Bobtailsquid.ink



Federal animal welfare regulations require that the Institutional Animal Care and Use Committee (IACUC) must review and approve all activities involving the use of vertebrate animals prior to initiation of such use. Once approved by the IACUC, any change(s) to the following protocol must be submitted in a written amendment for review and approval by the IACUC prior to implementation of the change(s). Protocols and related amendments are active for 1 year from protocol approval date and must be renewed yearly.PLEASE DO NOT REMOVE SECTION DIRECTIONSTitleProvide a descriptive title for the proposed study that includes the common name of the species to be used.Effects of modeled microgravity on the beneficial symbiosis between the bobtail squid Euprymna scolopes and luminescent bacterium Vibrio fischeri2. Principal Investigator(The ONE individual responsible and accountable for the design, conduct and monitoring of the protocol.)Provide the following information on the Principal Investigator (PI) and Responsible Point-of-Contact for this protocol. Please identify all other personnel (including Co-PIs) who will perform experimental manipulations on animals in section 19.PIName: Jamie S. FosterTitle: Full ProfessorAffiliation: University of FloridaMailing Address: Space Life Science Lab, 505 Odyssey Way, Merritt Island, FL 32953Email: jfoster@ufl.eduPhone Number: 321-525-1047 (cell phone only; no office or lab phone)Fax Number: noneEmergency Contact Number: 321-525-1047Responsible Point-of-ContactName: Jamie S. FosterTitle: Full ProfessorAffiliation: University of FloridaMailing Address: Space Life Science Lab, 505 Odyssey Way, Merritt Island, FL 32953Email: jfoster@ufl.eduPhone Number: 321-525-1047 (cell phone only; no office or lab phone)Fax Number: noneEmergency Contact Number: 321-525-10473.Protocol InformationProtocols are approved for one year. Please indicate if this is a new or renewal protocol.3A. FORMCHECKBOX New ProtocolIf this is a new protocol, please move on to section 4 FORMCHECKBOX Renewal ProtocolIf this is a renewal protocol, please complete the following:3B.Previously Approved Protocol Number 3C.Problems/Adverse Events: Have there been any unanticipated problems that have affected animal use, welfare, morbidity, or mortality? FORMCHECKBOX Noformcheckbox YesIf YES, provide a summary of the problems, the cause(s), if known, and how these problems were resolved.3D.Progress Report: Provide a brief statement of progress to date. State what was done in the previous approval period, how many animals were used, what was learned, and why additional animals are required to continue the study. If this work resulted in any changes in how you will proceed, please explain. 4.Nature of the Project/Study4A. Project Types:(indicate all that are applicable)Type of Procedures:(indicate all that are applicable)Hazardous Procedures:(indicate all that are applicable) FORMCHECKBOX Research/ Discovery Project FORMCHECKBOX Testing –Preclinical Development FORMCHECKBOX Training FORMCHECKBOX Other (specify): FORMCHECKBOX Behavioral/Neurobehavioral Studies FORMCHECKBOX Blood/Tissue/Embryo Collection FORMCHECKBOX Breeding Colonyformcheckbox Prolonged Restraint (>15min) FORMCHECKBOX Biohazardous/Infectious Agents FORMCHECKBOX Rodent Cell Lines/Biological Materials FORMCHECKBOX Human Cell Lines/Biological MaterialsSpecies:Indicate all applicable: FORMCHECKBOX Mice FORMCHECKBOX Rats FORMCHECKBOX Guinea Pigs FORMCHECKBOX Rabbits FORMCHECKBOX Nonhuman Primates FORMCHECKBOX Other (specify): Cephalopods FORMCHECKBOX Compound Dosing FORMCHECKBOX Single Dose FORMCHECKBOX Multiple Doses FORMCHECKBOX Induction of Acute Disease FORMCHECKBOX Induction of Chronic Disease FORMCHECKBOX Surgeryformcheckbox Vendor performed FORMCHECKBOX Multiple Survival FORMCHECKBOX Single Survival FORMCHECKBOX Terminal FORMCHECKBOX Tumor Inducement/Xenograftformcheckbox Expts. done (partially) at another institution FORMCHECKBOX Other (specify): Animals inoculated with natural mutualistic symbiont found in wild FORMCHECKBOX Recombinant DNA FORMCHECKBOX Chemical Hazards FORMCHECKBOX Radioactive Materials/ Radioisotopes FORMCHECKBOX X-Ray Machine Usage FORMCHECKBOX Irradiator Usage 4B.Is this a Pilot/Feasibility Study? (Small-scale discovery trial of limited duration and animal numbers designed to evaluate. refine, or modify devices, techniques, methodology and/or study design prior to submitting a protocol for a full-scale project.. Duration of study Is usually limited to six months or less and fifty animals or less) FORMCHECKBOX Yes FORMCHECKBOX No4C.Federal Funding Source(s) and Peer ReviewIs this project currently or proposed to be funded by either federal or other funding agencies? FORMCHECKBOX No. Please move on to section 5. FORMCHECKBOX Yes. Please complete the following:Current and Proposed Federal (or other) Funding Sources:NASATitle(s) of Grant Submittal: 80NSSC19K0520 - Effects of modeled microgravity on the induction of bacteria-induced apoptosis during animal development Has this proposed activity undergone peer review? FORMCHECKBOX Yes FORMCHECKBOX NoIf YES, who provided peer review and when? The grant was peer-reviewed through the NASA Space Biology program. Awarded 6/1/2018(For new protocols include one copy of the grant proposal with protocol submission.) A copy of the grant is attached to the document5. Location of Work Please mark all areas where work pertaining to this protocol will be conducted. If you will be doing work outside of the Contract Research Services (CRS) Facility, please list the institution’s full name under ‘other’. Please note all areas used for animal housing or procedures must first be approved by the IACUC and will be included in all facility inspections. FORMCHECKBOX Contract Research Services Facility At Ames Research Center FORMCHECKBOX Building N236 (Main Vivarium) FORMCHECKBOX Building N239 (list room number): FORMCHECKBOX Other (list buildings/ room number): (Please complete the attached form IACUC PERSONNEL SIGNATURE PAGE) FORMCHECKBOX NASA Facilities FORMCHECKBOX Johnson Space Center (list buildings/ room number): FORMCHECKBOX Kennedy Space Center (list buildings/ room number): FORMCHECKBOX Other (Including Field Studies. List facility or location, address, and room number):Field Collection: Adult breeder squid are collected in the shallow waters of O’ahu, Hawaii Breeding colony: Animal breeding colony is maintained at the University of Florida (UF) growth chamber (CEC2) in the Space Life Science Lab (SLSL), Merritt Island, FLSquid incubations and experiments: UF SLSL labs 233 and 234.6. Project OverviewUsing layperson terms, describe the purpose of the study and its intended benefits to science, medicine, or mankind. Please avoid the use of technical jargon and abbreviations that would not be understood by a non-scientist. This project examines the effects that modeled microgravity has on beneficial interactions between animals and their natural microbiome. The bobtail squid (Euprymna scolopes) has a specialized organ called the light organ - the site of the symbiosis with the beneficial bacterium Vibrio fischeri. The bacteria cause a normal developmental remodeling of the light organ and we are assessing how modeled microgravity impacts that normal bacteria-induced development. The Foster lab is using molecular techniques to examine the processes by which the bacteria and squid communicate as part of the normal health of the squid.7.Animal InformationPlease provide the specifications for all of the animals requested for use in this protocol. Please list each species separately. Please put ‘any’ if no preference. Species:Common Name/Scientific NameBreed/StrainPreferred SexPreferred Age RangePreferred Weight RangePreferred Vendor or SourceTotal Number RequestedBobtail squid/Euprymna scolopesWild caughtM and FReproducing adults and paralarvaeanyNone – field caught squid by PI15 field caught adult squid/yr ; 