Biofluorescent response in lumpfish Cyclopterus lumpus to a therapeutic procedure as assessed by hyperspectral imaging

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Biofluorescent response in lumpfish Cyclopterus lumpus to a therapeutic procedure as assessed by hyperspectral imaging

https://doi.org/10.21203/rs.3.rs-3348858/v1

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published 05 Feb, 2024

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The demand for lumpfish (Cyclopterus lumpus) as a biological control for salmon lice is increasing. However, lumpfish welfare is considered a limiting factor within aquaculture operations. Identifying a noninvasive parameter that measures subclinical stress in lumpfish is a key goal for improving their welfare. The lumpfish is amongst many marine species documented to biofluoresce within the blue shifted light of their environment. Lumpfish (n = 80) underwent a 3-hour freshwater bath therapeutant to evaluate whether fluorescence spectra produced by the species responds to external stimuli. Lumpfish were quickly scanned under a hyperspectral camera (400–1000 nm spectral range) prior to and after treatment. Here we show that lumpfish fluorescence responds to freshwater exposure within a controlled experiment. All three experimental groups increased their fluorescence in response to treatment, though the level of change depended on whether the averaged, red, or green spectra was analyzed; the control group remained consistent. The mean emission spectra differ between groups, particularly between 690 to 750 nm. All lumpfish emit green fluorescence while a segment (49%) produced red fluorescence. As lumpfish fluorescence shifts in response to the applied therapeutant, this study provides insight into how fluorescence may be incorporated into the welfare management of an aquatic species.

Biological sciences/Biophysics/Biological fluorescence

Biological sciences/Zoology/Ichthyology

The demand for lumpfish (Cyclopterus lumpus 1758 Linnaeus) to control sea lice in salmon farms is rising due to increasing resistance of Lepeophtheirus lice to licensed therapeutants¹. Sea lice infestations in salmon farms significantly impact the welfare of farmed fish, which in turn have repercussions for the viability of such operations. However, lumpfish welfare concerns, such as systemic infections, are considered a limiting factor within aquaculture operations for incorporating this species to combat sea lice². The difficulty of identifying parameters which successfully link together husbandry, health, and allostatic responses to environmental factors has slowed the formation of operational welfare indicators (OWIs) that can be used as an overall application for this species. A protocol developed by Eliasen et. al. to address this issue focused on weight-length relationship, external physical damage scores, stomach content analysis and calibrated liver colour scores. Though useful, this OWI remains a representative sampling technique of a population that requires invasive (lethal) techniques. As lumpfish are typically kept in high density cohorts or in commercial salmon pens, finding a way to study lumpfish en masse would be ideal for effective management.

Biofluorescent organisms absorb the shorter wavelengths of the dominant ambient blue light of the ocean to reemit them as longer wavelengths of green, orange, or red light³. Biofluorescence within the physiology of a broad assemblage of fish species is a well-documented phenomenon. Scorpaeniformes such as the false stonefish Scorpaenopsis diabolus were documented to produce fluorescent emissions in both the red and green parts of the spectra. As the lumpfish family Cyclopteridae is part of the Scorpaeniformes, it should come as no surprise that this red and green fluorescence has been documented within this cyclopterid. The related Pacific Spiny Lumpsucker Eumicrotremus orbis emitted sexually dimorphic fluorescence, with males producing red whilst females produced mainly green⁴. Juvenile lumpfish reared in an aquaculture facility produce a bright green fluorescence⁵. With approximately 29 species in 7 genera being recorded in Cyclopteridae⁶, this dimorphic fluorescence may be widespread. Lumpfish have a unique physiology that may allow fluorescence to change quickly. The bodies of gravid females have extensive subcutaneous jelly and a loose-fibred dorsal musculature with high water content that makes the animal similar in density to seawater while mature males contain a higher lipid content^7,8. This loosely structured, aqueous physiology may allow fluorophores to be transported quickly within the body. The fluorophore(s) responsible for fluorescence in lumpfish has not been isolated; however, they have been identified in other fish as either metabolites or fluorescent proteins^9,10. What is further unknown is whether lumpfish fluorescence responds to internal or external stimuli on the animal. Understanding the physiology of biofluorescence in lumpfish requires technology that can quickly analyze changing parameters in a living organism.

