A Novel Application of Bubble-eye Strain of Carassius Auratus for Ex Vivo Fish Immunological Studies

doi:10.21203/rs.3.rs-273301/v1

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A Novel Application of Bubble-eye Strain of Carassius Auratus for Ex Vivo Fish Immunological Studies

https://doi.org/10.21203/rs.3.rs-273301/v1

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In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at high temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and high rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

Applied & Industrial Microbiology

General Microbiology

Carassius auratus

ex vivo

bubble-eye

novel application

fish immunological studies

Infection control is key in aquaculture (Lafferty et al. 2015). Failure to control infection can lead to colony collapse (i.e., death), which can be very damaging to the local industry. Despite significant investments in infection control, including the use of antibiotics, the aquaculture industry is still affected by infectious diseases ^1–4. High temperatures, often caused by heat waves, increase the risk of infection through a combination of accelerated pathogen growth and suppression of host immune defenses ^1,5,6. In addition, aquaculture will experience unprecedented impacts from climate change, as global average temperatures are likely to increase by 3ºC by 2050 compared to the second half of the 20th century ⁷ and the number of heat waves is likely to increase. Therefore, understanding how temperature increases will affect infectious diseases in cultured aquatic species is important to prevent potential deterioration of the industry.

Temperature alteration impairs fish immunity and develops infections as evidenced by increased infection rates and shortened lifespan in aquatic species ^5,8−14. Most of those studies reported that a warm temperature is associated with increased infection resistance, while it is not always true for some contexts. Increased temperature triggers a stress response that suppresses the immune system of fish species ¹, and such hyperthermia may impact the fishery. To prevent potential damage to the industry, immunostimulants and vaccination may be effective, if not complete ^15,16. To overcome the effects of current and future infections, good laboratory models for screening immunostimulants are needed. For such immunostimulant screening, it is essential to use both live animals raised in laboratory tanks and ex vivo analysis using freshly harvested immune cells. Ex vivo systems allow high-throughput screening of potent immunostimulant candidates, while in vivo confirmatory experiments are required for extensive testing at the industrial level. Lack of either approach will delay the research. In this study, we propose the use of bubble-eye goldfish to fulfill this requirement.

The bubble-eye goldfish is a commercially available strain of goldfish in East Asia. Bubble-eye goldfish have large eye sacs (ocular sacs) under each eye. The ocular sacs are filled with fluid containing lymph and immune cells and can be retrieved with a needle without sacrificing the animal. The ocular sacs can be sampled repeatedly, allowing for time-course studies that track the same set of individuals over time. The lymph of the eye sac contains growth-promoting factors that act on fish cell cultures¹⁷, and cells in the eye sac fluid may play an immunological role. In this study, we will demonstrate the expression of immune-related genes in eye sac cells in response to immune challenge with heat-killed bacteria to elucidate their immune function. We will also demonstrate how temperature elevation affects immune function in goldfish, both in vivo and ex vivo, using a bubble eye goldfish model.

Temperature rise promotes infection in the common goldfish

As shown in Fig. 1, the common goldfish injected intraperitoneally with P. aeruginosa died earlier when kept at high temperature than when kept at the normal temperature (p = 0.021, Log-rank test). No death was observed for the saline-injected groups (Fig. 1).

Inflammatory responses to P. aeruginosa in the bubble-eye goldfish eye-sacs

Eye sac cells (Fig. 2) can be harvested and cultured ex vivo. In the response to immune challenge by heat-killed P. aeruginosa, redness (prominent blood vessels) was observed in the ipsilateral eye sac membrane, but not in the contralateral membrane (Fig. 3), which represents an inflammatory response induced in the eye-sac. We further examined the gene expressions of pro-inflammatory cytokines in the eye-sac immune cells (see Materials and Methods for technical details). As shown in Fig. 4, the mRNA level increased by 800 fold for IL1β1 (p = 0.0001), 200 fold for IL1β2 (p = 0.0005), 10 fold for TNFα1 (p = 0.0090), and 300 fold for TNFα2 (p = 0.0470) in response to the immune challenge (Fig. 4).

Temperature rise suppresses the pro-inflammatory cytokine expressions in the eye-sac immune cells both in vivo and ex vivo

In the eye-sac immune cells in vivo, mRNA levels of induced pro-inflammatory cytokine genes were lower when the fish were kept at high temperature (33ºC) than when kept at the normal rearing temperature (25ºC) (Fig. 5). We then harvested the eye-sac immune cells from untreated bubble-eye goldfish to obtain an ex vivo culture at 25℃ (see Materials and Methods for details), where induction of pro-inflammatory cytokine genes by heat-killed P. aeruginosa was observed in adherent cells but not apparent in non-adherent cells (Fig. 6). Using the ex vivo culture, we examined the effect of temperature rise on the pro-inflammatory cytokine expressions in the eye-sac cells induced by heat-killed P. aeruginosa cells. Compared with the normal temperature, high culture temperatures resulted in reduced cytokine expressions (Fig. 7, and Fig. S1-3).

