Impedanspnomografi til minimalt invasiv måling af puls i sene stadier hvirvelløse dyr
Summary
Måling af puls under en termisk udfordring giver indsigt i organismers fysiologiske reaktioner som følge af akutte miljøændringer. Ved hjælp af den amerikanske hummer (Homarus americanus) som en model organisme, denne protokol beskriver brugen af impedans pneumografi som en relativt noninvasive og nonlethal tilgang til at måle puls i sene fase hvirvelløse dyr.
Abstract
Temperaturerne i havene stiger hurtigt som følge af omfattende ændringer i verdens klima. Da organismefysiologi er stærkt påvirket af miljøtemperatur, har dette potentiale til at ændre termisk fysiologisk ydeevne i en række marine organismer. Ved hjælp af den amerikanske hummer (Homarus americanus) som model organisme, denne protokol beskriver brugen af impedans pneumografi at forstå, hvordan hjerte ydeevne i den sene fase hvirvelløse dyr ændringer under akut termisk stress. Protokollen præsenterer en minimalt invasiv teknik, der giver mulighed for real-time indsamling af puls under en temperatur ramping eksperiment. Data er let manipuleres til at generere en Arrhenius plot, der bruges til at beregne Arrhenius pause temperatur (ABT), den temperatur, hvor pulsen begynder at falde med stigende temperaturer. Denne teknik kan anvendes i en række sene stadier hvirvelløse dyr (dvs. krabber, muslinger eller rejer). Selv om protokollen udelukkende fokuserer på temperaturens indvirkning på hjertets ydeevne, kan den ændres for at forstå potentialet for yderligere stressorer (f.eks. hypoxi eller hyperkapni) til at interagere med temperaturen for at påvirke fysiologiske præstationer. Metoden har således potentiale til vidtrækkende anvendelser for yderligere at forstå, hvordan hvirvelløse havdyr reagerer på akutte ændringer i miljøet.
Introduction
I de seneste årtier har øget tilførsel af drivhusgasser (dvs. kuldioxid, metan og lattergas) i atmosfæren resulteret i udbredte mønstre for miljøændringer1. Verdenshavene er hurtigt opvarmning2,3, en tendens, der kan have alvorlige konsekvenser for organisme fysiologi. Temperaturen har stor indflydelse på fysiologiske hastigheder, og organismer har et optimalt temperaturområde for ydeevne4,5,6. Som sådan, enkeltpersoner kan støde på vanskeligheder med at opretholde ordentlig ilt levering til væv som temperaturer omstrejfende uden for dette interval. Dette har potentiale til at føre til fald i aerob ydeevne i lyset af opvarmning havtemperaturer5,7.
I et laboratorium indstilling, en metode til at forstå de fysiologiske virkninger af miljømæssige forandringer er at undersøge hjerte ydeevne i forbindelse med termisk stress. Dette giver indsigt i , hvordan eksponering for forudsagte opvarmningsforhold kan ændre præstationskurverne5,6 samt potentialet for akklimatiseringsplasticitet8. En række forskellige metoder er blevet gennemført med succes til tidligere at måle puls i marine hvirvelløse dyr. Men mange af disse teknikker indebærer kirurgisk fjernelse eller større manipulation af exoskelet og langvarig implantation af måleudstyr9,10,11, som indfører yderligere stress til forsøgspersonen og øger den nødvendige tid til en vellykket genopretning før eksperimenter. Desuden kan mindre invasive teknikker (f.eks. visuel observation, videografi) begrænses til tidlige livshistoriske stadier, hvor organismer kan være fuldstændig ellerhalvgennemsigtige 12. Desuden kan der præsenteres yderligere udfordringer for forskere, der ikke er velbevandret i mere teknologisk avancerede metoder (f.eks. observationer via infrarøde transducers eller Doppler perfusion8,11).
Denne protokol bruger den amerikanske hummer (Homarus americanus) som en sen fase marine hvirvelløse model til at demonstrere brugen af impedans pneumografi til at vurdere ændringer i puls under en temperatur ramping eksperiment. Impedanspnomografi indebærer passage af en oscillerende elektrisk strøm (AC) på tværs af to elektroder placeret på hver side af hjertesækken til at måle ændringer i spænding som hjertet kontrakter og slapper13,14. Denne teknik er minimalt invasiv, da den anvender små elektroder (dvs. 0,10-0,12 mm diameter), der forsigtigt implanteres lige under exoskeletet. Endelig giver det real-time vurderinger af både puls og vandtemperatur under rampen ved hjælp af en datalogger.
