To date there has been significant scientific publications that introduce methods on maintaining crayfish in a laboratory setting; However, there a far fewer studies that present life support systems in a biomedical vivarium. General crayfish life support literature has specifically investigated best practices for stocking densities and basic environmental parameters to house crayfish (i.e., day:light photoperiod, ionized water calcium carbonate concentrations, and pH).1,67,75,79,105,107,111,112 The authors' present a description of housing, husbandry, and selected clinical techniques in P. clarkii housed in a biomedical research setting, allowing for future evaluation of practices. The husbandry and housing described is economical, feasible, and reproducible in most laboratory animal facilities. Aquatic ecology experts were recruited to further investigate practical methods on assessing overall physical health, and behavior of the crayfish while housed in the life support systems. This setup provides a static system with individual tank aeration and basic bio-filtration, replicating the crayfish’s natural environment, while in a vivarium. Commercial alternatives for bio-filtration and aeration are available throughout the industry if future investigators want to purchase pre-made foam filters instead of manufacturing them individually. The primary containment system ensured no crayfish escaped from their enclosures. This is especially important, as crayfish can and will escape if not properly housed. Researchers must be informed that currently there is no information available on reports of these specific clear polypropylene containers used in the study leaching toxic or immunosuppressive chemicals in the water over time. There were no adverse effects attributed to the containment system material during the duration of this study.
As explained in the introduction, this and many other species of crayfish is robust and thus can be invasive.5,40,46,49,78,82,101 Prevention of non-native crayfish species entrance into local waterways is imperative during housing within a vivarium, as well as with disposal of animals. Before bringing crayfish into any research environment it’s critical that investigators consult with local authorities and agencies who provide oversight for the movement of aquatic species (i.e., State and Country Departments of Agriculture, FDA). This is to ensure that if required the necessary permits and consent is obtained. Investigators must be aware that depending on the region requirements may vary.
Individually housed benthic animals have been shown to grow larger due to increased water volume available, and accurate control of individual environments.26,61,75,93, 105–107,113 Additionally, it is well known that P. clarkii are highly cannibalistic which is typically why individual housing is a necessity for this species.6,26,45,56,71 For biomedical experimental trials, where reproducibility and proper biosecurity is paramount for validity, individual housing is imperative with novel specimens.13,63
Substrate would prove an optional aspect of the study. It allows for natural explorative behaviors as compared to a bare-bottom tank, since it simulates the natural environment. The use of substrate has been shown to promote increased survival rates in recirculating systems.32,33 Investigators must be aware that substrate can be difficult to clean with a siphon, which could result in detritus material building up over time, altering water quality parameters.
All subjects displayed no adverse clinical signs throughout the entirety of the housing and husbandry study arm. A 50% water change every 7 days proved efficacious during the study period. In fact, normal molting behavior and egg production were seen in several of the crayfish, both species-specific behaviors supporting efficient replication of their natural environment (unpublished results). Future studies however, are required to further investigate the effects of the presented life support system on breeding, fecundity, and best stocking densities during longer experimental periods similar to Rodriguez-Serna et al. 2000.79 Additionally, various levels of calcium carbonate in the water should be examined to determine optimal levels for molting, especially in young crayfish.34,60,91 Although not investigated in this study, weak light intensities (38 lux) has been shown to improve the welfare of Astacus astacus, favoring exploration of lighted environments.1
Invertebrates are currently not covered under the Animal Welfare Act and many other countries currently exclude invertebrates in their regulations.39,66,74 Many believe this stems from a lack of public concern for invertebrate welfare and the inability to conclusively show that distress occurs in invertebrates due to nociceptive stimuli. No matter what public opinion is, nociception has been seen in invertebrates and thus the welfare of these animals must be considered in the laboratory.24,35,80,83,109 Further reading is encouraged through Harvey-Clark (2011) for interested audiences.35 During the early development of this study our institution’s IACUC office was consulted. It was determined that a protocol was not warranted based on current regulations within the USA and to decrease regulatory burden. RSCHA used as a mechanism to quantify and assess welfare. Close collaboration was encouraged and maintained throughout the entire project to ensure that humane treatment was provided for these subjects. The institutions laboratory animal veterinarians were additionally consulted to provide guidance and oversight.