In the present study, we established a model system to study immune responses using the bubble-eye goldfish. As demonstrated in the present study, this model is suitable for both in vivo and ex vivo analyses of immune cells. For the ex vivo experiments, the eye sacs allow us to collect immune cells without sacrificing the animal, and the harvested immune cells can be cultured on a plastic dish. The ex vivo cultured cells expressed cytokine genes in response to the bacterial challenge, which is consistent with the cytokine gene expressions triggered by an immune challenge observed in vivo. The immune cells showed attenuated expressions of cytokine genes in response to temperature rise as observed in vivo and ex vivo, which sheds light on the underpinning mechanism of the increased infection risks in aquaculture upon temperature rises.

Among the cytokines, IL1β and TNFα play important roles both in innate and acquired immunity, such as the activation of phagocytic cells and the promotion of immune-related gene expressions in a series of immunoreactive cells both in mammals and fishes ¹⁸, indicating that whether an animal is capable of inducing the expression of IL1β or TNFα in response to immune challenges reflects its infection resistance. In this sense, the attenuated induction of these cytokines upon temperature rise may explain the enhanced bacterial infection in the goldfish upon the experimental temperature rise tested in this study.

The microscopy suggests that most of the eye-sac cells are mononuclear cells, but heterogeneous in multiple histological properties such as cell size and nucleocytoplasmic ratio. It should be noted that the adhesion rate to the plastic dish was approximately 50%, and those cells that did not adhere to the dish may represent distinct population from the adherent cell populations. At least, the adherent population showed apparent expressions of pro-inflammatory cytokines in response to bacterial challenge, suggesting its role in the pro-inflammatory cytokine production. Also, mammalian studies revealed that dish-adhering cell populations are rich in monocyte/macrophage lineage cells ^19,20, while goldfish macrophages express IL1β and TNFα ²¹. These are consistent with our finding, and the dish-adhering eye-sac immune cells are most likely monocyte/macrophage lineage cells that respond to the immune challenge by expressing the pro-inflammatory cytokines.

Regarding the temperature sensitivity of cytokine expressions, cytokine productions in human monocytes are reduced by high temperature in vitro²². However, empirical knowledge in fish immune cells is slim. A recent in vivo study in the crucian carp demonstrated that the host viability and the gene expression of pro-inflammatory cytokines after a bacterial infection was reduced by high temperature ²³, supporting the present study in the bubble-eye goldfish. In the bubble-eye goldfish, the eye sac enables immune cell analyses as it provides up to 1 mL of lymphoid fluid from each individual, even without sacrificing the animal. This feature is the major advantage of this model using the bubble-eye goldfish and will accelerate the molecular study of fish immunity and contribute to improved aquaculture productivities.

Goldfish strains (Carassius auratus)

The common goldfish (‘Wakin’) and the bubble-eye goldfish were obtained from a local supplier Kingyo-Zaka (Tokyo, Japan). Goldfish were fed with a commercial diet for goldfish (Kyorin, Hyougo, Japan). All experiments were done after an acclimation period (more than a week) in the laboratory after each purchase, where we kept the goldfish at 25ºC in a fish tank (30 x 45 x 23 cm). The ranges of body weight were 7–10 g for the common goldfish, and 20–28 g for the bubble-eye goldfish upon the start day of each experiment. The research protocol was approved by Animal Welfare Ethics Committee of Genome Pharmaceuticals Institute Co., Ltd. and was conducted in compliance with all relevant guidelines and regulations applicable at the time and place of the experiments, including the ARRIVE guidelines.

Temperature rise paradigms

Temperature rise paradigms were given to the goldfish following the acclimation period at the normal temperature (25ºC). For the infection experiments and the sterile immune challenge experiments, goldfish were kept at either 25ºC or 33ºC for 24 hours before treatments (i.e., infection or sterile challenge). For ex vivo experiments using harvested eye-sac immune cells, cells were harvested from bubble-eye goldfish reared at 25ºC, and the harvested cells were cultured either at 25ºC and 33ºC.

Bacteria (Pseudomonas aeruginosa)

P. aeruginosa, strain PAO1 ²⁴ was aerobically cultured overnight at 37℃ in LB10 medium. In infection experiments, the live P. aeruginosa cells were washed and suspended in saline (0.9% NaCl aqueous solution). For the heat-killed P. aeruginosa cells used in this study, we washed the live cells with saline, and then autoclaved the cells at 121℃ for 20 min.