12,000 embryo-paralarvae squid/yr7A.Justification of SpeciesPlease explain why the species and/or strain(s) requested is/are the most appropriate for this research. Statements that the planned species is traditionally used for the proposed research are not sufficient.For 30 years, the bobtail squid has served as a model organism to understand symbiosis for four primary reasons: 1) the symbiosis is binary in that there is only one symbiont and one host, thereby enabling the effects of the bacterium on the host development to be more easily studied; 2) the squid have a very short developmental time line (~20 days) enabling rapid turnover of experiments; 3) adult female squid lay approximately 1000-3000 eggs in their lifetime enabling numerous experiments from a minimal number of squid; and 4) the paralarvae (i.e., newly hatched juveniles) are small (~3 mm) in size helping to reduce costs associated with modeled microgravity studies.7B. Justification of Animal NumbersProvide a detailed justification for the number of animals requested. Include number of animals/group X number and composition of groups/study X number of studies. Whenever possible, the number of animals requested should be justified statistically. These numbers are the annual maximum that will be used, inclusive of breeding, animals used for maintaining colony size, animals used for developing and practicing techniques, or animals required for unforeseen circumstances.Animal TypeNumber (3-yr period)UseAdult Female36Breeding ColonyAdult Males9Breeding ColonyHatchling Squid (i.e. paralarvae)36000ExperimentalTypically, we maintain eight female squid at a given time, as not all of the females will lay eggs. When we collect squid in the wild we do not know the age of the squid and therefore, we try to collect cohorts of between 6-8 females and 1-2 males each collection trip to try and ensure that we collect a few females that are of a reproductive age (~ 3-6 months old). Females older than six months typically no longer lay viable eggs. We typically collect adult squid twice a year to maintain an active breeding colony throughout the year.For experiments involving hatchling squid, also known as paralarvae, a typical simulated microgravity experiment uses 30-80 hatchlings; therefore, our maximum egg production would generate 12,000 hatchlings each year enabling approximately 200 experiments annually.7C. Special Animal CareIf housing or care of animals is different from standard Animal Facility procedures (e.g., individual housing of rodents; change in caging type or light cycle or diet or cleaning schedule), provide detailed description and scientific justification.Source of squid:Adult bobtail squid (2-5 cm in size) are collected from the shallow sand flats in O’ahu, Hawaii by the PI at night. The squid are collected along the shore in approximately 1-2 feet of seawater. A flashlight is used to spot the squid and a dip net is used to scoop up the squid. Each adult squid is then transferred to an individual 1-gallon zip lock bag containing 2.5 liters of local seawater without touching them. The bags are then placed in a 5-gallon bucket and then transported to the Kewalo Marine Lab, which is part of the University of Hawaii, located in Honolulu. The adult squid are released into running seawater tanks at the Kewalo Marine Lab for the duration of the collection field trip and fed local shrimp each day.For shipment back to the University of Florida labs at the Space Life Science Lab (SLSL), each squid is transferred to a polypropylene bag containing 1.5 liters of seawater, topped with pure oxygen and then the bag is closed with elastic bands. A secondary containment bag is added and sealed. The bags of squid are then placed in a padded shipping crate and transferred to Delta Cargo where they are flown to Orlando airport and then transported to the SLSL.The squid are acclimated to the closed recirculating aquarium system at the SLSL (Figure 1) using the “drip method”, which has had a 100% survival rate since it was implemented in the Foster lab in 2009. Using the “drip method”, squid are removed from the bags and placed in a Pyrex dish containing 1 liter of the water they were transported in. The ammonia levels are measured and are typically > 4 ppm upon arrival. Every 15 min, approximately 50 mL of water from the bowl is removed and 50 mL of new aquarium water is added to the Pyrex bowl containing the squid. This process is repeated until the ammonia levels are below 0.5 ppm (approximately 3 hours). The “drip method” occurs inside the environmental growth chamber so that temperature of the water in the Pyrex dish containing the squid is also slowly acclimating to the temperature of the chamber. Once ammonia levels are below 0.5 ppm the animals are transferred to individual compartments within the closed aquarium system (Figure 1). The squid are closely monitored for typical behavior (e.g., swimming to the bottom and burying in the sand, feeding on shrimp). 397510000If for some reason a squid shows signs of osmotic shock (flipping over on its back) the squid is quickly returned to the Pyrex bowl and water is flushed across its gills using a plastic pipette. This response has happened only once in the past 10 years and the squid was revived and the drip method was continued for another hour until the squid behaved normally and acclimated to the aquarium seawater conditions.Squid breeding areasThe adult squid are maintained in a closed recirculating aquarium system kept in the Environmental Growth Chamber area of the SLSL (Figure 1). The University of Florida has a dedicated chamber for the squid culturing facility. The PI and trained students monitor the squid at least once each day. The adult aquarium is on a 12h light/12 h dark cycle (lights on 2300 h; lights off 1100 h). This interval is equivalent to what the nocturnal squid experiences in the wild, and provides ample time for lab maintenance and cleaning without disturbing the squid. The air temperature is ambient for the lab space, about 24°C, which is also an environmentally natural temperature for the squid. The natural temperature range of the water is from 20 to 28°C in Hawaii.The egg-incubation aquaria are also maintained in the same growth chamber on the same the same light/dark schedule and temperature. The eggs clutches are laid on 4” half PCV pipe are maintained in 2.5-gallon aquaria (max four clutches per tank) with aerators and are located on acrylic tabletops. Note: the bobtail squid are very sensitive to anything metal. So we use only acrylics, glass, and plastic for housing and working with the squid.Aquarium parametersThe main squid aquarium in the SLSL is a 300-gallon system for re-circulating seawater and has nine individualized compartments to keep the adults separated. A 100-gallon reservoir tank is connected to the system, which harbors the microbial-enriched sand to help remediate the water (specifically enables nitrogen cycling). Water temperature is ambient, about 24°C. The system contains a sump with a skimmer, through which the seawater passes. The seawater also runs through a UV filter before returning to the upper compartments. The filtered, natural seawater used in the system comes from the University of Florida, Whitney Marine Lab in St. Augustine, FL. Water bottles are filled monthly and transported to the SLSL. Ammonia and nitrite levels are monitored in the adult aquarium weekly and kept at zero (below detection). Nitrates are kept as low as possible using the live sand cultures to remediate the water, and has always been <10 ppm. Each of the adult squid is maintained in a separate compartment labeled with its own number. On occasion squid are put together if special cleaning or maintenance is required. The squid show no stress or aggressive activity when placed together in a tank.The adult aquarium system is made of blue, opaque acrylic. The bottom of each tank is covered with approximately 