News

Thu, Apr 04 2024

Fish resting respirometry 101

Image: Male toadfish showing strong paternal care for 10 DPF brood. Image by Martin Grosell, RSMAS, University of Miami.

If you are looking for a solid fish respirometry and hypoxia paper with thorough explanations on methods, protocols, and data analysis, look no further than this 2023 publication by LeeAnn Frank, Joseph Serafy, and Prof. Martin Grosell from the Rosenstiel School of Marine, Atmospheric, and Earth Science, University of Miami.


A large aerobic scope and complex regulatory abilities confer hypoxia tolerance in larval toadfish, Opsanus beta, across a wide thermal range

Frank, LeeAnn; Serafy, Joseph; Grosell, Martin (2023)

Science of The Total Environment

Link to article


The team of researchers provides an easy-to-follow methodical description on how to measure standard metabolic rate (SMR), routine metabolic rate (RMR), maximum metabolic rate (MMR), and aerobic scope (absolute and factorial) as well as how to assess the regulation index, P50, and Excessive Post Hypoxia Consumption (EPHOC) data in larval toadfish (Opsanus beta) over a range of their environmental temperatures. In addition to the detailed materials and method section, the authors supply the article with a manageable overview on how to analyze such respirometry data and which data parameters to include. The data are effectively illustrated using an assortment of data graphs, plots, and tables.

If you have any questions about the paper, you are welcome to contact LeeAnn Frank at leeann.frank@earth.miami.edu.

Want to know more about the respirometry system used in this paper?

Tue, Jan 23 2024

USER CASE – Testing microplate system against traditional gas analysis

In a peer-reviewed publication by Earls et al. (2023), a team of researchers from North Dakota State University and USDA compares the Loligo® microplate respirometry system with a traditional gas analyzer setup in a study measuring the metabolism of using alfalfa leafcutting bees (Megachile rotundata) and effects of temperature. The paper can be found here:

Effects of temperature on metabolic rate during metamorphosis in the alfalfa leafcutting bee | Biology Open | The Company of Biologists

Earls, Kayla N; Campbell, Jacob B; Rinehart, Joseph P; Greenlee, Kendra J

 

Daisy-chained microplate reader with glass plate on top.

The Loligo® microplate respirometry system used in this study featured two daisy-chained microplate readers using glass plates with 940 µl wells for simultaneous measurements of up to 48 bees at a time. Both readers were controlled using our MicroResp™ software, which was also used for calculating the V̇O2 slope and to randomly assign bees to the different wells.

 

    

A mix of brood cell and pupae M. rotundata in 940 µl wells. Photo by Kayla Earls.

 

The 48-chamber microplate setup was compared to a traditional closed respirometry setup with manual injections of air samples into a CO2 analyzer (Li-Cor-7000, LI-COR Biosciences, Lincoln, NE, USA) and O2 (Oxzilla FC-2 Differential Oxygen Analyzer, Sable Systems International, Las Vegas, NV, USA) gas analyzer.

The scientists found no significant differences between the V̇O2 data from the two different setups when comparing mass-specific metabolic rate calculations:

 

Copied from Fig. 1. in the publication.

 

But the setup comparison shows a significant difference in the time (and resources) spent on collecting the data:

Pupal bees in the traditional closed respirometry system remained in the [individual] syringes for longer periods (9h) than bees tested in the microplate system (3h) due to the differences in chamber size (3ml syringes versus 940 µl wells) and detection limits of the oxygen analyzers.


It is also worth mentioning that a microplate system can analyze up to 240 organisms at a time, while the bees in the traditional closed respirometry had to be analyzed one at a time. In other words, the time spent on collecting data is substantially shorter in the microplate system.

Most impressively, the researchers present a vast dataset on oxygen consumption rates in the leafcutting bees across a large range of temperatures (6 – 48 °C) and developmental stages and between sexes:

 

Copied from Fig. 2. in the publication.

 

Copied from Fig. 4. in the publication.

 

If you are interested in learning more about this project or its team, you are welcome to contact Kayla N. Earls (Kayla.Earls@wsu.edu).

 

Want to learn more about the microplate respirometry system?

