Zebrafish as Versatile Model for Assessing Animal Venoms and Toxins: Current Applications and Future Prospects

Abstract

Animal venoms and toxins hold promise as sources of novel drug candidates, therapeutic agents, and biomolecules. To fully harness their potential, it is crucial to develop reliable testing methods that provide a comprehensive understanding of their effects and mechanisms of action. However, traditional rodent assays encounter difficulties in mimicking venom-induced effects in human due to the impractical venom dosage levels. The search for reliable testing methods has led to the emergence of zebrafish (Danio rerio) as a versatile model organism for evaluating animal venoms and toxins. Zebrafish possess genetic similarities to humans, rapid development, transparency, and amenability to high-throughput assays, making it ideal for assessing the effects of animal venoms and toxins. This review highlights unique attributes of zebrafish and explores their applications in studying venom- and toxin-induced effects from various species, including snakes, jellyfish, cuttlefish, anemones, spiders, and cone snails. Through zebrafish-based research, intricate physiological responses, developmental alterations, and potential therapeutic interventions induced by venoms are revealed. Novel techniques such as CRISPR/Cas9 gene editing, optogenetics, and high-throughput screening hold great promise for advancing venom research. As zebrafish-based insights converge with findings from other models, the comprehensive understanding of venom-induced effects continues to expand, guiding the development of targeted interventions and promoting both scientific knowledge and practical applications.

Introduction

Animal venoms and toxins have captivated the attention of researchers for centuries due to their remarkable diversity and potential applications.^1–3 These natural compounds, honed through millions of years of evolution, serve various purposes for their hosts, ranging from prey capture and defense to intraspecific competition.⁴ Beyond their ecological roles, animal venoms and toxins have garnered increasing interest for their potential in medical and biotechnological advancements.⁵ However, harnessing their potential necessitates a comprehensive understanding of their effects and mechanisms of action, which requires robust and ethically sound testing methods.

As the pursuit of novel drug candidates, therapeutic agents, and biomolecules derived from animal venoms and toxins gains momentum, the need for reliable and ethical testing methods becomes essential. Traditional assays often involve the use of rodents, raising ethical concerns and posing limitations in terms of scalability and cost-effectiveness.^6,7 Moreover, rodent models struggle to replicate venom-induced coagulopathy, acute kidney injury, and thrombocytopenia observed in human envenoming due to the need for unrealistically high venom doses.⁷

Furthermore, the interplay of different cellular and physiological systems in response to venoms requires versatile testing platforms that can provide a holistic view of their effects. In this context, zebrafish (Danio rerio) has emerged as a powerful model organism for studying animal venoms and toxins.⁸ Zebrafish attributes, including its high genetic similarity to humans and rapid embryonic development, make it an ideal candidate for assessing the effects of venoms across different biological levels.⁹ This review delves into the current applications and future prospects of utilizing zebrafish as a model for assessing animal venoms and toxins.

Zebrafish as a Model Organism

Zebrafish has become a prominent model organism due to its unique biological characteristics and genetic makeup.¹⁰ Originating from the freshwater rivers of South Asia,¹¹ zebrafish have gained popularity across various fields, including developmental biology, genetics, neuroscience, toxicology, and ecotoxicology.^12–18 This popularity is attributed to their distinctive advantages, such as rapid development, transparency, and genetic tractability.^11,19,20 These features also make zebrafish an ideal candidate for studying animal venoms and toxins, offering promising avenue for scientific investigation.

The rapid development of zebrafish embryos allows for real-time observation of developmental processes. Zebrafish transparency facilitates visualizing their internal structures and cellular responses.^21,22 These attributes enable researchers to monitor the effects of venoms and toxins on embryogenesis, organogenesis, and overall morphological changes efficiently.

Moreover, leveraging zebrafish well-characterized genome and genetic manipulation tools, scientists can introduce specific modifications to study venom interactions with precise cellular targets or pathways.²³ Furthermore, zebrafish share physiological and genetic similarities with higher vertebrates,^10,21,22 making them suitable for studying the toxic effects on cardiovascular, nervous, and immune systems.

Zebrafish also can be used to assess the impact of venoms and toxins on both early developmental stages and mature individuals. Therefore, it enables the investigation of acute and chronic toxicity, organ-specific damage, cellular responses, and potential therapeutic interventions. The ability to visualize and quantify toxicological endpoints within a living organism enhances the accuracy and relevance of toxicity assessments. Thus, it may contribute to a comprehensive understanding of the mechanisms underlying animal venom-induced pathologies.