Hyperspectral imaging technology has the capacity to provide a non-destructive method to monitor changing conditions within live fish. Detailed spectral bands of emitted light captured by hyperspectral imaging exceed the recording capacity of traditional cameras¹¹ The light measured for each pixel is recorded across the electromagnetic spectrum, reflecting the chemical and physical composition of the imaged object^12–14. To our best knowledge, hyperspectral imaging on live fish has been limited. A study by Lindberg et. al. used this technology on anesthetized Atlantic salmon Salmo salar smolts¹⁵. Further studies used freshly euthanized Atlantic salmon smolts in successfully auditing hemorrhaging in the dorsal fin region¹⁶. Our study aims to determine whether lumpfish biofluorescence is responsive to a standard veterinary therapeutic treatment through hyperspectral analysis.

General fluorescence

Lumpfish within this study produce 2 distinct fluorescence peaks, a broad green peak (~ 580–690 nm), and a pronounced red emission maximum (~ 690–715 nm). The documented fluorescence spectra are defined as green dominant with only the broad green peak fluorescence and red dominant that emits both green fluorescence and a sharp emission peak in the red spectra (Fig. 1). A portion of the measured lumpfish have both (49%), while the rest fluoresce green only (51%). A visual representation of the red and green fluorescence produced by lumpfish can be seen in Fig. 2.

Fluorescence spectra analysis

The fluorescence of the four experimental groups before and after the therapeutic treatment was compared first. The mean and the standard deviation of the fluorescence spectra for the experimental groups in the spectral range from 570 to 800 nm were classed as a combined single average of the fluorescence spectra (Fig. 3a-d). The mean emission spectra differ between groups, particularly between 690 to 750 nm. (Fig. 3e-h). A difference in the mean and a lower overlap of the standard deviation can be observed in this spectral region for the groups after the stress treatment; The mean fluorescence is higher after the treatment (Fig. 3f-h). For the control group, the mean spectra before and after the treatment are unchanged (Fig. 3e).

There is a high overlap between the standard deviation of the fluorescence spectra before and after the treatment (Fig. 3). For a more precise analysis, we computed the area under the curve (AUC) of the fluorescence spectra, which is the radiance over the spectral range, and used violin plots to represent their distribution before and after the treatment (Fig. 4). When all the spectral range (570 to 800 nm) is considered, we can observe different distributions of the AUC between the experimental groups (Fig. 4a). Additionally, we can observe that the distribution of the data corresponding to the control group, group 2, and group 3 present a main peak but a long tail for high values of the AUC. For the green spectral range (570 to 690 nm), there is a change in the AUC distribution for all the groups before and after the treatment (Fig. 4b). For the groups under stress treatment (G1, G2, and G3), the change in the shape of the distribution is more evident, and an increase in the mean AUC is observed. Additionally, a bimodal distribution can be observed for G1. Finally, the violin plots of the AUC in the red region of the spectra (670 nm to 800 nm) do not show evident differences in fluorescence before and after treatment (Fig. 4c).

Florescence type analysis

Since two distinct fluorescence signals were present in the lumpfish, each signal was analyzed separately. The mean and the standard deviation of the fluorescence spectra for all lumpfish (Fig. 5a), and for green dominant (Fig. 5b) and red dominant lumpfish (Fig. 5c) are shown in Fig. 5. Peak emission signals were reviewed separately due to detection limits. For the green dominant samples, a difference in the mean fluorescence spectra for the treatment groups can be observed, with a higher fluorescence after treatment, showing an increase in the mean spectral radiance after treatment of 11%, 4% and 16% for groups G1, G2, and G3, respectively. Those differences are more pronounced in the spectral range from 570 to 690 nm and for groups G1 and G3 (Fig. 5b). For the control group, the mean spectra before and after 3 hours are similar (with a 1% increase in the mean spectral radiance after the treatment), and the variations of the spectra are entirely overlapped. A similar trend can be observed for red dominant samples, with an increase of 5%, 10%, and 15% in the measured mean spectral radiance for groups G1, G2, and G3, and only a 1% increase for the control group. The mean fluorescence spectra are higher after treatment for all the treatment groups in the spectral range from 670 to 750 nm (Fig. 5c). Groups G1 and G3 also showed differences in the fluorescence spectra between 570 to 690 nm. For the control group, there are no visible differences between the fluorescence before and after 3 hours.