Infection experiments

To know the effect of temperature on the goldfish immunity, we intraperitoneally injected P. aeruginosa live cells (3 x 10⁷ CFU/fish) to the common goldfish (Wakin). The injected fish were then kept at either 25℃ (normal rearing temperature) or 33℃ (high temperature) and monitored for their survival.

Sterile immune challenge to the eye-sac using heat-killed bacterial cells

An overnight culture of P. aeruginosa was spun and the pellet was resuspended in a tenth volume of saline. We autoclaved this suspension at 121ºC for 20 minutes to obtain heat-killed P. aeruginosa cells. We injected 50 µL of the heat-killed P. aeruginosa cells into the eye-sac of one side, and 50 µL of saline into the eye-sac of the other side. The immune challenges were done either at the normal rearing temperature (25ºC) or at the high temperature (33ºC).

Collection of eye-sac cells

Because the common goldfish is difficult to collect their immune cells without sacrificing the animal, we used the bubble-eye goldfish in the following part of this study to investigate the molecular responses of the goldfish immune system to temperature rises. Eye-sac cells floating in the eye-sac fluid can be easily collected from the eye sacs of bubble-eye goldfish ( ≧ 2 x 10⁵ cells/mL). Eye-sac cells consisted predominantly of mononuclear cells of various sizes, ranging from 5 to 20 µm, and different nucleocytoplasmic ratios (Fig. 2). Some of the cells showed adhesion to plastic dishes in vitro (data not shown). From the bubble eye-goldfish, the eye-sac fluid (containing the eye-sac cells) was collected from the eye sacs using a disposable plastic syringe with a 21-Gauge sterile needle.

Gene expression analysis of eye-sac immune cells

Twenty-four hours after the injection of heat-killed P. aeruginosa into the eye sac, we collected the eye-sac cells as described in the ‘Collection of eye-sac cells’ section. We then isolated the mRNA and performed qRT-PCR analyses (details describe in the following section).

Ex vivo culture of the eye-sac cells

We used a disposable syringe (5 mL) with needle (21 Gauge) to harvest the eye-sac immune cells. The harvested cells (typically 4 mL) were suspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated calf serum (SAFC Biosciences, USA, 3% autologous eye-sac fluid, 20 mM HEPES buffer and antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) and cultured in 24-well plastic plate (catalog# 3820-024, AGC Techno Glass) (8 x 10⁴ cells/0.8 mL/well) for at least 2 hours. The medium had been preincubated at 25 ºC. In this condition, approximately 50% of the harvested cells adhered to the plastic dish. To give an immune challenge, 1.5x10⁸ cells of heat-killed P. aeruginosa were added in the medium when the cells were suspended in the culture dish.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from eye-sac cells by using TRIzol Reagent (ThermoFisher Scientific), treated with DNase I (Promega), and reverse transcribed to obtain cDNA using the High Capacity RNA-to-cDNA Kit (ThermoFisher Scientific) following the manufacturer’s protocol. Using the cDNA, gene expressions were analyzed by qRT-PCR. The qRT-PCR was performed using 7500 Fast Real-Time PCR System (ThermoFisher Scientific) using Fast SYBR ® Green Master Mix (ThermoFisher Scientific). Primers for each target gene were described in the literature ²¹. We chose the elongation factor 1α (EF1α) gene as an internal control as done in the preceding study in the goldfish ²¹.

Statistical analysis

To test the differences between survival curves, log-rank tests were performed using GraphPad Prism version 8.4.3 (GraphPad Software Inc.). To test the differences between mean values, Student’s t-tests were performed using Microsoft Excel 2013.

Acknowledgements

We thank Genome Pharmaceuticals Institute and Kingyozaka (the local goldfish supplier) for providing materials, animals, and technical advice. This work was supported by JSPS KAKENHI for Early-Career Scientists for AM (Grant# 20K16253).

Author contributions

Study conception and design: KS

Acquisition of data: HN, KS

Analysis and interpretation of data: HN, AM, HH, KS

Drafting of manuscript: HN

Critical revision: HN, AM, HH, KS

Competing interests

The authors declare no competing interests.

