Many people don’t think of invertebrates as lab animals, but they are used a lot for studies in toxicology, developmental biology, cellular and molecular biology, and radiation biology with euthanasia as the endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia. Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp (Artemia franciscana). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2. it was mixed with 5% eugenol, 4% g/L tricaine methanesulfonate (TMS), and then it was added to euthanasia solutions containing 20% alcohol, 29% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2. 5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 20% alcohol did make people sleepy, the time it took to fall asleep was different for each replicate group, and exposure led to more abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Even though all three methods of euthanasia worked, more research is needed before any suggestions can be made about how to best kill this species.
Brine shrimp (Artemia spp. ) are branchiopod crustaceans found along coastlines and in salt lakes. They are used in studies of radiation, toxicology, development, cellular and molecular biology, and they are also fed to aquatic lab animals like zebrafish. Two experiments at our institution showed that twenty-five percent alcohol was an effective euthanasia agent but caused abnormal behavior in Artemia. However, twenty-five percent alcohol, which is recommended by the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition (AVMA Guidelines) as a first-step agent for euthanasia of aquatic invertebrates, was not effective at producing anesthesia. 8 Information regarding euthanasia techniques for this species is sparse currently. As people become more aware that invertebrates may feel pain and distress, rules should be made for how to treat them humanely and how to end their lives.
The AVMA Guidelines provide euthanasia recommendations for both terrestrial and aquatic invertebrates. Eighteen recommendations include a two-step process that starts with anesthesia or assuming death and then adds a way to chemically or physically damage the brain or major ganglia. The use of an adjunct method alone is described as not acceptable. In the first step, solutions like eugenol, 1% to 5% ethanol, and magnesium salts are suggested. In the second step, additional methods like freezing, boiling, or plumbing are suggested. It is not okay to take invertebrates out of water to dry out, leave them in water without oxygen to become hypoxic, or use harsh chemicals or traumatic methods on them. 8.
We chose alcohol, eugenol, and tricaine methanesulfonate (TMS) as the three first-step (that is, anesthetic) solutions for testing because they are useful. There aren’t any published data on how much of these chemicals to give to Artemia, and there are only a few, very different sets of data on how much to give to other invertebrate species.
Alcohol may be used as an anesthetic in invertebrates. 10,16,24 The mechanism of action is not fully known but is likely multifactorial. 19 In mollusks, alcohol inhibits neuronal sodium and calcium channels. 21 In crustaceans, there is evidence of neuromuscular junction depression of the excitatory postsynaptic potentials. 3,19 Alcohol is cost-effective and easily available. The AVMA Guidelines say that ethanol concentrations between 1% and 5% should be used as the first step in a two-step euthanasia process, and concentrations above 70% should only be used as the second step. 8% ethanol has been used as an anesthetic on Penaeus monodon (a type of giant tiger shrimp). 23.
Eugenol is commonly used as an anesthetic in both fish and crustaceans. 5 This organic phenol is what clove oil is mostly made of, and it doesn’t pose many health risks or harm. Researchers think that it works by blocking vanilloid receptor 1 in fish. It has also been shown to bind to GABAA and NMDA glutamate receptors. 17 Eugenol is cost-effective and readily available. The AVMA Guidelines recommend using 0. 125 mL/L (125 mg/L) for euthanasia or anesthetic induction; lower concentrations should be used for anesthesia alone. 8 For anesthesia, a dose range of 0. 03 to 1 mL/L (30 to 1000 mg/L) has been recommended for crustaceans. 24. A safe amount of eugenol to put white Indian shrimp postlarvae to sleep was 1 to 2 milligrams. 3 mg/L; other doses tested were 2. 5 and 3. 7 mg/L. 5 For Norway lobsters (Nephrops norvegicus), a level of 900 μL/L (900 mg/L) of eugenol has been found to work. 12.
TMS, which is also called MS222, is an FDA-approved drug used to put fish, amphibians, and other cold-blooded aquatic animals to sleep. It is a sulfonated isomer of benzocaine. The exact way it works isn’t known, but it’s thought to be like how benzocaine blocks action potential conductance through voltage-gated sodium channels. 27 Although TMS has been suggested to be ineffective in crustaceans,9,13,24 successful anesthesia was shown in ostracods. 29. In the ostracod Eucypris virens, the lowest dose of anesthetic that worked was 500 mg/L, and the induction times ranged from 20 s to 2 s. 5 min depending on concentration. 29. It was found that 2500 mg/L in a 20-minute bath was the best dose for cherry shrimp, Neocardinina denticulate. 20 A well-known reference work recommends a dose of 100 mg/L for the anesthesia of aquatic invertebrates. 17 A small amount of TMS could be used to kill a lot of Artemia, but it would cost more than the other drugs.