Although invertebrates are not considered to be a “covered” species in many of the animal research regulatory documents in the USA, authors urge that care must be taken when performing non-terminal hemolymph procedures.74,96 (the guide, blue book, AWA) Current nonterminal bleeding standards in mammals limit to blood draws to 10% of total circulating volume, once every 3 to 4 weeks, if no replacement fluids are provided. Repeated blood samples cannot exceed 1% of the animal’s blood volume, once every 24 hours.72 Blood volume compared to weight is a negative relationship with increasing crayfish size, and inconsistencies have been seen when assessing carapace length to wet weight. Studies have shown that crayfish blood volume to weight ratio can range from 9–43%.76,77 In Limulus Polyphemus 20–40% hemolymph collection has shown varying mortality from 0–30% depending upon the study.7,41,54 We based our hemolymph collection limits and total blood volume off of mammalian studies, but further studies are required to evaluate the maximum total volume for nonterminal hemolymph collection in crayfish.72,73 Of utmost importance while performing hemolymph collection (or giving injections through the abdominal segment) while at the VCHCZ, is that ventral midline is avoided. The ventral midline houses the ventral nerve cord, which is synonymous to the vertebral column of mammalian species.8,78,83 The middle also houses the abdominal section of intestine which could cause bacterial translocation if aspirated. If the needle is inserted too cranial, the needle has the potential to enter the cephalothorax, and cause damage to internal organs.27,38,44,82
Authors chose VCHCZ site for hemolymph collection due to the restraint requirements of crayfish, allowing for clear visualization of anatomic landmarks. Previous works have noted collection of hemolymph from the ventral abdomen.12
Additional options for hemolymph collection are described in the literature for a number of different aquatic invertebrates. P. clarkii hemolymph collection can also be performed through pericardial cavity puncture, and inter-joint membranes.16,69,86 Albores et al. (1993) have presented the following as a collection protocol: “The haemolymph was extracted from the pleopod base of the first abdominal segment near the genital pore” as an acceptable way to obtain 100uL of haemolymph in Penaeus californiensis.99 Shields (2017) states that any crustacean with a > 15mm carapace (width or length) can have hemolymph collection performed at the leg joint arthrodial membranes, or at the junction of the abdomen and carapace with a 27 gauge needle.81 In Litopenaeus vannamei, hemolymph can be collected with a 26 gauge needle from the first abdominal segment venous sinus.85 In Limulus Polyphemus, blood collection is typically from the heart itself at the joint where the prosoma and opisthosoma meet.11 The varied collection techniques for hemolymph have not been investigated formally in a study for efficacy or translational ability, and would provide useful information on guidance for best practices.
Commercially available alternatives, that include sodium thiosulfate, are also appropriate for dechlorination of water from laboratory sources. Typically these solutions are around 3.175g/L Sodium thiosulfate.68,98
MS-222 is currently the only FDA approved drug for use in anesthesia in fish and other cold-blooded aquatic animals, and thus commonly used, indicating the need for further investigation in crayfish.9,25,95Immersion of MS-222 did not have any anesthetic effects on the crayfish used in this study. This is in agreement with many published works.17,23,88 Further investigation with immersion at higher doses or direct injection into the hemolymph may be warranted. Propofol was utilized for sedation methods within this study, as it has been shown to be safe and have neuro-protective effects.2,3,48,52,93 Propofol is well researched throughout the literature.18,31,52,59,64,97 Propofol must be used under veterinary supervision, but it is not a controlled drug under DEA guidance.4 This makes propofol easy obtained in a biomedical environment that has laboratory animal services department, as veterinary staff is familiar with the handling, use, and disposal of this commonly used and inexpensive drug. Other options for sedation including clove oil, cooling, MgCl2, and CO2 were not investigated in this study.28,30
Although mortality rate observed with injection was 15.8%, this was due to investigator specific errors in three of the tested crayfish. One crayfish had a handling induced thorax fracture in association with recent ecdysis. Two other crayfish experienced multiple traumatic needle insertions during propofol administration into musculature and nerve cord due to improper injection technique. Thus avoiding these mistakes is imperative for the appropriate collection of hemolymph. Future studies on VCHCZ utilization would be useful for optimizing this approach. Propofol injected into coelomic hemolymph provided effective anesthesia for non-invasive or experimental techniques and research procedures, with the longest duration of action at 100 mg/kg when compared to propofol immersion. With an anesthetic effect lasting 164 minutes, investigators would have sufficient time to perform procedures that may require lengthier anesthesia periods for experiments (i.e., imaging modalities: PET-CT, ultrasonography). This duration of time also creates a long post-operative period, which could be inappropriate for some study protocols. Future studies are indicated to determine if this dose is appropriate for surgical interventions and if the dose could be optimized for more succinct time management. Propofol immersion provided enough sedation, or light anesthetization, for safe and effective handling. Immersion could be utilized in preventing injury to the crayfish and the investigators during personnel training sessions. Future work is needed to optimize the propofol concentrations and dosing.
Our team was able to successfully demonstrate effective techniques on obtaining for cytological evaluation without any animal losses. It is important to note that authors recommend analyzing fresh collected samples within a 2-hour period using EDTA tubes. Previous works have demonstrated the effectiveness of EDTA solutions for hemolymph storage, and adaptions of protocols were used in this experiment.12,47,65,99
In summary authors also present practices and data that could be incorporated when housing other crayfish in the genus Procambarus and other related species used not only in biomedical research, but also in aquaculture settings if warranted.