Tue, Dec 12 2023

New MicroResp™ version 1.1.0

We have updated the MicroResp™ application to version 1.1.0. You can download this latest version here:

 

Download MicroResp™ 1.1.0

 

MicroResp™ 1.1.0 introduces some exciting new features including MO2 measurements in in air/gas for scientists working with air-breathers like adult insects (e.g., Drosophila, mosquitoes, solitary bees, midges, etc.) or terrestrial invertebrates (e.g., amphipods, isopods, etc.). Simply enable this feature in the Settings tab, while running an experiment, and enter the relative humidity below.

 


Another requested feature is that you can now specify the number of organisms per well. Previously, MicroResp™ assumed that you had the same number of organisms in each well, but you can now set the individual number for each of the 24 wells, and get MO2 values per organism. Enable “Set individual” under Organisms per well in the Settings tab, and then type in the number of organisms for each well.

 

 

Finally, we have added a timestamp to each MO2 value in the data file.

 

Download MicroResp™ 1.1.0

Fri, Oct 13 2023

New update – AutoResp™ v3.1.0

We have released the latest update to AutoResp™ v3. Version 3.1.0 includes several new features, user interface changes, and some minor bug fixes.

 

DPTV flow tracking is now part of AutoResp™ v3
The most important new features is the implementation of digital particle tracking velocimetry used for automated calibration of water velocities in our Blazka-type swim tunnels 170 mL and 1500 mL, so the need for a second software (the DPTV 2 software) is no longer necessary. Now, calibration data is saved directly inside AutoResp™ v3 making it easier than ever to perform those water velocity calibrations.

For more information on how to calibrate your swim tunnels in AutoResp™ v3.1.0, please watch this tutorial video:

 

 

Manual assignment of pump relays

We usually recommend setting up respirometry chambers independently for increased throughput, but it requires that each flush and recirculation pump is plugged in to their own separate relay on the PowerX4 power bar. However, in the new AutoResp™ v3.1.0, you now have the option to assign multiple pumps to a specific (single) relay to run multiple chambers synchronously thereby saving on the number of required PowerX4 relays.

Please notice that this way of powering the pumps will prevent you from using some of the new fancy respirometry modes based on chamber oxygen levels or R2 values.

Watch this tutorial video on how manual assignment of power relays work in AutoResp™ v3.1.0:

 

 

Other features

  • You can now analyze old files from the discontinued AutoResp™ version 2.2 - 2.3 log files directly in the analysis menu in AutoResp™ v3 to make use of the many new useful analysis tools and statistics. Simply select the AutoResp 2.x files (.txt) as the file type upon clicking the Load button in the analysis menu, and then choose your AutoResp™ 2 “_raw data files”.

  • A new notification feature has been added. Upon starting AutoResp™ v3, you may receive a notification directly inside AutoResp™ telling you about relevant information, like when a new update is available. Your computer must be connected to the internet to receive notifications.

  • Recently, we have upgraded our motor controller for the Blazka-type swim tunnels (170 mL and 1500 mL) and you will be prompted for which motor controller you are using (old vs new). This is done in the lower left corner of the screen, when accessing a DAQ-BT tab in the Calibration menu.

Wed, Sept 20 2023

AutoResp™ v3 usercase: DTU Aqua, Hirtshals, Denmark

Upon launching the new AutoResp™ v3 software for Windows 11, we had the opportunity to visit Dr. Peter Vilhelm Skov at DTU Aqua’s facilities in Hirtshals, Denmark.

Peter and his post doc, Dr. Tilo Pfalzgraff, had rigged up an acrylic resting chamber system ready to use with 21-33 g juvenile rainbow trout. The trout were each placed inside 500 ml acrylic chambers connected to flush and recirculation pumps. Each pump was controlled independently for using two PowerX4 power strips, and each chamber setup was fitted with an oxygen sensor connected to the Witrox 4 oxygen instrument. All the chambers were submerged in a water bath with climate-controlled freshwater running through it to keep everything at a stable 15.3 °C.

 

 

Upon completing the setup and oxygen sensor calibration, and making sure each fish had a steady supply of fully air saturated water, Peter and Tilo received a thorough introduction on how to use the many new features in AutoResp™ v3.

 

 

One of the new features introduced in AutoResp™ v3 is the Min/Max intermittent respirometry mode. This mode allows the user to set a minimum and maximum % air saturation level while running the standard intermittent phases of flush, wait, and measure. In the rainbow trout experiment here, we wanted to measure MO2 values while the % air saturation was dropping from 94 % air saturation down to 85 % air saturation after which the flush pump would turn on and increase the % air saturation level in the chamber back up to 94.