Some of the extra drugs that the AVMA suggests for the second step of the euthanasia process are alcohol (200%), alcohol (2095%), and neutral buffered formalin (NBF). 8 All of these chemicals are common lab preservatives that don’t cost much and should work with post-euthanasia histology. Because researchers at our institution need to look at euthanized Artemia histologically, we did not look at other methods suggested by the AVMA, like boiling, freezing, and pithing. 8.
The goal of this study was to find a two-step method for euthanizing Artemia that would work well. Following the testing of various first-step solutions for anesthetic effectiveness, we thought that, based on our previous experience with 20% alcohol, 20% alcohol would most likely cause abnormal behavior and would not be suitable as a first-step agent. We also thought that both TMS and eugenol would work as anesthesia, but that eugenol would work more consistently than TMS. Finally, after Artemia were put to sleep using a first-step solution, we thought that 27% alcohol would not be enough for euthanasia, but that 29% alcohol and 10% NBF would work.
Adult A. franciscana (The Aquatic Critter, Nashville, TN) were maintained in 7. 5 L artificial seawater (made by using tap water and Instant Ocean [catalog no. SS15-10, Spectrum Brands, Blacksburg, VA]) at 25 °C, salinity of 1. 030 g/dL (40 parts per thousand), and pH 8. 0. Three times a day, the Artemia were fed spirulina from Whole Foods in Nashville, TN. To get rid of chlorine and chloramines, API Stress Coat (85A) from Mars Fish Care North America in Chalfont, PA was added to the water. LED lighting was provided on a 12:12-h light:dark cycle, and moderate aeration was provided at all times. The current study followed the rules and policies for using animals at Vanderbilt University Medical Center, even though our institution doesn’t need an IACUC-approved protocol for using invertebrates. Concentrations of the anesthetic solutions were determined according to the results of the titration trials (described later).
Brine shrimp, also known as Artemia, are small crustaceans that live in salty bodies of water. They are an important part of many aquatic ecosystems and are commonly used as live feed for fish in aquaculture. But an important question arises – do these tiny creatures actually experience pain?
In this article, we’ll examine the scientific evidence surrounding pain perception in brine shrimp and other small aquatic invertebrates.
What Are Brine Shrimp?
Brine shrimp are primitive arthropods that belong to the genus Artemia. There are several different species, with the most common being Artemia salina and Artemia franciscana.
These small animals have segmented bodies, large compound eyes, and 11 pairs of leaf-like appendages used for swimming and feeding Brine shrimp obtain their energy through filter feeding, consuming microalgae, yeasts, bacteria, and other organic particles suspended in the water
There are very high salt concentrations in brine shrimp’s water, which is why they do so well in salt lakes, coastal lagoons, and inland seas all over the world. Their ability to tolerate such hypersaline environments is one of their defining characteristics.
Do Brine Shrimp Have a Nervous System?
For an organism to be able to perceive and respond to painful stimuli it must have some form of nervous system. So do brine shrimp have the anatomical structures necessary?
Studies have shown that brine shrimp do have a basic nervous system made up of
- A dorsal brain or supraesophageal ganglion
- A double ventral nerve cord with segmental ganglia
- Sensory nerves throughout the body
Even though it’s not as complex as the nervous systems of vertebrates, brine shrimp can sense and react to things in their environment, such as possible threats. Because brine shrimp have sensory nerves and ganglia, it’s likely that they can sense and react reflexively to things that hurt them.
However, whether they can actually experience the subjective sensation of pain remains unclear.
Responses to Noxious Stimuli
Several studies have examined how brine shrimp behaviorally and physiologically respond to noxious stimuli, such as extreme heat or harsh chemicals.
Researchers have observed that when exposed to noxious stimuli, brine shrimp display:
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Protective motor reactions – contractions or tail flicks to move away from threat
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Physiological stress responses – increased heart rate and oxygen consumption
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Avoidance learning – avoiding stimuli after previous negative exposure
These studies demonstrate that brine shrimp detect and react to harmful stimuli using reflexive responses mediated by their nervous system. Their physiological changes and avoidance behaviors suggest a capacity for nociception.
However, the question remains whether these reflexive responses entail any conscious experience of pain or suffering.
Do Brine Shrimp Have Nociceptors?
Nociceptors are specialized sensory receptors that detect potential tissue damage and generate signals interpreted by the brain as pain in vertebrates. They are responsible for the sensory-discriminative aspect of pain.