Green dominant fluorescence

The violin plots for the green dominant samples show a change of distribution and an increase in the fluorescence AUC for the groups after the treatment when all the spectral range is considered (Fig. 6a). We can also observe a change in the distributions before and after treatment when only the green region of the spectra (570 to 690 nm) is considered (Fig. 6b). These differences are more pronounced for groups G1 and G3. In the red parts of the spectra (from 690 to 800 nm), we observe that the range of the AUC values is similar to those within the green spectra (Fig. 6c). It is possible to additionally observe an increasing trend of the fluorescence AUC for the experimental groups after treatment, which is also shown to be higher for groups G1 and G3. According to this information, we can conclude that green dominant lumpfish show an increase in fluorescence after treatment in both the green and red regions of the spectra. Moreover, it is possible to observe bimodal distributions for the green and the red spectral ranges, which suggests that there are also subpopulations within the green dominant lumpfish. Regarding the control group, there is a slight change in the AUC distribution before and after 3 hours, but the increase in fluorescence is not evident.

Red dominant fluorescence

The violin plots of the red dominant lumpfish for all the spectral range show a subtle increase in the AUC for the groups under treatment (Fig. 7a). When the AUC distributions are calculated for the green and the red spectral ranges separately (Fig. 7b and 7c), it can be observed that the higher contributions to the total AUC come from the red region of the spectra. However, it can be observed that the most significant changes before and after treatment are shown in the green region of the spectra, where an increase in fluorescence can be observed for groups G2 and G3. Also, in that region of the spectra, the distribution of the AUC is changed after 3 hours for all the groups. On the other hand, in the red region of the spectra, the difference in AUC before and after 3 hours is not as evident, which indicates that the main changes in fluorescence spectra after treatment for red dominant lumpfish are in the green region of the spectra. Bimodal AUC distributions can be observed for both green and red spectra.

The production of biofluorescence by lumpfish is not new to science. A study of lumpfish serum (F = 8; M = 4) found female lumpfish producing a blue-green blood serum that fluoresced a pale blue under long-wave UV light (~ 350 nm), with the red-colored male serum emitting a magenta-orange fluorescence¹⁷. This coloration matches the distinct external fluorescence produced by the lumpfish in this study. This difference in fluorescence is caused by the sexual dimorphism within the species. The serum of both sexes contained a chromoprotein with biliverdin prosthetic group; however, the male serum contained a red pigment such as phycoerythrin at a much higher rate than in females. The sex-based fluorescence matches lumpfish fluorescence documented in this study as all lumpfish were found to produce green fluorescence but only 49% were producing red fluorescence. Sexually dimorphic lumpfish do not feed during the breeding season; such color produced by the species at this time cannot be linked to diet¹⁸.

In this experiment we show that lumpfish fluorescence responds to freshwater exposure within a controlled experiment. This therapeutic treatment was selected as the stress inducing factor as it is a routine treatment in the aquaculture industry for lumpfish suffering from amoebic gill disease. Lumpfish are known to physiologically tolerate freshwater therapeutic baths well. As their tolerance to this treatment is typically higher than other fish species, the fact that a change in their baseline fluorescence levels can be observed from such a short-term impact means that their fluorescence responds quickly to their physiological state. This responsiveness was supported by the differences between the three experimental groups and the control group. The mean fluorescence emission spectra (690–750 nm) differ between these groups. This fact can be associated with the presence of varying numbers of lumpfish showing different intraspecific fluorescence types in the groups. On the other hand, the red region (670–800 nm) within the average fluorescence spectra shows a high difference between the groups. Such changes within the experimental groups may be caused by individual male lumpfish exhibiting different levels of red fluorescence. In this region of the spectra, only group 3 shows a higher fluorescence after the treatment. This may mean that the red fluorophore is actively manipulated by male lumpfish. The fluorescence emission spectra were consistent in the control group between the pre and post treatment period. As the fish underwent the same procedures as the three control groups, the only external variable that could explain the limited change in fluorescence was that their 3-hour period was spent in salt water. This supports our hypothesis that freshwater treatment causes observable changes in the fluorescence spectra within the experimental groups.