 

 

After entering the required setup information like intermittent respirometry mode settings, fish weight, atmospheric pressure etc. in AutoResp™ v3, the data logging was initiated in each chamber. Another new feature is that since each chamber had its flush and recirculation pump controlled from individual relays on the PowerX4, each chamber was controlled and logged individually. And after about an hour of data logging, the experiment was ended and the three AutoResp™ v3 data files were loaded directly into the AutoResp™ v3 Analysis menu.

 

 

Selecting an individual chamber in the legend panel on the right provides useful visual information on the flush [F], wait [W], and measure [M] phases throughout the experiment (image above). And when selecting multiple channels, each data graph is represented with just a data graph line for clean output.

 

 

Loligo® Systems would like to thank Peter and Tilo for hosting us in Hirtshals, and for their kind feedback on using AutoResp™ v3. We are looking forward to a second visit in October.

Want to learn more about AutoResp™ v3?

Tue, Aug 22 2023

Antarctic insect metabolism

The microplate respirometry system just outside of Palmer Station, Antarctica.

 

Antarctica - In a bid to better understand how insects can adapt to extreme environments, a team of researchers has been studying the Antarctic midge, Belgica antarctica. The team, led by Nicholas Teets (Associate Professor, University of Kentucky), includes Jack Devlin (PhD Student, University of Kentucky), Cleverson de Sousa Lima (PhD Student, University of Kentucky), Yuta Kawarasaki (Associate Professor, Gustavus Adolophus College), JD Gantz (Assistant Professor, Hendrix College), and Vitror Pavinato, (Postdoctoral Associate, Ohio State University) is looking into the molecular and physiological processes mechanisms that underlie the insect's extreme stress tolerance and how it can survive in the harsh Antarctic conditions.


As part of their research, the team recently completed a field season in Antarctica cruising around the Antarctic Peninsula, where they collected terrestrial insects from various islands and conducted physiological experiments on the research vessel and station. One of the team's primary interests was how these insects manage to cope with the energetic challenges of stress.


Cleverson Lima using the microplate respirometry system with the MicroResp™ software. The microplate glass plate is placed inside an acrylic water bath (near Cleverson’s left hand), and a recirculating bath provides 10 °C water to run through the water bath to maintain a stable temperature.


Using the microplate respirometry system from Loligo® Systems, the team was able to measure real-time oxygen consumption rates in individual, terrestrial midge larvae following various environmental stresses – data that the team did not have the capacity to obtain in their past projects. Their new tool, the microplate respirometry system, thus allowed the researchers to measure metabolic rates in real-time, providing crucial data on how the insects are affected by different stressors.


MicroResp™ v1.1, the software that comes with the microplate system, supports real-time and time stamped MO2 calculations for air/gas as well as in water measurements.


One team member, Jack Devlin, was interested in the potential impact of microplastics contamination in Antarctica. Also using the microplate respirometry system, he studied whether exposure to microplastics caused any changes in metabolic rates. These research findings could prove vital in understanding the potential effects of microplastic pollution on the continent's delicate ecosystem.


MicroResp™ data example created by Jack Devlin of MO2 and Oxygen vs Time data graphs for each individual glass plate well containing Antarctic, terrestrial midge larvae. Blacks are blanks, grey is control group, and blue are insects exposed to a high concentration of microplastics. The humidity level was maintained by adding a piece of moist filter paper to each well.


The team is also comparing the stress tolerance and genome of Belgica antarctica to several close relatives to identify the key mechanisms that allow this insect to thrive in Antarctica. The study could provide a better understanding of how insects adapt to extreme environments and offer insights into the resilience of species in the face of changing environments.


If you are interested in learning more about this project or its team, you are welcome to contact Nicholas Teets (n.teets@uky.edu) or visit his website: Insect Stress Biology - Home (teetslab.com)

Fri, Aug 04 2023

New “Fish gym”: Shubin and Di Santo Explore Fish Locomotion

Loligo® Systems is thrilled to announce that our treadmill swim tunnel is being utilized at University of Chicago’s MBL (Marine Biological Laboratory) by Professor Neil Shubin of the University of Chicago, and Dr. Valentina Di Santo from Stockholm.

The treadmill allows Shubin and Di Santo to create controlled flow conditions, simulating a wide range of water velocities, while separately controlling a motorized, transparent belt underneath the fish. Their research aims to provide critical insights into how fish adapt, move, and interact with their environment, contributing to the understanding of ecology, evolution, and conservation.