2 cm of calcium-carbonate (marine) sand in which the squid bury in during the day. Two halved 4” PVC pipes are placed in the tanks (called “caves”), on which the females lay eggs. The lids of each compartment are transparent acrylic and allow light to penetrate, so that the squid experiences a natural light/dark cycle and can be easily inspected.MonitoringAll squid are monitored daily including holidays and weekends. Adult squid are fed daily as well.Environmental parameters including salinity, nitrate, nitrite, and ammonia are monitored weekly. Light levels are on a 12h light/dark cycle and levels, along with temperature are recorded automatically every 5 min.If a squid looks sickly, the information is recorded. If a squid has died the squid is removed immediately and treated as biological waste.Typically, lab personnel rotate weekly from Monday through Sunday including holidays.Emergency Preparedness Plan The SLSL follows KSC’s emergency preparedness protocols. These protocols, along with the SLSL emergency plan, are found on a public server that all SLSL employees have access to. Each SLSL employee must attend a safety-training workshop before starting work at the SLSL. Emergency Protocols for Squid Rearing Facility: The environmental growth chamber is maintained on emergency power. There is a generator for the SLSL building that can last several days in case of an emergency (e.g. hurricane). However, should a power outage occur that lasts longer than the generator has fuel for, the volume of water on the tables is sufficient to keep the squid immersed and oxygenated for several days.FeedingLive freshwater glass shrimp (Palaemonetes kadiakensis or P. paludosus) are fed ad libitum to squid each morning, right before the squid’s subjective “dark” cycle. The glass shrimp are obtained from Ekk Will Waterlife Resources, a licensed wholesale fish farm in Ruskin, Florida. The shrimp are maintained in two separate aerated 50-gallon aquaria just outside the environmental growth chamber area.Cleaning of adult breeder squid aquariumOn a daily basis, dead and dying feeder shrimp are removed from the compartments first thing in the morning and the lids are rinsed to prevent salt build up. This daily cleaning prevents buildup of nitrogenous waste in the system and in the sand layer. Also, the salinity of the water is checked daily using a refractometer and DI water is added as needed to the reservoir tank to maintain a constant 33 - 36 ppt salinity.Each individual compartment is cleaned between squid collections or when noticeably dirty (e.g. alga build-up). The squid are transferred to a separate unused compartment or holding tank (5-gallon aquarium) for the duration of cleaning (< 20 minutes). Sand is removed and washed with very hot tap water, followed by a thorough rinsing with deionized water. Algae, leftover feed, and other detritus is removed from the sand layer through this process. The sides, bottom, and top of the tanks are also scrubbed to remove any accumulating alga or cyanobacteria.Approximately, once a month a 10% water change is conducted by partially draining 30 gallons from a built-in drain port in the reservoir tank and adding new seawater collected from the UF Whitney Marine Lab in St. Augustine, FL.Annually, the aquaria compartments are completely drained and the UV light bulbs are changed and the sand in the upper compartments is autoclaved. The compartments are then refilled with seawater and allowed to run for at least a week before a new shipment of adult squid arrives.The water in egg tanks are changed daily with fresh natural seawater. The egg aquaria are scrubbed and rinsed with tap water daily before new water is added. As a general note for all aquarium, materials, and glassware no soap or detergents are used as that has been shown to be detrimental to the health and development of cephalopods. Breeding of adultsThe mantle lengths of all squid are measured in the first week of arrival after collection. A single female is matched with a similarly sized (or slightly smaller) male. During each breeding event (once every two weeks for each female), the male is moved into the female’s tank for 12 h overnight, and returned to its own tank the next morning for recovery. Each male has at least two days of recovery between mating events.Record KeepingElectronic records are maintained in the Foster lab for ease of input and access. Facility records, such as water parameters and purchasing receipts, are maintained in paper and electronic form. Temperature and light levels are recorded daily automatically. Nitrate, nitrite and ammonia levels are checked weekly or more often if a squid display signs of distress. Squid identifiers are designated by number and monitored for the duration of the squid’s life. Squid death dates are recorded, and if the squid is euthanized, that is recorded as well. Mean longevity in captivity can thus be calculated.Each egg clutch is assigned a unique identifier. For each clutch, the lay date, the approximate number of eggs, the female number, and the PVC cave identifier are recorded.From each clutch, the number of hatchlings is recorded. This information includes the number of paralarvae that hatched overnight and the number of paralarvae that hatched at known times. Any dead, sickly, or premature hatchlings are also noted. From these data, we can compute the number of hatchlings per clutch total, the number of hatchlings per time period, the number of hatchlings per female, and the clutch’s range of incubation time.Training of personnel to work with bobtail squidAll personnel, including postdoctoral fellows, graduate and undergraduate students, are required to be trained by the PI in all husbandry and squid care protocols prior to working with the adult and hatchling squid. The PI has more than 27 years working with the bobtail squid and will provide one-on-one training on all methods described in this protocol. Additionally, all staff are required to take AALAS courses “Pain Management in Laboratory Animals” and “Working with the IACUC” through the UF IACUC office. Recognition of illness, senescence, or stressAdults are observed at least once daily (typically first thing in the morning during feeding and cleaning). Signs of illness or aging include failure to bury in the sand during daytime hours or failure to eat. Squid deemed to be ill are euthanized (see below). The life span of the bobtail squid is approximately nine months. Towards the final weeks of a squid’s life they can exhibit whitening of the eye and mantle as well as loss of motor and chromatophore control. At this point, the squid are euthanized using protocols described below.During cleaning, breeding, and tank maintenance, it is sometimes necessary to remove the squid from its sand cover. This may evoke a temporary stress response (e.g., inking), but care is taken to minimize these occurrences, and to proceed as carefully and gently as possible. Because the viscous ink may clog gills, it is removed immediately from the water with a turkey baster.Termination of experiments and euthanasiaAdult breeder squid die either naturally or are euthanized due to old age. For all adult squid, euthanasia is performed through over-anesthetization. Anesthetization and euthanasia is performed by exposing the squid to either 2% ethanol or 0.12-0.15M MgCl2, in filtered sterilized seawater (FSW) for 20 min. This protocol is the current standard for working with cephalopods as described in the article, which serves as a standard in the field for cephalopod laboratory care.Fiorito, G., Affuso, A., Basil, J., Cole, A., de Girolamo, P., D’angelo, L., Dickel, L., Gestal, C., Grasso, F., Kuba, M. and Mark, F., 2015. Guidelines for the Care and Welfare of Cephalopods in Research–A consensus based on an initiative by CephRes, FELASA and the Boyd Group.?Laboratory animals,?49(2_suppl), pp.1-90.Paralarvae are typically anesthetized and euthanized by