High differences in the average fluorescence spectra were observed between all experimental groups. We documented that the number of lumpfish per group showing variations in fluorescence (green dominant or red dominant) was different. This variation in fluorescence may relate to the sex, dominance, or sexual maturity of the lumpfish within each experimental group. However, the lumpfish were randomly selected by an independent facility technician to prevent sex-based bias in the study. Hence, these different fluorescence types may influence the mean fluorescence spectra. For this reason, we performed the same analysis independently for the different fluorescence signal types. Large individual variations of fluorescence signals were observed between the fish, and we find it reasonable to assume that the fluorescence in lumpfish depends on more than their treatment. Documenting this variation produced by lumpfish alone has provided a contribution towards understanding the dynamics of fluorescence within this species. In retrospect it would have been advantageous to track the individuals during the experiment and note other relevant information that could have contributed to explaining the behavior of the data. However, the welfare-based time constraint set for handling fish out of water was a limiting factor in our experimental design. The group method for assessing fluorescence was further chosen as tracking individual fluorescence in an aquaculture setting with thousands of fish is not currently feasible with the technology available. As it stands, a group average seems inadequate for making a sound statistical inference on a treatment effect. This may be further influenced by the physiological tolerance of lumpfish to freshwater therapeutic baths received by the species. Finding a more precise method for tracking changes in fluorescence that matches the documented variation within lumpfish biology should be prioritized. The challenges related to the group average may be leveraged when using hundreds of individuals per trial, where the unexplained variation might be lower.

Several potential advantages of incorporating fluorescence level monitoring within lumpfish operations exist. This automated approach for monitoring lumpfish welfare includes increased sensitivity to subclinical changes within lumpfish, assessing stress levels on a large scale within production cohorts, and producing quantitative data that can help producers make informed husbandry decisions. Validation through further testing is needed to determine the feasibility of integrating these results into the existing welfare auditing programs for aquaculture operations raising lumpfish. Underwater camera technology, imaging analysis programs, and artificial intelligence are rapidly making automated fish OWI monitoring a feasible alternative in aquaculture operations¹⁹. Fluorescence monitoring in combination with other OWI monitoring standards can provide comprehensive, non-invasive monitoring within production units on a scale that has not been attempted to date. To the best of our knowledge, this is the first example of using hyperspectral imaging for monitoring biofluorescence as a potential welfare standard in aquaculture. Understanding stress in lumpfish prior to the manifestation of clinical signs has the potential to improve their welfare prior to the onset of either morbidity or mortality.

Experimental outline

The experimental component of this study is based on an accepted veterinary therapeutant (freshwater bath) to control the amoebic gill disease (AGD) pathogen Paramoeba perurans in lumpfish aquaculture operations²⁰. The standard therapy for AGD in lumpfish is a three-hour freshwater bath, with a salinity of less than three parts per thousand (ppt)²¹. The histological results, good tolerance of lumpfish to freshwater, and the lack of morbidity/post treatment mortalities during clinical trials indicate that treatment of lumpfish with freshwater baths can be successful with either short- or long-term treatments. Each trial cohort was placed in a flow through a 1,000-litre freshwater bath for 3 hours. The freshwater bath was chlorine free, oxygenated, and matched in temperature to seawater prior to flowing through the trial tank. A total of 3 iterations were conducted of the treatment with cohorts of 20 fish each plus a control group of 20 (n = 80). The lumpfish were imaged before and after the treatments with a hyperspectral camera.

Lumpfish

The 80 lumpfish (931 ± 50 g, 27 ± 0.5 cm) utilized for the stress test were maintained at the Aquaculture Research Station located in Kårvik, Norway. The fish originated from within the same production system and were the same age/size class. Three groups of 30 fish were acclimated for 1 month in 3 500-liter flow through tanks supplied with filtered sea water. Tanks were maintained at ambient seawater temperatures and salinity (36 ppm) with lights maintained at 24 hours with 75% intensity. Fish were starved for 24 hours prior to utilization in the stress test. The experimental cohorts of lumpfish were humanely euthanized after the experiment using an overdose of the anesthetic benzocaine (Benzoak vet., ACD Pharmaceuticals AS, Oslo, Norway). The use of lumpfish for this experiment was authorized by the Norwegian Food Safety Authority (FOTS) ID 29929.