Researchers have found some evidence that brine shrimp and other crustaceans may possess nociceptor-like cells:
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Their sensory nerves express genes resembling those involved in nociception in vertebrates
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Some sensory neurons exhibit sensitization after injury, reminiscent of nociceptor behavior
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They show reduced responses to noxious stimuli when given anesthetics or analgesics
However, the presence of nociceptor-like cells alone does not confirm the capacity to consciously experience pain. More research is needed to understand how brine shrimp process nociceptive input.
The Role of Opioids
Endogenous opioid chemicals act as natural pain modulators in many animals, including humans. If brine shrimp’s brains contain opioids, it could indicate an evolutionary need to regulate pain.
Interestingly, several studies have detected opioids like enkephalins, endorphins, and dynorphins in crustacean nervous systems. In vertebrates, these opioids bind to specific receptors to block pain signals.
Researchers have identified opioid receptors in crabs and crayfish and have shown opioids induce analgesia (pain relief) in these crustaceans. The presence of an opioid system in brine shrimp suggests they may have some capacity for managing nociceptive signaling. However, the role of opioids in invertebrates is still poorly understood.
Limitations of Brine Shrimp Research
Despite these intriguing findings, there are several limitations to research on pain perception in brine shrimp:
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Their simple nervous system makes studying nociception difficult
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Reflexive responses may be mediated unconsciously
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We lack methods to assess subjective pain experiences in brine shrimp
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Opioid systems may serve functions beyond pain modulation
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Findings in other crustaceans may not extend to brine shrimp
Ultimately, we cannot definitively conclude from this evidence whether brine shrimp feel pain consciously or not. We can infer a capacity for nociception, but not necessarily phenomenal pain.
The Bigger Debate Around Pain in Crustaceans
The question of pain in brine shrimp fits into a larger debate around invertebrates in general. Do any invertebrates actually feel pain, or are their responses just unconscious reflexes?
For crustaceans like crabs and lobsters, some compelling evidence indicates they could experience pain:
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They have complex nervous systems with sensory integration
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They exhibit flexible nocifensive behaviors beyond reflexes
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Their behavior changes when given analgesics
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They avoid electric shocks when given a choice, suggesting aversive experience
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They meet criteria proposed to indicate capacity for pain
However, the debate continues due to difficulties studying consciousness and the diversity of invertebrate species. Overall, the possibility that some crustaceans may experience pain should not be dismissed.
Animal Welfare Implications
If brine shrimp and other small aquatic invertebrates are capable of experiencing pain, it would have implications for how they are ethically treated in various contexts, such as:
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Aquaculture – Billions of brine shrimp are harvested annually as live feed for farmed fish. Methods should aim to minimize any potential suffering if they feel pain.
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Research – Using brine shrimp in experiments that may cause pain should involve precautions to avoid inhumane treatment.
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Environmental Protection – The possibility of pain perception supports conservation efforts to protect ecosystems where brine shrimp live.
Though the evidence is limited, the capacity for brine shrimp to suffer should not be completely ignored in practices that impact them. A precautionary approach is reasonable.
The presence of opioids and nociceptor-like cells suggests it’s plausible brine shrimp feel some form of discomfort or nociceptive urge. However, their simple nervous system makes studying pain perception difficult.
Given the ethical implications, we should give the possibility of pain experience the benefit of the doubt. More research on brine shrimp nociception and behavior could help shed light on this complex issue. Though uncertain, the capacity for these tiny aquatic creatures to suffer warrants careful consideration.
Titration of first-step solutions.
Using 95% reagent alcohol (85% ethyl alcohol, 5% isopropyl alcohol, 4% methanol; product no. Based on %209500-1,%20StatLab%20Medical%20Products,%20McKinney,%20TX) and tap water, we made solutions with varying amounts of alcohol (5%) and water. These solutions had ethanol concentrations of 53% in total. 7% for the 60% alcohol solution. Our goal was to keep the salinity of all the anesthetic solutions the same to reduce stress, but salt dissolved in solutions with 30% alcohol or more in the tank water. In these cases, the dissolved salt formed a gel-like consistency that was hard for the Artemia to swim through. So, we mixed alcohol with tap water. In early tests, Artemia did not react differently to tap water or tank water. We pipetted 1. A 24-well plate had two wells that were filled with 5 mL of solution each. Five artemia were then put into each well. The time to anesthesia and any abnormal behaviors were recorded. Anesthesia was defined as not being able to move forward or respond to a probe’s stimulation. Euthanasia was defined as a lack of thoracopod movement for 10 s of observation.