placing the paralarvae in 0.12-0.15M MgCl2, in filtered sterilized seawater for 5 min prior to dissection and imaging.For those experiments in which ribonucleic acid (RNA) will be extracted, the paralarvae are either flash-frozen in liquid nitrogen or euthanized in RNAlater solution, which kills the animal in approximately 2-3 seconds.Carcass disposition and storageAdult squid that have been euthanized are typically placed in a sealed plastic bag and disposed of in the biological waste stream of the SLSL.After an experiment with paralarvae, the squid is either preserved in RNAlater or flash frozen and stored for future use. 8. Description of Animal UseProvide a complete description of the proposed use of the animals, including the approximate time period the animals will be on study. Include descriptions of: animal identification methods; radiation (dosage and schedule); use of restraint devices (note that prolonged restraint of greater than 15 minutes must be justified); sites, volumes, and frequency of collections of bodily fluids; names/type, dosages, and routes of administration of compounds and other materials administered; animal manipulations (such as centrifugation, microgravity exposure, etc.); summary of surgical manipulations; and similar details. If surgical manipulations are to be included in the protocol, details must be provided in Section 9.This description should allow the IACUC to understand the entire experimental design, from the arrival of an animal at the Vivarium, through the experiment and its endpoints, and final disposition of the animal(s). A diagram or chart may be helpful to explain what is being done.HatchingMost eggs (70-80%) hatch within 2 h of dark, that is, between 1100 and 1300 h. Squid are collected regularly from the egg table using a 2 mL disposable glass pipette to transfer them to a sterile glass Pyrex dish. During the day, squid are collected within 30 min of hatching and are placed into filter-sterilized (0.22 ?M pore-sized filter membrane) seawater (FSW). Squid are washed three times in FSW to prevent any contamination from the egg table water. For the first seven days after hatching, bobtail squid paralarvae feed off of an internal yolk sac and are not fed. In the rare instance the paralarvae are kept longer than one week then they are fed mysid shrimp.Ground controls and inoculation with symbiotic Vibrio fischeriTo maintain squid in the aposymbiotic state after collection (non-colonized), the squid are transferred to individual 20 mL scintillation vials containing 5 mL FSW. Hatchlings used in colonization experiments are transferred to 20 mL scintillation vials containing 5 mL of FSW inoculated with between 1,000-1,000,000 CFU/mL of Vibrio fischeri (concentrations depends on the experimental question). In the wild, V. fischeri concentrations range from 500-1000 cells/mL in the water column and 10,000-100,000 cells/mL in the sand in which they bury. Scintillation vials are placed in cardboard racks and loosely covered with shrink-wrap plastic film to limit evaporation. Racks are stored in a Percival growth incubator maintained at a constant 24°C incubation room, so that the hatchlings experience the same light/dark cycle and temperature as they did as eggs during incubation. On a daily basis, or until the experiment is complete, hatchlings are checked for colonization and luminescence. The scintillation vial containing the 3 mm paralarvae in FSW is put into an adenosine triphosphate (ATP) photometer (Promega GloMax), and the light produced by the bacteria that have colonized the squid’s light organ is recorded without harm or stress to the squid.Simulated Microgravity ExperimentsParalarvae are placed in 50 mL High Aspect Ratio Vessel (HARV) bioreactors, which are used to simulate the low shear environment of microgravity. The squid are added through a 1 cm port in the HARV using a wide-mouth glass pipette. The HARVs are rotated at 13 rpm in either a vertical position (to mimic microgravity) or in a horizontal position (as a gravity control). Between 1 and 5 paralarvae are co-incubated in a single HARV vessel. The Foster lab owns 16 HARVs, therefore the maximum number of squid per simulated microgravity experiments is n = 80 paralarvae. Typically, paralarvae are incubated for between 2 and 24 h in the HARVs then examined for colonization state using the non-invasive ATP photometer. On occasion paralarvae have been maintained up to 72 h in the HARV bioreactors, with daily seawater changes. The paralarvae are removed from the HARVs by unplugging the 1 cm stopper and gently pouring the water (and paralarvae) out of the HARV into a sterile 50 mL beaker. The paralarvae are then transported between vessels using a wide-mouth glass pipette.After exposure to the simulated microgravity experiments, the paralarvae are anesthetized and euthanized in 0.12-0.15M MgCl2 using the previously described protocol For those experiments that examine gene and protein expression of a particular treatment, the paralarvae are immediately processed without anesthesia using the following:1. Flash freezing in liquid nitrogen to immediately inhibit transcription and translation and to preserve tissue samples; or2. Preservation in RNAlater to immediately inhibit transcription and translation and to preserve tissue samples.9. Surgery (To be completed only if surgery is involved.)All surgical procedures must be performed in compliance with the relevant IACUC guidelines for surgery. Check the statements that describe your project: FORMCHECKBOX Non-survival surgery (animals are euthanized under anesthesia without regaining consciousness) FORMCHECKBOX Vendor conducted FORMCHECKBOX Major survival surgery (penetration and exposure of a body cavity, or resulting in a permanent impairment of physical or physiologic functions) FORMCHECKBOX Minor survival surgery FORMCHECKBOX Multiple survival surgical procedures (provide the timeframe between surgeries, describe any differences in surgical procedures, and provide a scientific justification for conducting multiple survival surgical procedures, if applicable)9A.Location (where surgery will be performed—building and room number)Any dissection of adult or paralarvae occurs in the Space life Science Lab room 234.9B.AnestheticsList the anesthetic(s), including dosage(s), frequency of dosing, and route(s) of administration that will be used, and describe how you will monitor the depth/quality of anesthesia to ensure it is adequate. Anesthetization and euthanasia is performed as described above (see section 7C): exposure to 0.12-0.15M MgCl2, in FSW. Anesthesia is considered complete when the squid stops swimming and begins to drift or become ataxic. Chromatophore control will also relax and the squid will appear pale. This process typically takes 3-5 minutes in paralarvae.9C.Preoperative CareDescribe the preoperative care of the animal (e.g., withholding of feed for 18 hours prior to surgery or administration of prophylactic antibiotics).There is no preoperative care before dissection.9D.Minimization of ContaminationDescribe the methods employed to minimize microbial contamination of the surgical site. Include brief descriptions of the preparation of the animal, surgeon, and instruments.The paralarvae are euthanized prior to the surgical dissection of the light organ and therefore, microbial contamination is not a concern.9E.Surgical ProceduresDescribe the surgical procedure. Include descriptions of methods and materials for ligatures and wound closure.Once anesthetized and euthanized in a scintillation vial, the paralarvae are removed with ethanol- (or RNAse-) treated forceps and transferred to a clean depression glass slide under a dissecting microscope.Paralarvae are anesthetized and euthanized by placing the animal in filtered seawater containing 0.12 – 0.15 M MgCl2 for 5 min. The euthanized paralarvae can then be dissected open, the funnel is removed, and the paralarvae is