Instrumentation

The hyperspectral imaging setup was a custom imaging platform consisting of a VNIR-1800 hyperspectral camera (Hyspex, Oslo, Norway) mounted above a conveyor belt with a custom illumination setup. The VNIR-1800 is a push-broom hyperspectral camera, which means that each frame consists of the spectral information of a thin spatial line across the field of view (x-axis) captured by this camera. The second spatial dimension (y-axis) is collected by spatially scanning the samples. The lumpfish were placed in a white tray partially filled with water on a conveyor belt for spatial scanning. The VNIR-1800 camera works in the Visible and Near Infrared (VNIR) spectral range (from 400 to 1000 nm), with a spectral resolution of 5.5 nm, and 1800 spatial pixels. The working distance between the camera and the samples was 100 cm, resulting in a FOV of 30.6 cm across the conveyor belt (x-axis), with a spatial resolution in the x direction of 0.17 mm. The light source used in this experiment was LED lighting (G5 XR30 Pro Radion, Ecotech, Bethlehem, PA, USA) and the excitation wavelength selected was Royal Blue (wavelength peak at ~ 445 nm). Since the fluorescence signal is expected to be weak, a high exposure time was configured for the hyperspectral measurements (7.52 ms). The spatial resolution in the y-axis on push-broom cameras depends on the exposure time and the speed of the conveyor belt. The speed of the conveyor belt was configured to 6 cm/s resulting in a spatial resolution of 0.45 mm in the y-axis.

A digital single lens reflex (DSLR) camera (D5100 /Nikon AF-S 60 mm f/2.8G IF-ED Micro lens, Nikon, Minato City, Tokyo, Japan) was used for photographing lumpfish presented in Fig. 2. A seawater filled aquarium was illuminated under full spectrum LED lighting Royal blue illumination (~ 445nm) to document biofluorescence while the ambient light photographs were taken with cool white light. A yellow barrier filter (Tiffen 62DY15 62 mm Deep Yellow 12 Filter) was utilized when capturing RGB to help block reflected blue light.

Hyperspectral analysis

When it comes to conventional hyperspectral image applications that use broadband light, a common practice is to conduct a flat field calibration. This involves using a material with high and known reflectance in the spectral range of interest to convert the input radiance into reflectance, while also correcting for the spectral response of the instrumentation^22,23. Unfortunately, there is no standard procedure for calibrating fluorescence spectral data. For that reason, we perform a radiometric calibration in this work to produce spectral radiance values in radiance over the spectral range \(W\bullet {m}^{-2}\bullet s{r}^{-1}\bullet n{m}^{-1}\). The radiometric calibration was performed using the HySpex Rad v2.5 software (Norsk Elektro Optikk, AS, Oslo, Norway).

The radiometrically calibrated hyperspectral data was loaded into the Breeze analytical software (Prediktera, Umeå, Sweden) to segment the lumpfish. The segmentation of the fish consisted of selecting a spectral channel near the excitation wavelength showing a high contrast between the background and the fish, followed by the manual identification of the contour of the fish. The mean spectra from 570 to 800 nm were extracted for each sample and processed using MATLAB 2022b and the Seaborn Python package²⁴.

Statistical analysis

There is, to the best of our knowledge, no published literature on this topic, which makes it rather tricky to calculate a minimum sample size for statistical inference. It is also not given that the distributions of the variables of interest are sufficiently well behaved for the most common hypothesis tests (ANOVA, t test etc.) to be applicable. Nevertheless, the sample size was decided based on a power analysis for an ANOVA test assuming a small to medium Cohen’s d²⁵. However, the distribution of the data made an ANOVA test unsuitable, so the decision was made to keep the statistics on a descriptive level.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author.

Acknowledgements

I would like to thank Evan Durland of Nofima for helping make this project happen. We would also like to thank Eirik Mack Eilertsen (sea lice Eirik) of Tromsø Aquaculture Station for his help during the experimental trial. This project was supported by the Deep Vision Project of Nofima with aadditional funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 956697.

Author contributions statement

Conceived the experiment(s) TJD. Conducted the experiment(s), TJD, SO, AK. Analyzed the results, SO, TJD, SL. Original draft written by TJD, SO. Review and editing SO, SL, TJD.

Competing interests
The author(s) declare no competing interests.

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No competing interests reported.

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You are reading this latest preprint version

Biofluorescent response in lumpfish Cyclopterus lumpus to a therapeutic procedure as assessed by hyperspectral imaging

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Journal Publication

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Abstract

Figures

Introduction

Results

General fluorescence

Fluorescence spectra analysis

Florescence type analysis

Green dominant fluorescence

Red dominant fluorescence

Discussion

Methods

Experimental outline

Lumpfish

Instrumentation

Hyperspectral analysis

Statistical analysis

Declarations

Acknowledgements

Author contributions statement

References

Additional Declarations

Status:

Journal Publication

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