Protokollen indeholder også instruktioner til beregning af Arrhenius-pausetemperatur (ABT), den temperatur, hvor pulsen begynder at falde med stigende temperaturer13,15. ABT fungerer som en ikke-dødelig indikator for den termiske grænse for kapaciteten hos forsøgspersoner, der kan være begunstiget frem for at måle det kritiske termiske maksimum (CTmax, den øvre grænse for hjertefunktion5,6), da dødbringende grænser ofte er ekstreme og sjældent forekommende i det naturlige miljø5.
Protocol
Representative Results
Discussion
Denne protokol beskriver brugen af impedanspneumografi til at måle ændringer i hjertefrekvensen for sene stadier hvirvelløse dyr under et temperaturrampingseksperiment. Den primære fordel ved denne teknik i forhold til andre laboratoriebaserede tilgange9,10,11 er, at det er minimalt invasiv og ikke indebærer større kirurgisk manipulation af exoskelet, hvilket reducerer mængden af restitutionstid, der er nødvendig før ek…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Paul Rawson for laboratoriebistand og National Science Foundation tildeling IIA-1355457 til Maine EPSCoR ved University of Maine for midler til at købe udstyr. Dette projekt blev støttet af USDA National Institute of Food and Agriculture, Hatch projektnummer MEO-21811 gennem Maine Agricultural and Forest Experiment Station, samt NOAA National Marine Fisheries Service Saltonstall Kennedy Grant #18GAR039-136. Forfatterne takker også tre anonyme anmeldere for deres kommentarer til en tidligere version af dette manuskript. Maine Landbrugs- og Skovforsøgsstation Offentliggørelse nummer 3733.
Materials
| 1.6 mm (1/16 in) drill bit | Milwaukee Tool at Home Depot | 1001294900 | This is for a 1.6 mm (1/16 in) diameter drill bit. This item can be found at most home-improvement stores. |
| 38 AWG Copper Magnet Wire | TEMCo | MW0093 | This wire is used to make the wire electrode leads that are implanted into the test subjects. This listing is for a 4 oz coil of 38-gauge magnetic wire. TemCo also has 36-gauge magnetic wire that is also suitable for use in constructing wire electrodes. |
| Cyanoacrylate glue | Loctite | 852882 | This item includes a brush tip, which makes it easier to control the amount of glue used to secure electrodes to the carapace. |
| Ethanol, 70% Solution, Molecular Biology Grade | Fisher BioReagents | BP82931GAL | This reagent is used in combination with the sterile cotton balls to disinfect the carapace prior to electrode implantation. |
| Excel | Microsoft | N/A | This program is used in the protocol for organizing, manipulating, and analyzing data. It is compatible with both PC and Mac operating systems. |
| Fisherbrand 8-Piece Dissection Kit | Fisher Scientific | 08-855 | This kit includes the forceps, scissors, dissecting knife (and blades), and dissecting needle needed to accomplish the electrode implantation steps in the protocol. |
| Fisherbrand Isotemp Refrigerated/Heated Bath Circulators: 5.4-6.5L, 115V/60Hz | Fisher Scientific | 13-874-180 | This is a complete system that consists of an immersion circulator and a bath. It can be used as a temperature controlled bath or to circulate fluid externally to an application. Temperature range of this water bath is -20 to +100 °C, and the unit heats/cools rapidly and is easy to drain upon conclusion of use. |
| Fisherbrand Sterile Cotton Balls | Fisher Scientific | 22-456-885 | These swabs should be soaked in 70% ethanol before being used to disinfect the carapace prior to electrode implantation. |
| Fork Terminal, Red Vinyl, Butted Seam, 22 to 16 AWG, 100 PK | Grainger | 5WHE6 | Terminals are soldered to the magnetic wire to construct the wire electrodes. These can be purchased from a variety of home-improvement vendors. |
| Impedance converter | UFI | Model 2991 | Measures impedance changes correlated with very small voltage changes, ranging from 0.2 ohm to over 5 ohms. This model can convert impedance changes that stem from resistance, capacitance, or inductance variations, as well as a combination of all three. |