mounted in FSW for imaging under a compound epifluorescent microscope. To then collect the light organs, the anesthetized and euthanized paralarvae are placed on a depression glass slide under a dissecting microscope, with ethanol- (or RNAse) treated forceps, the mantle and funnel are removed and the light organ is plucked from the paralarvae.The light organ (500 μm in size) is immediately placed in liquid nitrogen, RNAlater, or RNA extraction buffer.After imaging or light organ removal, the remaining tissues are disposed of in the biological waste stream at the Space Life Science Lab.9F.Postoperative Surgical CareDescribe the post-surgical care. Include information regarding the use of pain-relieving drugs (give the drug(s), dosages, route(s) of administration, frequency), monitoring of animals for normal recovery from anesthesia and wound healing, and provision of supportive care, such as supplemental heat and fluid or antibiotic therapy. Describe who will perform post-surgical observations, and the frequency and duration of observations.There are no postoperative surgical care procedures.Reminder: Documentation of the surgical procedure and post-surgical care is required and is the responsibility of the Principal Investigator. Copies of the surgical/post-surgical records must be readily available to the veterinary staff, the IACUC, and regulatory officials.10. Minimizing Pain and Distress- Clinical Outcomes and Humane EndpointsDescribe any pain, distress, or clinical outcomes (e.g.,: tumors/ lesions, weight loss, behavioral abnormalities, etc.) that an animal may experience as a result of this study Please indicate any treatment/ procedures designed to ensure that discomfort and injury to animals will be limited to that which is unavoidable in the conduct of this project. Describe objective criteria/ parameters (e.g.,: weight loss of 20%, loss of mobility, etc. and endpoints, as well as the frequency/ schedule of monitoring of animals during the entire experiment. Clearly indicate when animals will be euthanized should any of these endpoints be reached.Adult Breeding Colony: Adult bobtail squid are observed at least once daily. Signs of illness or aging include failure to bury in the sand during daytime hours or failure to eat. Squid deemed to be ill are euthanized. The life span of the bobtail squid is approximately nine months. Towards the final weeks of a squid’s life they can exhibit whitening of the eye and/or mantle, we well as loss of motor and chromatophore control. At this point, the squid are euthanized using protocols previously described (section 7C).During cleaning, breeding, and tank maintenance, it is sometimes necessary to remove the squid from its sand cover. This may evoke a temporary stress response (e.g., inking), but care is taken to minimize these occurrences, and to proceed as carefully and gently as possible. Because the viscous ink may clog gills, it is removed immediately from the water with a turkey baster.Simulated microgravity experiments:Paralarvae placed in the rotating HARV vessels give few outward indications of their physiological state. Most experiments include exposure to the simulated microgravity environment for less than 24 h; however, for those multi-day experiments, the squid are observed daily and those squid that have prematurely died are removed to prevent distress to the other paralarvae. At the end of simulated microgravity experiments all paralarvae are anesthetized and euthanized using the previously described protocols.11. Pain, Discomfort, and Distress11A.USDA Pain/Distress ClassificationCheck the category that indicates the highest level of pain/distress the animals will experience during the course of these studies (use the reference chart below for determination). FORMCHECKBOX C FORMCHECKBOX D FORMCHECKBOX E*Category C: No Pain or Distress (Use of Pain Relieving Drugs Is Not Indicated). Procedures that may result in only slight or momentary pain such as routine injections, blood collections, or other minor procedures are included in this category. Category D: Tests or Procedures Involving the Potential for Pain or Distress (Appropriate Anesthetic, Analgesic or Tranquilizing Drugs are Used). Animals in Type D studies have the potential to experience pain/discomfort, but the necessary drugs to alleviate the symptoms are provided. This includes terminal bleeding performed under anesthesia or retro-orbital sinus bleeding of rodents under general anesthesia, because these procedures would result in pain if anesthetics were withheld. All surgical procedures where anesthesia is used to alleviate pain or distress, including studies on anesthetized animals that do not regain consciousness are included in this category.Category E*: Pain or Distress Without the Benefit of Pain Relief These are procedures (e.g., efficacy studies of novel pain therapeutics) or situations (induction of chronic illness/disorder such as arthritis or liver failure) for which the use of analgesics, anesthetics or tranquilizing drugs would adversely affect the procedures, results or interpretation of data. *For all E category classifications complete the Category E Explanation Sheet at the end of this form.12. Alternatives to Potentially Painful/Distressful Procedures12A.Written StatementProvide a written statement of the methods and sources used to determine that alternatives to potentially painful/distressful procedures are not available. Reduction of animal numbers and Refinement of procedures to eliminate or minimize pain and distress must be considered, as well as Replacement of animals with non-animal alternatives. If alternatives to painful or distressful procedures exist, but were not chosen, explain the reasons for not using the alternatives. Reduction:Adult Breeding Colony: The PI has more than 27 years experience rearing the Hawaiian bobtail squid. Experience has shown that adult female bobtail squid will lay between 10 and 15 viable cultures in her lifetime. However, not all females are fertile. Therefore, to reduce the unnecessary collection of adult female bobtail squid we try to keep the annual collection number to 12 females per year to minimize the over production of eggs. Additionally, it is estimated that between 50 and 75% of the collected adult females have been previously mated in the wild and often lay viable cultures before in-house mating occurs. The females can store the sperm packages for several months and therefore; the need for males in the breeding colony is low. The PI only collects 2-3 males per year to only breed with those females that don't produce viable clutches within the first two weeks of being transported back to the SLSL facility.Paralarvae experiments:Due to the high variability between clutches and individual hatchlings, we often need between 5 and 10 replicate paralarvae per treatment to generate statistically robust data. By keeping the number of breeding females low, we can try to produce the minimal number of egg clutches needed to provide statistically viable experimental results.Refinement:The PI has restricted her research questions to examine early developmental events in the squid and then can minimize the length of the paralarvae exposures to various experimental treatments (e.g., microgravity).Replacement:At this time, no virtual or non-animal model exists for these symbiotic relationships and the squid-bacteria model is one of the best systems to study the influences of beneficial bacteria on animal development.12B.Alternatives SearchDescribe your consideration of alternatives to procedures listed for categories D and E that may cause more than momentary or slight pain or distress to the animals, and your determination that alternatives were not available. Delineate the methods and SPECIFIC SOURCES used in the table below. Examples of appropriate sources include Biological Abstracts, Index Medicus, the Current Research Information Service, and the Animal