Eugenol was tested at 1. 3 and 2. 5 mg/L. For a stock solution, we diluted 99% eugenol (product no. They mixed a substance (AC119110050, Acros Organics, Morris, NJ, USA) with 29% alcohol, then with tank water, and then they put the mixture in an amber bottle and left it at room temperature. The final concentration of alcohol in the 2. 5-mg/L dose was 2. 4%. We used a pipette to put 1 mL of each solution into two wells of a 24-well plate. Then, we put five Artemia in each well. The time to anesthesia and any abnormal behaviors were recorded.
TMS (product no. Western Chemical’s NC0135573, Tricaine-S, was diluted to 10 g/L with tank water and then buffered to pH 7 to 7. 5 with sodium bicarbonate (product no. S233-500, Fisher Scientific, Hampton, NH). The solution was then weakened even more with tank water until it was 1, 2, or 4 g/L. The resulting solutions were kept at 4 °C in amber bottles. Each solution was allowed to warm to room temperature prior to exposure. We used a pipette to put 1 mL of each solution into two wells of a 24-well plate. Then, we put five Artemia in each well. The time to anesthesia and any abnormal behavior were recorded.
Artemia were put into four groups, with n = 2030 cells per group, and were mixed with alcohol solutions 4% g/L and TMS solutions 2%. 5 mg/L eugenol, or tank water (control). Each of the six 24-well plates had 20 wells that were randomly assigned to either 1 mL of an anesthetic solution or tank water. We then used a transfer pipet to add a single Artemia to each well. To keep the solution from getting too weak, no more than 50 μL of tank water was moved with each shrimp. A person who wasn’t aware of the treatment used a wooden probe to confirm anesthesia, which was defined as the animal not moving forward or responding to the probe. Time to anesthesia was recorded for each animal, with a cut-off time of 60 min. After 5 min of anesthesia, the Artemia were transferred to a euthanasia solution.
In 2010, anesthetized Artemia were split into subgroups. Each subgroup was then sent to a different euthanasia solution, such as alcohol (product no. 7070-1, StatLab), 95% alcohol (product no. 9500-1, StatLab), or 10% NBF (product no. 28600-5, StatLab). Time to euthanasia, defined as a lack of thoracopod movement for 10 s of observation, was recorded. 28.
Behavior during the first 5 min in the anesthetic solution was scored by a treatment-blinded observer. One point was given for each of three types of abnormal behavior: strange posture, hyperactivity, or seizure-like behavior. This made a scale from 0 to 3: 0 meant no abnormal behavior, 1 meant mild behavior, and 2 or more meant severe behavior.
Anesthesia, euthanasia, and behavior scoring were tested in triplicate on separate days.
Artemia (n = 10) from each anesthetic group were anesthetized in a 24-well plate as described earlier. After being put to sleep for 5 minutes, they were rinsed by putting them in a well of tank water and then moved right away to a second well of tank water to see how well they would recover over the next two hours. Recovery was considered to be achieved on regaining forward motion. This experiment was repeated in triplicate (total, n = 90).
During the original experimental period, eugenol consistently induced anesthesia, as shown in the Results section. About 4 months later, we tried to induce Artemia again with anesthesia but couldn’t get the same results with 2 5 mg/L eugenol. Troubleshooting was performed, including purchasing a new bottle of eugenol. We started measuring eugenol in a series of titrations to find the concentration that would give us the same results as the last experiment. Four titrations of eugenol were performed over a 7 mo period to account for possible seasonal variation. Following the steps previously explained, eugenol was made: 99% eugenol was first diluted with 29% alcohol, and then it was diluted even more with tank water. Pipetting 1 mL of each solution into two wells of a 24-well plate made two copies. Five artemia were then put in each well. Concentrations tested included 0. 125, 1. 3, 2. 5, 13, 25, 75, and 130 mg/L; the total alcohol concentration in the 130-mg/L dose was 12. 4%. As a control, 2 wells of 60% alcohol each containing 5 Artemia were tested also. The time to anesthesia was recorded.
We made Kaplan–Meier survival and cumulative morbidity curves for each treatment group. For anesthesia and euthanasia, we used log-rank statistics to look for differences. Proportional odds logistic regression was used to determine cumulative odds ratios of abnormal behavior between groups. The Kruskal–Wallis ANOVA was used to compare behavior scores between groups. Logistic regression analysis was used to compare the significance of anesthetic recovery rates among the 3 anesthetic groups. One-way ANOVA was used to determine significance of replication anesthetic times of alcohol. A P value of less than or equal to 0. 05 was considered significant. Statistical analyses were performed by using Stata version 14 (StataCorp, College Station, TX) or Prism 7. 03 (GraphPad Software, La Jolla, CA).
In the study, one Artemia from the eugenol group and three from the TMS group were taken out because of mistakes in the recording.
How Do Brine Shrimp Survive In Packaging For Years?
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