| LabChart software | ADInstruments | N/A | Purchase of the PowerLab datalogger includes the LabChart software, but a license for the software can also be directly downloaded online. LabChart allows the user to record data, open and read LabChart files, analyze data, as well as save and export files. There is a free version of the software, LabChart Reader, but users can only open and read LabChart files and analyze them (i.e., it cannot be used to record, save, or export data files). One also has the option of selecting LabChart Pro, which includes LabChart teaching modules that can be used for educational purposes. |
| LED Soldering Iron | Grainger | 28EA35 | This is a generic soldering iron that can be used to solder the magnetic wire to the fork terminals to create the wire electrodes. |
| PowerLab datalogger | ADInstruments | ML826 | There are a variety of models of the PowerLab. This catalog number is for the 2/26 model that is a 2 channel, 16 bit resolution recorder with two analog input channels, independently selectable input sensitivities, two independent analog outputs for stimulation or pulse generation and a trigger input. The PowerLab features a wide range of low-pass filters, AC or DC coupling and adaptive mains filter. This unit has a USB interface for connection to Windows or Mac OS computers and a sampling rate of 100,000 samples/s per channel. |
| Prism8 | GraphPad | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature through its “Segmental linear regression” data analysis option. This program does not require any programming and is compatible with both Mac and Windows operating systems. |
| R | R Project | N/A | This is free software for statistical computing that is compatible with UNIX platforms, as well as Windows and Mac operating systems. This program can also be used to calculate the Arrhenius Break Temperature using the “segmented” package. There are a number of tutorials and user guides available online through the r-project.org website. |
| Rosin Core Solder | Grainger | 331856 | This product has a diameter of 0.031 in (0.76 mm) and is ideal for use in soldering speaker wire (similar gauge as magnetic wire used for electrodes). |
| SAS | SAS Institute | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature. However, it does require programming and is not compatible with Mac operating systems. |
| SigmaPlot | Systat Software, Inc. | N/A | This is the authors’ preferred program for statistical determination of the Arrhenius Break Temperature. The “Regression Wizard” is easy to use and does not require any programming. One can obtain a free 30-day trial license before purchase. However, it is compatible only with PC computers. |
| T-type Pod | ADInstruments | ML312 | Suitable for measurement of temperatures from 0-50 °C using T-type thermocouples. |
| T-type Thermocouple Probe | ADInstruments | MLT1401 | Compatible with the T-type Pod for connection. Measures temperature up to 150 °C, and is suitable for immersion in various solutions, semi-solids, and tissue (includes a needle for implantation). This product is a 0.6 mm diameter isolated probe that is sheathed in chemical-resistant Teflon and a lead length of 1.0 m. |
| UV Cable Tie, Black | Home Depot | 295813 | This is for a 100-pack of 8-inch (20.32 cm), black cable ties. However, based on the size of test subjects, smaller or larger cable ties may be needed. This item, and others like it, can be purchased at any home-improvement store. |
References
- Stocker, T. F., et al. . Climate Change 2013: The Physical Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. , (2013).
- Pershing, A. J., et al. Slow adaptation in the face of rapid warming leads to collapse of the Gulf of Maine cod fishery. Science. 350 (6262), 809-812 (2015).
- Smale, D. A., et al. Marine heat waves threaten global biodiversity and the provision of ecosystem services. Nature Climate Change. 9 (4), 306-316 (2019).
- Pörtner, H. O., Farrell, A. P. Physiology and climate change. Science. 322 (5902), 690-692 (2008).
- Pörtner, H. O., Bock, C., Mark, F. C. Oxygen- and capacity-limited thermal tolerance: bridging ecology and physiology. Journal of Experimental Biology. 220 (15), 2685-2696 (2017).
- Somero, G. N., Lockwood, B. L., Tomanek, L. . Biochemical adaptation: response to environmental challenges, from life’s origins to the Anthropocene. , (2017).
- Sokolova, I. M., Frederich, M., Bagwe, R., Lanning, G., Sukhotin, A. A. Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates. Marine Environmental Research. 79, 1-15 (2012).