Welfare Information Center. You must use at least two different databases. The key words “Alternative” and/or “Alternatives to Animal Testing” and common name(s) of species must be included and combined with the potentially painful procedures.Date Literature Search Conducted:12/23/19Date Range Used in Search:From:anytimeTo:presentKeywords Used in Search:Cephalopod, welfare, husbandry, care, painDatabase(s) Consulted:Google Scholar, PubmedOther Information Source: (provide details)Attendance of annual squid-vibrio science meeting. UF IACUC meetingsSummary of findings:The primary reference publication used for cephalopod husbandry, care, and welfare is derived from an initiative by several private entities in collaboration with international researchers to improve care of cephalopods used in research. The article was a direct response to the change by the European Union to include ‘live cephalopods’ in their regulation to protect cephalopod used in scientific research.Fiorito, G. et al. (2015) Guidelines for the care and welfare of cephalopods in research – a consensus based on an initiative by CephRes, FELASA and the Boyd group. Laboratory Animals 49(52) 1-90.Additionally, as keyword search of PubMed to minimize pain in cephalopods yielded a few results worth noting:1. A leader in this field of how cephalopods sense pain is Robyn Crook and she recently published a paper assessed neural and behavioral correlates with different anesthesia and euthanasia in cephalopods. This study suggested ethanol and magnesium chloride caused loss of neurological pain signals and were recommended for both anesthesia and euthanasia.Butler-Struben, H. M., Brophy, S. M., Johnson, N. A., & Crook, R. J. (2018). In vivo recording of neural and behavioral correlates of anesthesia induction, reversal, and euthanasia in cephalopod molluscs.?Frontiers in physiology,?9, 109.2. Crook's earlier work in 2011 primarily described the likelihood of molluscs feeling pain and suggested the use of magnesium chloride to relax muscles of cephalopods.Crook, R. J., & Walters, E. T. (2011). Nociceptive behavior and physiology of molluscs: animal welfare implications.?Ilar Journal,?52(2), 185-195.3. In this 2018, paper below provides a nice review of the organization of neurological structures in molluscs. The paper discusses presence of nociceptors and that molluscs have the capacity to feel pain. The paper also reviews the current understanding of anesthetics on mollusks, including cephalopods, but the conclusions state there is still a need for future studies and makes the recommendation for using magnesium chloride.Winlow, W., Polese, G., Moghadam, H. F., Ahmed, I. A., & Di Cosmo, A. (2018). Sense and Insensibility–An Appraisal of the Effects of Clinical Anesthetics on Gastropod and Cephalopod Molluscs as a Step to Improved Welfare of Cephalopods.?Frontiers in physiology,?9.4. In 2019 the paper below discusses the challenges of detecting pain in cephalopods but again recommends using anesthesia such as magnesium chloride. This feature article talks about how there is a need for additional studies in these areas.Neff, E.P. (2019) Considering the cephalopod.?Lab Animal?48, 19-22.12C.Assurance of Non-duplicationProvide a written statement that the experiments covered under this proposal do not unnecessarily duplicate previous experiments.The experiments derived from these protocols would address new previously unaddressed research questions regarding the impacts of microgravity on beneficial symbiosis. On occasion it may be necessary to replicate treatments for control purposes, but efforts will be made to minimize unnecessary experiments.13. Method of Euthanasia or Disposition of Animals Provide details on method(s) of euthanasia or final disposition of animals. Euthanasia methods must comply with the current recommendations of the American Veterinary Medical Association's Guidelines on Euthanasia (). Justification must be provided for any proposed alternative methods. If injectable agents are used, provide agent name, dose, and route of administration. (Check All Applicable Boxes) FORMCHECKBOX CO2- followed by secondary method (e.g., bilateral thoracotomy, cervical dislocation) FORMCHECKBOX Isoflurane overdose FORMCHECKBOX Cervical Dislocation (rodents < 200 gm) w/ sedation FORMCHECKBOX Decapitation/Guillotine w/ sedation FORMCHECKBOX IV Euthanasia Solution (Specify agent, route, dose): FORMCHECKBOX IP Euthanasia Solution (Specify agent, route dose): FORMCHECKBOX Excess/Deselected Animals may be transferred per approved process to another protocol, if applicable FORMCHECKBOX Other (Specify method and provide justification): All squid are euthanasia by exposure to either 2% ethanol or 0.12-0.15M MgCl2, in filtered sterilized seawater (FSW). The use of ethanol or magnesium chloride is the accepted methods of euthanasia for cephalopods according to a consensus guideline generated by the cephalopod research community as described in:Fiorito, G. et al. (2015) Guidelines for the care and welfare of cephalopods in research – a consensus based on an initiative by CephRes, FELASA and the Boyd group. Laboratory Animals 49(52) 1-90.For those paralarvae being examined for altered gene expression under modeled microgravity conditions the animals will be placed directly in either liquid nitrogen or RNAlater to mirror concurrent spaceflight conditions and minimize changes transcriptional changes.14.Use of Hazardous Agents or Biological MaterialsWill animals be exposed to any of the following agents? If yes, specify the agent, including CDC biosafety level, as applicable. AgentName, Type or Description of AgentRadioisotopesNoneChemical HazardsRNAlater, ethanol, liquid nitrogen, magnesium chlorideBiohazardsNoneRecombinant DNANoneBiological MaterialsVibrio fischeri, the natural symbiont of the squid, which poses no hazard to the squid or researchers; caspase inhibitor peptides.OtherNoneFor specific agents used, provide special handling instructions for animals, caging and equipment, and other special precautions (, special housing, personal protective equipment requirements and any decontamination procedures). Use of radioactive materials will take place in designated areas only.Chemical Hazards:All of the listed molecules will be used only in SLSL lab 233 and will be used to euthanize the squid, therefore no special handling conditions are required to protect the squid. Ethanol will be stored in a flammable cabinet until use. RNAlater and magnesium chloride are non-hazardous salts that are stable at room temperature. Liquid nitrogen will be stored in a special dewar. Biological Materials: Vibrio fischeri symbiont: No special handling protocols will be used when inoculating paralarvae with the natural symbiont V. fischeri. The adult breeders naturally shed the V. fischeri into the surrounding seawater as it is part of their natural microbiome. The paralarvae take up the V. fischeri added to the water and use it in their normal anti-predator behavior.Caspase inhibitor peptides: The caspase inhibitors are short chains of amino acids that will be incubated with paralarvae between 2 and 24 h. Pharmacological grade peptides will be added to the FSW. The exposures will be conducted within the HARV bioreactors for simulated microgravity treatments (located in SLSL lab 233). According to the safety data sheets (e.g., Millipore Sigma) the peptides are non-flammable, stable, and there are no known toxicity issues with this material.15. Safety PrecautionsProtocols involving radiation or biosafety hazards must be approved by the appropriate official before IACUC approval will be granted.Environmental Health & Safety Representative (Print and Sign Name, Date)Biosafety Officer (Print and Sign Name, Date)Radiation Safety Officer (Print and Sign Name, Date)16. Photo DocumentationAs per the NASA Photo-documentation Policy, images involving animals may be obtained for purposes of animal welfare, scientific