Want to know more about how Shubin and Di Santo are using our treadmill swim tunnel? Dive into the full story at Welcome to the Fish Gym - Shubin Di Santo Lab.

Tue, Jun 13 2023

AutoResp™ v3 in under 3 min.

AutoResp™ v3 is here. Learn more about this major upgrade by watching this short video:

 

 

New features in AutoResp™ v3 are:

  • High Throughput – up to 20 resting chambers or 10 swim tunnels
  • NEW hardware for either LAN/Ethernet (cabled) or Bluetooth (wireless) PC communication
  • NEW Analysis Outputs with Aerobic Scope, Cost of Transport, EPOC, Maximum Metabolic Rate, etc.
  • NEW Allometric Scaling Function
  • NEW Temperature Normalization Function
  • Independent control of chamber pumps for increased throughput when working with multiple chambers
  • Multiple respirometry modes, e.g., time, min/max O2 levels, min R2.
  • Re-designed user interface with animated setups and guides
  • Comprehensive analysis dashboard with across experiments, treatments, and blanks statistics
  • Windows 11 compatible

 

Learn more about AutoResp™ v3.

Wed, Mar 01 2023

LoliTrack 5.2.0 is out!

The latest version of LoliTrack 5 has been released. Version 5.2.0 contains several new features, improvements, and some bug fixes.

 

IMPORTANT - Loligo® license dongle. LoliTrack 5.2.0 requires a Loligo® USB license dongle. Without the dongle, LoliTrack 5.2.0 will only run in demo mode (i.e., no data export). Previous turquoise Wibu dongles will not unlock LoliTrack 5.2.0. Contact us if you need a Loligo® dongle.

 

 

The frequency analyzer. A frequency analyzer allows you to calculate frequencies from video recordings. This is a helpful tool for automated measurements of heart rate, tail beat, ventilation, and other types of frequencies. The Frequency analyzer also allows for volume calculations, which enables you to estimate stroke volume, pumping volume, etc.:

 

 

You will find the Frequency analyzer in the main menu.

 

 

Another way to utilize the frequency analyzer is in the Analysis tab in Tracking 2D. This tool is especially useful for tail beat frequency analysis. Under the Bend graph, you will find the analyzer in the lower right corner:

 

 

Batch processing. You now have the option of batch tracking multiple videos in 2D. This is especially useful if you have multiple videos recorded in the same setup. Simply, track your first video like you normally would, and then apply the same settings to multiple videos. The batch processing window will tell you how much time is left:

 

 

Custom object color. To help discriminate between similar looking organisms (e.g., zebrafish) you can now specify the exact color of each tracked object and label it. Click the color box to customize the color according to your liking. The selected color will be reflected on the video preview:

 

 

Other features

  • LoliTrack 5 now supports more camera models for video recording.
  • You can now save arena and treatment setups.
  • “Convex hull” area estimation is now a data parameter that can be exported for object in 2D tracking.
  • You can now change the thickness of rendered trajectories in 3D.
  • You can now choose from three different spine tracking options in the Tracking tab in Tracking 2D.
  • Added distortion correction for when videos are distorted, e.g., for wide-angle videos.

 

Improvements

  • Overall speed improvement of tracking and analysis
  • Improved performance of the video recorder
  • Improved filter options and speed with integration of filter tools from ShuttleSoft 3.

 

Bug fixes

  • Fixed bug which prevented image export in 2D.
  • Fixed a bug where exporting videos may take up a lot of memory and crash.
  • Fixed a bug where video could not be exported as .avi

Wed, Feb 22 2023

RespFest 2023

RespFest is a conference focused on respirometry. The aim of the meeting is to discuss the latest advances, the difficulties encountered, and optimization of methodologies in model organisms to learn more about this aspect of physiology. RespFest 2023 is free of charge, but requires registration which can be done here:

 

Création d'un compte (univ-lr.fr)

 

The 2023 meeting takes place in the Coutant amphitheatre of La Rochelle aquarium in the city of La Rochelle, France, from the 2nd to 3rd of May.

 

More information about the program: RespFest - Respirometry for physiologically oriented questions (univ-lr.fr)

 

More information about the social events: RespFest - Respirometry for physiologically oriented questions (univ-lr.fr)