sand. Water Artificial pond water is tap water that has been was dechlorinated for at least 24 hours. Crayfish were exposed to conditions identical to those outlined above in the Microcystin-LR Exposure section.in one of the five treatment groups (refer above for methods of dosing).. After exposure, lLive crayfish were placed in the freezer (-20 °C ) for approximately 10 minutes before removal of antennules. Antennules were immediately fixed in 4 % paraformaldehyde. Crayfish were placed back in the freezer until they stopped moving. Crayfish were euthanized through rapid decapitation. 100 uL haemolymph was collected from the heart and was added to 150 uL heparin sodium and van-harrervalds solution mixture (acting as an anticoagulant). Lastly, the hepatopancreas was removed and weighed. The hepatopancreas was then cut and separated into three 0.2 g pieces. One piece was saved for a TUNEL assay and two pieces were saved for flow cytometry
Flow Cytometry
Each piece of hepatopancreatic tissue tissue was resuspended in phosphate-buffered saline (500 µL (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)) supplemented with 5% fetal bovine serum (BioSpa) and 5.5 mmol ethylenediamine tetraacetic acid (EDTA). The tissue was manually mottled with a small manual homogenizer to separate the cells from the tissue. Cells were filtered using a100 µm mesh filter. After filtration, cells were resuspended in 250 µL PBS, and centrifuged for 10 min at 467 × g at 4 °C. The supernatant was subsequently removed and 500 µL of the appropriate stains were added. Fluorescent stains were added to quantify cell viability. The three stains used were fluorescein diacetate (1 mg/mL) to quantify the number of alive cells, propidium iodide (1 mg/mL) to quantify the number of dead cells, and SYBR Green I (1 mL in a 1.5 mL tube) to quantify the number of all cells. For each organism, one tube will contain fluorescein diacetate and propidium iodide. A second tube will contain propidium iodide and SYBR green. After staining, samples were stored overnight at (4 °C). Blank samples were prepared in the same way but without stains. Samples were processed for flow cytometry using channels FL1 (green fluorescence, 530/30) and FL3 (red fluorescence, 670 Long Pass).