Lafferty, K. D. et al. Infectious diseases affect marine fisheries and aquaculture economics. Ann Rev Mar Sci 7, 471-496, doi:10.1146/annurev-marine-010814-015646 (2015).
Moran, J., Whitaker, D. & Kent, M. A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries. Aquaculture 172, 163-196 (1999).
Vendramin, N. et al. Viral Encephalopathy and Retinopathy in groupers (Epinephelus spp.) in southern Italy: a threat for wild endangered species? BMC Veterinary Research 9, 1-7 (2013).
Traxler, G., Roome, J., Lauda, K. & LaPatra, S. Appearance of infectious hematopoietic necrosis virus (IHNV) and neutralizing antibodies in sockeye salmon Onchorynchus nerka during their migration and maturation period. Diseases of Aquatic Organisms 28, 31-38 (1997).
Jiravanichpaisal, P., Soderhall, K. & Soderhall, I. Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish. Fish Shellfish Immunol 17, 265-275, doi:10.1016/j.fsi.2004.03.010 (2004).
Ndong, D., Chen, Y.-Y., Lin, Y.-H., Vaseeharan, B. & Chen, J.-C. The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures. Fish & Shellfish Immunology 22, 686-694, doi:https://doi.org/10.1016/j.fsi.2006.08.015 (2007).
Rowlands, D. J. et al. Broad range of 2050 warming from an observationally constrained large climate model ensemble. Nature Geoscience 5, 256-260, doi:10.1038/ngeo1430 (2012).
Vidal, O. M., Granja, C. B., Aranguren, F., Brock, J. A. & Salazar, M. A profound effect of hyperthermia on survival of Litopenaeus vannamei juveniles infected with white spot syndrome virus. Journal of the world aquaculture society 32, 364-372 (2001).
Guan, Y., Yu, Z. & Li, C. The effects of temperature on white spot syndrome infections in Marsupenaeus japonicus. Journal of invertebrate pathology (Print) 83, 257-260 (2003).
Amend, D. F. Control of infectious hematopoietic necrosis virus disease by elevating the water temperature. Journal of the Fisheries Board of Canada 27, 265-270 (1970).
Castric, J. & De Kinkelin, P. Experimental study of the susceptibility of two marine fish species, sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus), to viral haemorrhagic septicaemia. Aquaculture 41, 203-212 (1984).
OSEKO, N., YOSHIMIZU, M. & KIMURA, T. Effect of water temperature on artificial infection of Rhabdovirus olivaceus (hirame rhabdovirus: HRV) to hirame (Japanese flounder, Paralichtys olivaceus). Fish Pathology 23, 125-132 (1988).
Kobayashi, T., Shiino, T. & Miyazaki, T. The effect of water temperature on rhabdoviral dermatitis in the Japanese eel, Anguilla japonica Temminck and Schlegel. Aquaculture 170, 7-15 (1999).
Dorson, M. & Touchy, C. The influence of fish age and water temperature on mortalities of rainbow trout, Salmo gairdneri Richardson, caused by a European strain of infectious pancreatic necrosis virus. Journal of Fish Diseases 4, 213-221 (1981).
Smith, V. J., Brown, J. H. & Hauton, C. Immunostimulation in crustaceans: does it really protect against infection? Fish & shellfish immunology 15, 71-90, doi:10.1016/s1050-4648(02)00140-7 (2003).
Lillehaug, A., Lunestad, B. T. & Grave, K. Epidemiology of bacterial diseases in Norwegian aquaculture--a description based on antibiotic prescription data for the ten-year period 1991 to 2000. Dis Aquat Organ 53, 115-125, doi:10.3354/dao053115 (2003).
Sawatari, E. et al. Cell Growth-Promoting Activity of Fluid from Eye Sacs of the Bubble-Eye Goldfish (Carassius auraitus). Zoological science 26, 254-258 (2009).
Zou, J. & Secombes, C. J. The function of fish cytokines. Biology 5, 23 (2016).
Mosier, D. E. A requirement for two cell types for antibody formation in vitro. Science 158, 1573-1575 (1967).
Nathan, C. F., Karnovsky, M. L. & David, J. R. Alterations of macrophage functions by mediators from lymphocytes. The Journal of experimental medicine 133, 1356-1376 (1971).
Grayfer, L., Garcia, E. G. & Belosevic, M. Comparison of macrophage antimicrobial responses induced by type II interferons of the goldfish (Carassius auratus L.). Journal of Biological Chemistry 285, 23537-23547 (2010).
Dinarello, C. A. et al. Inhibitory effects of elevated temperature on human cytokine production and natural killer activity. Cancer research 46, 6236-6241 (1986).
Jiang, M., Chen, Z.-g., Zheng, J. & Peng, B. Metabolites-enabled survival of crucian carps infected by edwardsiella tarda in high water temperature. Frontiers in immunology 10, 1991 (2019).
Miyashita, A., Takahashi, S., Ishii, K., Sekimizu, K. & Kaito, C. Primed immune responses triggered by ingested bacteria lead to systemic infection tolerance in silkworms. PLoS One 10, e0130486 (2015).

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A Novel Application of Bubble-eye Strain of Carassius Auratus for Ex Vivo Fish Immunological Studies

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