TITLE: Assessing Growth Parameters in amphipod, Parhyale hawaiensis: Weighing and Measuring Length
AUTHORS: Ibrahim Lawan

[DESCRIPTION]
The developed protocol provides a standardised method for precisely determining the weight and length of Parhyale hawaiensis, a crucial model organism in ecotoxicology and ecophysiology research. By employing precise techniques with analytical scales and imaging software, researchers can obtain reliable data to investigate the effects of environmental stressors on aquatic ecosystems.

[STEPS]
SECTION: Introduction
1. Parhyale hawaiensis is a circumtropical marine amphipod crucial for epibenthic communities in tropical marine ecosystems (Poovachiranon et al., 1986). This amphipod has gained recognition for its sensitivity to environmental stressors and ecological significance, making it a promising model organism for tropical marine studies (Dos Santos et al., 2022) and a valuable laboratory model for various disciplines (Paris et al., 2022). To fully utilise P. hawaiensis as a global model for ecotoxicology and ecophysiology research, it is essential to establish a standardised and precise protocol for accurately measuring its weight and length. Doing so will enable researchers to collect accurate data and investigate the impact of environmental stressors on aquatic ecosystems, ultimately advancing our understanding of the ecotoxicological effects of key environmental contaminants.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
2. To measure the weight of the amphipod, Parhyale hawaiensis, a Sartorius digital analytical scale (0.01 ± 0.02 mg) that can measure small masses with a high degree of accuracy was used. Here is ageneral protocol for weighing P. hawaiensis:
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
3. Obtain a small dry, clean weighing boat or petri dish.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
4. Turn on the analytical balance and allow it to stabilise for a few minutes.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
5. Place the small weighing boat on balance and tare it (i.e., set it to zero) or record the weight of the boat.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
6. Cut the tip of a 127-P330-000 liquipette pipette at 3 - 4 mm
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
7. Gently use the cut pipette and transfer the specimen (a group of 4 to 6 amphipods, based on preference) into the weighing boat on the scale.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
8. Use another liquipette pipette to remove all the water from the boat and around the amphipods.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
9. Record the weight of the amphipods. OR
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
10. Subtract the weight of the empty weighing boat from the combined weight of the boat and the specimen to obtain the weight of the amphipods.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
11. Expose the organisms for a specified experimental period, such as one to two weeks.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
12. Obtain a new specimen (a group of 4 to 6 amphipods, based on preference) of the organisms at the end of the exposure.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
13. Measure the weight of the specimen.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
14. Compare the weight of the specimen from the start and end of the exposure to analyse growth parameters. OR
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
15. Repeat the process at several time points to determine the overall growth rate of the amphipods over time.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
16. Repeat the process for the additional specimens if needed.
SECTION: Weighing Parhyale hawaiensis Wet Weight to Measure Growth Parameters
17. NB: Handling the amphipods gently and with care and keeping their environment as stable as possible are essential to avoid stress or causing any damage during the process. Also, it is crucial to keep the weighing boat and scale clean to avoid contaminating the specimen.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
18. To measure the body length of a curved amphipod crustacean such as Parhyale hawaiensis, ImageJ software was used in a slightly different approach when measuring a straight-line length. Therefore, the body length was calculated as the distance along the dorsal side from the base of the first pair of antennae to the base of the telson, as suggested by (Asochakov, 1994). Here are the steps to measure the length of P. hawaiensis using ImageJ:
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
19. Place a clean and dry petri dish on a graph sheet.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
20. Cut the tip of a 127-P330-000 liquipette pipette at 3 - 4 mm
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
21. Use the cut pipette to gently transfer the specimen (a group of 4 to 6 amphipods, based on preference) into the petri dish.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
22. Use another liquipette pipette to remove all the water from the petri dish and around the specimen.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
23. Take an image of the specimen and open it in ImageJ.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
24. Use the "Set Scale" function in ImageJ to set the correct pixel-to-length conversion. To do this, select the "straight line " tool from the toolbar, measure a known length of a reference object in the image (such as a 10 mm size from the graph in the picture) and select “Analyze”, then set the scale as 10 mm known size. ImageJ will then adjust the length measurements accordingly.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
25. Right-click and select the "segmented line " tool from the toolbar.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
26. Carefully trace the curved body of Parhyale from the base of the first pair of antennae to the base of the telson with the “segmented line” selection tool. Try to follow the curve body as closely as possible without including overlapping areas.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
27. Right-click at the last point to drop the “segmented line” selection tool and select “Analyze”, and then “Measure” to obtain the length of the amphipod.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
28. Repeat the process for the additional amphipods from the specimen.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
29. Once done with all the amphipods from the specimen, select "Analyze" from the top menu and then "Measure". This will open a window that displays various measurements of the selections, including the lengths.  Copy the length measurements of all the amphipods from the specimen into Excel and take the average as the length of a particular specimen. Or save the measurements by selecting the desired output format.
SECTION: Measuring the Length of Parhyale hawaiensis to Analyse Growth Parameters
30. NB: To avoid stress or damage, the amphipods must be handled gently and with care, and their environment must be kept as stable as possible.
SECTION: Conclusion
31. In summary, the developed protocol offers a robust and systematic approach for accurately determining the weight and length of Parhyale hawaiensis, an amphipod that is crucial for ecotoxicological research. By employing a Sartorius digital analytical scale with high precision and ImageJ software for weight and length measurements, respectively, this protocol ensures the reliability and consistency of data acquisition. The method highlights the importance of handling amphipods with care to minimise stress and maintain environmental stability throughout the procedure, thus reducing the likelihood of any potential harm to the organisms. Additionally, the protocol provides clear and concise step-by-step instructions, which facilitates the ease of replication and application in research settings. Overall, this protocol serves as a useful tool for scientists aiming to assess growth parameters and analyse the effects of environmental stressors on aquatic organisms, contributing to advancements in the field of aquatic ecotoxicology.