- Tepolt, C. K., Somero, G. N. Master of all trades: thermal acclimation and adaptation of cardiac function in a broadly distributed marine invasive species, the European green crab, Carcinus maenas. Journal of Experimental Biology. 217 (7), 1129-1138 (2014).
- Frederich, M., Pörtner, H. O. Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 279 (5), 1531-1538 (2000).
- Metzger, R., Sartoris, F. J., Langenbuch, M., Pörtner, H. O. Influence of elevated CO2 concentrations on thermal tolerance of the edible crab Cancer pagurus. Journal of Thermal Biology. 32, 144-151 (2007).
- Walther, K., Sartoris, F. J., Bock, C., Pörtner, H. O. Impact of anthropogenic ocean acidification on thermal tolerance of the spider crab Hyas araneus. Biogeosciences. 6 (10), 2207-2215 (2009).
- Styf, H. K., Sköld, H. N., Eriksson, S. P. Embryonic response to long-term exposure of the marine crustacean Nephrops norvegicus to ocean acidification. Ecology and Evolution. 3 (15), 5055-5065 (2013).
- Camacho, J., Qadri, S. A., Wang, H., Worden, M. K. Temperature acclimation alters cardiac performance in the lobster Homarus americanus. Journal of Comparative Physiology A. 192 (12), 1327-1334 (2006).
- Braby, C., Somero, G. N. Ecological gradients and relative abundance of native (Mytilus trossulus) and invasive (Mytilus galloprovincialis) blue mussels in the California hybrid zone. Marine Biology. 148 (6), 1249-1262 (2006).
- Stenseng, E., Braby, C. E., Somero, G. N. Evolutionary and acclimation-induced variation in the thermal limits of heart function in congeneric marine snails (Genus Tegula): implications for vertical zonation. Biological Bulletin. 208 (2), 138-144 (2005).
- Factor, J. . Biology of the Lobster: Homarus americanus. , (1995).
- Muggeo, V. M. Segmented: an R package to fit regression models with broken-lin relationships. R News. 8 (1), 20-25 (2008).
- Ryan, S. E., Porth, L. S. A tutorial on the piecewise regression approach applied to bedload transport data. General Technical Report RMS-GTR-189. , (2007).
- . . Prism8 Statistics Guide. , (2020).
- Cuculescu, M., Hyde, D., Bowler, K. Thermal tolerance of two species of marine crab, Cancer pagurus and Carcinus maenas. Journal of Thermal Biology. 23 (2), 107-110 (1998).
- Stillman, J. H. A comparative analysis of plasticity of thermal limits in porcelain crabs across latitudinal and intertidal zone clines. International Congress Series. 1275, 267-274 (2004).
- Maderia, D., et al. cellular and biochemical thermal stress response of intertidal shrimps with different vertical distributions: Palaemon elegans and Palaemon serratus. Comparative Biochemistry and Physiology, Part A. 183, 107-115 (2015).
- Padilla-Ramirez, S., et al. The effects of thermal acclimation on the behavior, thermal tolerance, and respiratory metabolism in a crab inhabiting a wide range of thermal habitats (Cancer antennarius Stimpson, 1856, the red shore crab). Marine and Freshwater Behaviour and Physiology. 48 (2), 89-101 (2017).
- Pörtner, H. O. Ecosystem effects of ocean acidification in times of ocean warming: a physiologist’s view. Marine Ecology Progress Series. 373, 203-217 (2008).
- Pörtner, H. O. Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. Journal of Experimental Biology. 213 (6), 881-893 (2010).
- Zittier, Z. M. C., Hirse, T., Pörtner, H. O. The synergistic effects of increasing temperature and CO2 levels on activity capacity and acid-base balance in the spider crab, Hyas araneus. Marine Biology. 160 (8), 2049-2062 (2013).
- Harrington, A. M., Hamlin, H. J. Ocean acidification alters thermal cardiac performance, hemocyte abundance, and hemolymph chemistry in subadult American lobsters Homarus americanus H. Milne Edwards, 1837 (Decapoda: Malcostraca: Nephropidae). Journal of Crustacean Biology. 39 (4), 468-476 (2019).
- Depledge, M. H. Photoplethysmography – a non-invasive technique for monitoring heart beat and ventilation rate in decapod crustaceans. Comparative Biochemistry and Physiology Part A: Physiology. 77 (2), 369-371 (1984).
