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In this article, we showcase how the microplate respirometry system provides an easy to use, high throughput respirometry tool for determining oxygen consumption rates in various life stages of terrestrial insects, here using fruit flies (Drosophila melanogaster and Drosophila littoralis). The project was performed at Aarhus University in Denmark in collaboration with Prof Jesper G. Sorensen at Aarhus University in Denmark.

The first part of the project looked at the respiration rates in eggs, third instar larvae, and pupae of D. melanogaster. The fruit flies were maintained at 19 °C, and were in the following life stages:

• melanogaster eggs: 0-12 hours old
• melanogaster larvae: 6-7 days since egg stage
• melanogaster pupae: 8-9 days since egg stage

The first trial had 10 x eggs + 10 x pupae (+ 4 x blanks). The eggs and pupae were transferred directly from their food solution/colony and placed individually in a well in the 80 µl glass microplate. The wells were then closed using gas-tight non-toxic sealing film, and placed onto the microplate reader. The microplate reader and glass plate were then covered with an opaque plastic box, to prevent ambient light from interfering with the optical oxygen sensor inside each well, and left at room temperature (21 °C). Here is a picture of the setup:

The MicroResp™ v1.1 software was used to calibrate the oxygen sensors, set up the plate and experiment settings, and to log data. Here are the exported data graphs:

Oxygen data graph for 10 x eggs (dark golden), 10 x pupae (purple), and 4 x blanks (black) measured over 2 hours and 18 min.

The next trial used 20 x third instar larvae (dark cyan) and 4 x blanks (black), and data was logged for 1 hour and 35 min.:

A data plot of the MO₂ values shows oxygen consumption rates in the pupae (second from right) and larvae (first from right) but no consumption was detected in the eggs and blanks (far left):

The next part of the project investigated the respiration rates in adult fruit flies, here D. littoralis. Besides determining the oxygen consumption rates of adult individuals, we also wanted to look at the effects on MO₂ from anesthetization with CO₂ – a common anesthetic used when handling live fruit flies. An individual adult fruit fly was transferred using an aspirator tube from their maintenance chamber at 21 °C and then directly into a well or anesthetized for 30 sec. with CO₂ prior to transfer. The 200 µl glass plate was then closed with sealing film and placed under a box.

The MicroResp™ v1.1 software was used to calibrate the oxygen sensors, set up the plate and experiment settings, and to log data. Here are the exported data graphs:

Oxygen data graph for 12 x adult flies (green), 8 x adult flies anesthetized with CO₂ (blue), and 4 x blanks (black) measured over 4 hours and 41 min. A data plot of the MO₂ values shows no apparent difference between non-anesthetized (mid) and anesthetized individuals (right):

In conclusion, the microplate respirometry system was able to dertemine oxygen consumption rates in various life stages of fruit flies in a relative short period of time.

Want to learn more about the microplate respirometry system?

• Microplate respirometry system product page
• Learn more about the MicroResp™ software
• User case – Testing microplate system against traditional gas analysis
• User case - Antarctic insect metabolism

We are excited to offer users of Loligo® software this new free and useful tool.

The LoliGO App is a smartphone application for Android and iOS that allows users to remotely monitor their experiments while having the option of getting push notifications in case of power outs in the lab, computer failures, or alarms based on water quality thresholds – like if the temperature gets critically high in your respirometry chambers.

The LoliGO App uses a secure cloud-based service to handle data sent from the Loligo® software and to the App. Users can set up a user profile, and multiple users can access the same user profile at the same time (if they know the password!) aka you can have a shared lab profile.

You cannot alter or stop experiments from the app, and you cannot export or otherwise gain access to the raw data from the app. Completed experiment data will be automatically deleted from the app after 14 days.

The LoliGO App is free to use, and is currently compatible with AutoResp™ v3, and the app will be available with future updates of other Loligo® software soon.

Download the latest version of the LoliGO App:

• LoliGO for Android (on Google Play)
  
  
• LoliGO for iOS (on App Store)

Once again, we are honored to be supporting the scientific community. In 2024, we are sponsoring a list of symposia, workshops, and meetings, and we encourage all interested to take a closer look at the following events:

• We have sponsored travel money for invited speakers at the very interesting SEB 2024 Symposium ”Balancing energy acquisition, expenditure, and allocation in an ever-changing world” through Dr. Amanda Pettersen (co-chairs: Neal Dawson, Ed Ivimey-Cook), and we hope to see you all for a great event in Prague, July 2-5!
  
  
• We have donated cash to support students going to the CSZ-SCZ annual meeting on May 6-10, 2024, in Moncton, NB.
  
  CSZ-SCZ 2024 - Home / Accueil (cszsczmoncton2024.com)
  
  
• Once again, we have accepted to be ICBF 2024 Student Travel Sponsors to help young people participate in this biennial meeting.
  
  International Congress on the Biology of Fish 2024 | 15th (michiganseagrant.org)
  
  We also support this meeting through a Lake Superior Sponsorship and participate as exhibitors as we did for nine (9) consecutive ICBF meetings
  
  See you in Ann Arbor Jun 23-27!
  
  
• We are proud sponsors of the 47th annual Larval Fish Conference in Huron, Ohio, on May 12-16, and hope that it will be a great event.
  
  47th Larval Fish Conference
  
  
• We are also proud sponsors of the upcoming Midwest Zebrafish Conference 2024 taking place June 7-9 at Washington University in St. Louis.
  
  Check out the meeting right here: Midwest Zebrafish Meeting 2024
  
  
• We give financial support to a workshop on metabolism in animal ecophysiology arranged by Dr. Christel LeFrancois, Dr. Marie Vagner (Brest) and Dr. Loïc Teulier (Lyon). This seminar will take place on October 2-3, 2024, in La Rochelle, France.
  
  For more details, please visit: https://www.rt-metabolica.cnrs.fr/

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?

• Glass chamber respirometry systems
• Read more about the intermittent respirometry software, AutoResp™ v3
• Learn how to calculate the respirometry parameters in AutoResp™ v3
• More published papers on aquatic respirometry

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

• Biol Open (2023) 12 (12): bio060213.
• https://doi.org/10.1242/bio.060213

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 (∼9 h) than bees tested in the microplate system (∼3 h) due to the differences in chamber size (3 ml 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?

• Microplate respirometry system product page
• Learn more about the MicroResp™ software
• Another microplate respirometry usercase featuring Antarctic insects

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

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.

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?

• AutoResp™ v3 main site
• How to videos on AutoResp™ v3
• Algorithms used in AutoResp™ v3