Larval development and gene expression were followed from hatch until the corresponding first-feeding stage in each temperature treatment. The first-feeding stage, as previously defined [3 ], was set as the time point when eye pigmentation, mouth and jaw formation was completed. Endogenously feeding larvae of European eel were anesthetized using tricaine methanesulfonate (MS-222) prior to digital imaging and euthanized post-sampling by using an MS-222 overdose. All images were taken using a digital camera (Digital Sight DS-Fi1, Nikon Corporation, Japan) attached to an objective microscope (Eclipse 55i, Nikon Corporation, Japan). NIS-Elements D analysis software (Version 3.2) was used to analyze the images of eggs, embryos, and larvae (Nikon Corporation, Japan).
Nis elements d analysis software version 3.2
Manufactured by Nikon
Sourced in Japan
NIS-Elements D analysis software (Version 3.2) is a comprehensive imaging software package designed for use with Nikon microscope systems. The software provides a suite of tools for image acquisition, processing, analysis, and management, catering to the needs of researchers and professionals working in various scientific and industrial fields.
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5 protocols using nis elements d analysis software version 3.2
1
Eel Larval Development and Gene Expression
2
Eel Larval Development and Gene Expression
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Larval development (biometry), mortality, and gene expression of selected genes, corresponding to specific molecular mechanisms were followed from hatch and throughout the endogenous feeding stage (2, 4, 6, 8, 10, and 12 dph). All endogenously feeding larvae of European eel were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging and euthanized post-sampling by using an MS-222 overdose [13 (link)]. All images were taken using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan), while NIS-Elements D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan).
3
Assessing Embryonic Survival and Development
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Dead embryos were removed from the bottom of the incubator and counted at 4, 24, and 48 hpf for assessment of embryonic survival. Embryos (n = ~20) from the floating layer from each replicate were randomly sampled at 4 hpf (Fig 2A ) and imaged using a digital camera (Digital Sight DS-Fi1, Nikon Corporation, Japan) attached to an objective microscope (Eclipse 55i, Nikon Corporation, Japan) and categorized according to irregular cell cleavage patterns following [35 (link)]. Subsequent samples were taken at 24 hpf (Fig 2B ) and digitally imaged for measuring chorion diameter and perivitelline space area. NIS-Elements D analysis software (version 3.2) was used to analyse the images (Nikon Corporation, Japan).
4
Larval Development Biometry in Fish
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In order to follow larval development (biometry) from hatch until the feeding stage, larvae were sampled at 0, 6, and 12 dph. From each replicate (n = 3), family (n = 3), and treatment (n = 4) ~10 larvae were randomly sampled and imaged using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan). All larvae were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging. Subsequently, NIS-Elements-D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan), where total body area (BA) and oil-drop area (ODA) were measured for each larva. Larval growth [(BA (day12) -BA (day0) ) / Age (12 days) ] and oil-drop utilization [(ODA (day0) -ODA (day12) ) / Age (12 days) ] rate were measured from the change in body and oil-drop area, respectively. Growth efficiency was then measured by dividing the increase in body area by the corresponding decrease in oil-drop area (Politis et al., 2014 b) . Moreover, the severity of pericardial edema was calculated using the ratio between edema/neurocranium heights (Okamura et al., 2009) (link).
5
Larval Biometry and Oil-Drop Utilization
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Larval development (biometry) was followed from hatch and throughout the endogenous feeding stage (0, 6, and 12 dph), where ~10 larvae from each replicate (n = 3), family (n = 3), and treatment (n =4) were randomly sampled and digitally imaged using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan). All endogenously feeding larvae were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging. NIS-Elements-D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan), where total body and oil-drop area were measured for each larva. Larval growth and oil-drop utilization rate were measured from the change in body and oil-drop area, respectively. Growth efficiency was then measured by dividing the increase in body area by the corresponding decrease in oil-drop area (according to Politis et al 2014) (link). Moreover, the severity of pericardial edema was calculated using the ratio between edema/neurocranium heights according to Okamura et al 2009.
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