data collection, operational verification of hardware or procedures, and/or education, training, and public outreach purposes. However, all photo-documentation must be approved in advance by the Institutional Animal Care and Use Committee (IACUC) and, in cases involving flight downlinks, by the NASA Chief Veterinary Officer (CVO) or designee. Only those purposes listed above provide sufficient justification for approval of photo-documentation. No unofficial or personal photos of animal activities are allowed. The IACUC or CVO may require, as a condition of approval, an opportunity to review, prior to their use, images that will be used in publications or presentations. This policy applies to all animal activities that are conducted at NASA facilities, use NASA personnel, and/or use NASA hardware or vehicles for any portion of the activity.It should be noted that the Public Affairs Office (PAO) must additionally review and approve release to the media of any images involving animals. The PAO may not approve the release of images that the IACUC and/or CVO have disapproved, but the PAO may prohibit the release to the media of images that the IACUC/CVO has approved for collection for approved purposes.Note that appropriate personal protective equipment (PPE) must be worn by all individuals working with the animals. Images that include people without the appropriate PPE will not be approved. FORMCHECKBOX I have read the photo documentation policy above. No photo-documentation will be conducted during this experiment. FORMCHECKBOX Yes, photo-documentation will be conducted during this experiment. I have completed, signed, and attached the photo-documentation approval sheet to this protocol for IACUC review for approval.Request for IACUC Approval:Photo-documentation of Animal ActivitiesAs per the NASA Photo-documentation Policy, images involving animals may be obtained for purposes of animal welfare, scientific data collection, operational verification of hardware or procedures, and/or education, training, and public outreach purposes. However, all photo-documentation must be approved in advance by the Institutional Animal Care and Use Committee (IACUC) and, in cases involving flight downlinks, by the NASA Chief Veterinary Officer (CVO) or designee. Only those purposes listed above provide sufficient justification for approval of photo-documentation. No unofficial or personal photos of animal activities are allowed. The IACUC or CVO may require, as a condition of approval, an opportunity to review, prior to their use, images that will be used in publications or presentations.This policy applies to all animal activities that are conducted at NASA facilities, use NASA personnel, and/or use NASA hardware or vehicles for any portion of the activity.It should be noted that the Public Affairs Office (PAO) must additionally review and approve release to the media of any images involving animals. The PAO may not approve the release of images that the IACUC and/or CVO has disapproved, but the PAO may prohibit the release to the media of images that the IACUC/CVO has approved for collection for approved purposes.Note that appropriate personal protective equipment (PPE) must be worn by all individuals working with the animals. Images that include people without the appropriate PPE will not be approved.1.Protocol Title and Number; Approval Date (if previously approved protocol)Effects of simulated and natural microgravity on the beneficial symbiosis between the bobtail squid Euprymna scolopes and luminescent bacterium Vibrio fischeri2.PI and Institutional AffiliationName: Jamie S. FosterTitle: Full ProfessorAffiliation: University of FloridaMailing Address: Space Life Science Lab, 505 Odyssey Way, Merritt Island, FL 32953Email: jfoster@ufl.eduPhone Number: 321-525-1047 (cell phone only; no office or lab phone)Fax Number: noneEmergency Contact Number: 321-525-10473.Description of Photo-documentation required. (Type of image – e.g., video, still, etc.; Subject – e.g., whole animal, dissection, etc.; How many images, etc.) Images of live squid within the testing equipment. These images would include photos of the adults in the aquaria, paralarvae within the HARV vessels, and paralarvae within the culture bags.Images of dissected squid to monitor the effects of experimental treatment. Epifluorescent or confocal micrographs will be taken to monitor the experimental results of a given experiment.Video may be taken of paralarvae in the simulated microgravity HARV vessels while rotating to monitor squid behavior during the experiments.4.Purpose of photo-documentation. (Must be consistent with approved purposes: animal welfare (e.g., document health status); data collection; operational verification of hardware or procedure; education, training, or outreach)The purposes of all images will be to monitor experimental research progress and to verify operational hardware procedures. Some images may be used to train incoming students for dissection training.5.How will the images be used? Briefly summarize. (For example, video of procedures shown to astronauts; attending vet. will review daily; activity level each day of centrifugation will be analyzed by lab staff and quantified to determine the effects of centrifugation; etc.) Images will be used in scientific presentations at conferences, for peer-reviewed research publications, and potential educational outreach presentations.6.Do you anticipate that any of these images will be released to the Media? If so, explain and work with your local public affairs office for additional approval. (For example: Images of adult rats in the new flight hardware will be provided to the media to help describe how scientific research is conducted within the unique environment of space.)It is possible that images of the live squid will be released to media to show what a bobtail squid looks like. The paralarvae are rather small but it is possible that the paralarvae within the HARVs may be released to the public and media.7.Do you anticipate that any of these images will be used in publications or presentations? If so, explain. (For example: Images of rat neonates taken before and after space flight will be presented at XYZ national meeting to demonstrate the effects of space flight on their posture.)Yes, we anticipate that many of the epifluorescent and confocal images taken during the experiments will be used in peer-reviewed publications.8.Will these images be downlinked from Space? If so, explain why this is necessary. (CVO must be contacted for additional approval)No, these experiments are all conducted under simulated microgravity ocnditions.9. Will the collection of photo-documentation have any impact on animal welfare? If so, this must be approved by the IACUC. (Summarize any impact on animal welfare here and fully describe this impact in the IACUC protocol, or its amendment.)It is not anticipated that taking any of the photos would have an impact on the squid welfare.- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - REVIEWED BY: O Photo-doc. Subcommittee O Full IACUC O CVO RESULTS: O Approved as requested O Approved with conditions (see below)O Revision requested O Referred to CVO O DisapprovedApproved by ________________________________________________ Date: _______ Name, Title, SignatureConditions of Approval:O Review prior to release Reviewed on __________, by __________________________________O Approved for release O Release not approvedComments: _____________________________________________________________ _____________________________________________________________ 17. Permits17A.Are Special Permits required? FORMCHECKBOX Yes FORMCHECKBOX NoIf Yes:Permit type: 17B.Does the NASA facility (or PI) already have this permit? FORMCHECKBOX Yes FORMCHECKBOX NoIf Yes:Permit Expiration Date: 17C.Are the animal numbers requested in Section 7 within the limits of the permit? FORMCHECKBOX Yes FORMCHECKBOX No18.Experience and QualificationsList the experience and qualifications of the PI and all personnel who will be supporting this protocol, along with their role in each procedure listed. Only personnel listed on the protocol are approved to work with animals, and only for the procedures indicated in the protocol. All personnel who perform any animal manipulations, including, but not limited to anesthesia or surgery, must be qualified through training or experience to accomplish these tasks in a humane and scientifically acceptable manner. For any personnel who require training to participate in a procedure, describe what training will be provided. Current training files documenting experience, continuing education and/or training must be provided for all personnel in conjunction with participation in an Occupational Health Program and maintained in the IACUC Office.NameTitle/ AffiliationEmail addressPhone NumberList all procedures this individual will perform and their experience for each procedure listed. Jamie S. FosterProfessor, University of Florida; jfoster@ufl.edu; 321-525-1047Foster is the PI of the project and oversees all squid husbandry and breeding of the adult breeders, egg care, and paralarvae experiments. She has had 27 years working with and raising the bobtail squid and using the paralarvae in scientific experiments. In addition to the 27 years working with the bobtail squid, Dr. Foster has taken the following courses “Pain Management in Laboratory Animals” (AALAS-03982) and “Working with the IACUC” (course AALAS-04155) through the University of Florida IACUC office. Documentation of training can be provided as needed.Natasha SngPostdoctoral Fellow, University of Florida; nsng@ufl.edu; 857-225-6244Sng only works with the paralarvae. She has been working with the bobtail squid for 1 year. Dr. Jamie Foster has trained Dr. Sng on the husbandry of all squid care activities. Dr. Sng has taken the following courses “Pain Management in Laboratory Animals” (AALAS-03982) and “Working with the IACUC” (course AALAS-04155) through the University of Florida IACUC office. Documentation of training can be provided as needed.Madeline VroomGraduate Student, Univ. of Florida; mmvroom@ufl.edu; 312-485-6337Vroom assists with the paralarvae experiments in the simulated microgravity simulations. She has 3 years experience working with the bobtail squid and was trained in all husbandry and experimental protocols by Dr. Jamie Foster. Additionally, Ms. Vroom has taken the following courses “Pain Management in Laboratory Animals” (AALAS-03982) and “Working with the IACUC” (course AALAS-04155) through the University of Florida IACUC office. Documentation of training can be provided as needed.19. Principle Investigator AssurancesI hereby certify that the foregoing information is complete and correct and that professionally acceptable, ethical and humane standards governing the care, treatment and use of animals will be followed.I affirm that all procedures involving animals will be carried out humanely and will be performed by qualified personnel, and that as the Principal Investigator, I am responsible for all work conducted under this protocol.I understand that federal regulations authorize the Attending Veterinarian to utilize his/her discretion in the implementation of the procedures herein described in order to assure the welfare of the animal subjects. I understand that any other variance from what is written in the protocol form would constitute a violation of regulatory guidelines. Any changes in this project will be forwarded promptly to the IACUC for review. Changes to protocols will not be implemented until IACUC approval has been obtained. I agree to abide by all applicable laws, regulations and guidelines for the care and use of animals. I agree to cooperate with the IACUC and the Attending Veterinarian to assure compliance with federal, state and institutional regulatory requirements and policies.I hereby certify that these studies do not unnecessarily duplicate previous experiments.Signature of Principal Investigator: Date _9_/_26_/_19__ANY INDIVIDUAL MAY CONTACT THE IACUC CHAIRPERSON, ATTENDING VETERINARIAN OR ANY MEMBER OF THE IACUC (ANONYMOUSLY, IF SO DESIRED) IF THERE ARE ANY COMPLAINTS OR CONCERNS REGARDING THE CARE OR USE OF RESEARCH ANIMALS AT OUR FACILITIES. PERSONNEL SHALL NOT BE DISCRIMINATED AGAINST OR BE SUBJECT TO ANY REPRISAL FOR REPORTING ANY CONCERNS.45466011684000NASA Management Approval and Assignment of Point Of Contact(NASA Protocols Only)I have reviewed this protocol and affirm that the use of animals for this protocol is necessary to achieve our organization’s scientific or engineering goals. The work is consistent with NASA’s guidelines on the ethical use of animals, and will be carried out in accordance with the relevant federal, agency, and institutional regulations and policies. Resources are available to complete these activities as described.1657354826000Manager’s Name and Title (Print) DateSignature1657356096000Point of Contact (Print) DateSignatureIACUC Chairperson SignatureThis protocol has been approved by the IACUC.16573513716000Chairperson (Print) DateSignatureFor IACUC Office Use OnlySpecies: USDA Category: Date Submitted:IACUC Approval Date: Date Notification of IACUC Decision Sent to PI:Protocol Number: Protocol Expiration Date: A copy of the signed protocol will be sent directly to the PI listed in Section 2 of this document.Category E Explanation FormThis form is intended as an aid to completing the Category E explanation. A Category E Explanation must be written so as to be understood by laypersons, as well as scientists.________________________________________________________________________Species (common name) of animals used in these studies: Euprymna scolopes (bobtail squid) paralarvaeExplain the procedure producing pain and/or distress:During simulated microgravity experiments that require RNA analysis (i.e., monitoring the effects of the treatment on transcription using NanoString or quantitative real-time PCR) the paralarvae will be euthanized immediately in liquid nitrogen or RNAlater. No anesthesia will be provided for these cohorts of paralarvae. Although pain is likely to occur, the process would take only 2-3 seconds to euthanize the paralarvae by direct submersion in liquid nitrogen or RNAlater.Provide scientific justification why pain and/or distress could not be relieved. State methods or means used to determine that pain and/or distress relief would interfere with test results:One of the key research questions examined by my funded NASA Space Biology proposals is to understand the impact that microgravity has on host-microbe interactions over time. A key methodology to examine the molecular mechanisms in which animals respond to microgravity is gene expression analysis, such as NanoString and quantitative real-time PCR. These RNA analysis approaches examine all of the expressed genes at a given moment in time by sequencing mRNA transcripts. It has been recently published in 2017 that the half-life of mRNA molecules (i.e., expressed gene transcript) is two minutes. That means potentially half of the transcripts will be degraded or turned over within 2 min of the end of a particular treatment. The citation for this recent publication, which used several different methods to assess mRNA turnover in eukaryotes, is listed below:Baudrimont, Antoine, et al. "Multiplexed gene control reveals rapid mRNA turnover."?Science advances?3.7 (2017): e1700006.Therefore, as anesthetizing the paralarvae may take up to 5 min to fully incapacitate the animal, we are requesting that for gene expression experiments no anesthesia be used as it may alter the RNA expression results and interpretation of the effects of modeled microgravity.2451735127000Signature of Principal Investigator: _______________________________Date: _9/26/19_____ ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download