Bioethical Guidelines for Zebrafish as an Animal Model

Zebrafish plays a crucial role in the ethical landscape of scientific research, adhering to a set of 10 ethical principles known as the “10 Rs”.^24,25 These principles encompass the classical 3 Rs for animal welfare—replacement, reduction, and refinement—augmented by 7 additional Rs pertaining to scientific principles: registration, reporting, robustness, reproducibility, relevance, responsibility, and respect.^24,25 Zebrafish, with its well-established position in scientific research, serves as an exemplary model system to replace traditional mammalian models, aligning with the ethical imperative of replacement.^26–29

In addition, zebrafish models contribute to reduction by minimizing the number of animals required, as the models offer greater informational and predictive potential with reduced resource demands compared with conventional models.²⁴ The refinement principle ensures optimal welfare for zebrafish through considerations such as suitable housing, water parameters, and anesthesia procedures during experimental manipulations.^30–33 In addition to classical 3Rs in bioethics, researchers implement additional scientific initiatives, such as registration, reporting, robustness, reproducibility, and relevance, to guide the ethical conduct of research with zebrafish.²⁴

The registration and reporting of studies involving zebrafish contribute to transparency and accountability, whereas the principles of robustness and reproducibility ensure the reliability of results, addressing the broader issue of the reproducibility crisis in scientific research. The relevance principle emphasizes the importance of choosing zebrafish based on its unique features and characteristics that align with the specific goals of the research, thereby avoiding unnecessary use of animals.

Finally, the principles of responsibility and respect underscore the ethical aspects throughout the care and utilization of zebrafish in research, highlighting the obligation to treat animals as intrinsically valuable.²⁴ Adhering to these 10 Rs enables researchers to advance the ethical and responsible utilization of zebrafish, fostering animal welfare, scientific integrity, and societal responsibility within the domain of scientific inquiry.

Literature Search, Database Selection, and Criteria Establishment

In this review, a systematic literature search was conducted on February 11, 2024, utilizing the Scopus database with the parameters set to include all publications up to December 2023. Scopus was selected as the exclusive database due to its broad coverage of scientific literature, enabling a thorough investigation into zebrafish-based studies on animal venoms and toxins. Importantly, unlike other databases, Scopus utilizes strict criteria and rigorous selection process to exclude predatory journals and gray literature, ensuring the inclusion of reliable sources.

The advantages of Scopus, including its expansive coverage, citation tracking, and indexing of reputable journals, were considered instrumental in ensuring a thorough and up-to-date review.^34–36 The search employed a predefined set of keywords to refine the scope of the inquiry, specifically targeting studies related to zebrafish and animal venoms or toxins. The keywords utilized for the search were (“zebrafish” OR “zebra fish”) AND (“venom” OR “toxin”).

The inclusion criteria for the identified literature encompassed studies that employed zebrafish (D. rerio) as a model organism in the study of animal venoms and toxins. Included studies needed to provide insights into the physiological responses, developmental alterations, or potential therapeutic interventions induced by venoms originating from diverse species, such as snakes, jellyfish, cuttlefish, anemones, spiders, and cone snails. Furthermore, studies utilizing zebrafish embryos, larvae, and adults were considered for inclusion to allow a comprehensive assessment of venom-induced effects at various developmental stages.

Conversely, exclusion criteria were established to refine the focus of the review. Studies that did not employ zebrafish as a model organism, those not published in English, and those lacking relevance to the overarching theme of animal venoms and toxins were excluded from consideration. In addition, exclusion criteria were applied to studies that did not provide sufficient detail on the methodology, results, or interpretation, as transparency and robustness were deemed essential for the inclusion of studies in this comprehensive review.

Applications of Zebrafish in Animal Venom and Toxin Research

To assess the annual productivity within the field, a bibliometric analysis was conducted focusing on articles related to the utilization of zebrafish as an animal model in venom and toxin research. A comprehensive search spanning from 1955 to 2023 was performed using the Scopus database, revealing a total of 703 studies published across various countries worldwide. Within this data set, 692 articles (98.4%), 5 book series (0.7%), 4 proceedings (0.5%), and several other document types were identified, reflecting the multidisciplinary nature of the research encompassed in this analysis. Among the identified documents, a significant majority, comprising 694 publications (98.7%), were composed in the English language.

This prevalence of English language publications could be attributed to the global dominance of English as the primary language for scientific communication, facilitating broader dissemination and accessibility of research findings. Figure 1 provides a visual representation of the progressive trend in publications within this field from 1955 to 2023. Notably, there is a consistent upward trajectory in the number of publications over the observed timeframe. Specifically, the year 2020 marked the pinnacle with the highest recorded number of publications (n = 72; 10.28%).

FIG. 1.

Absolute (black bars) and cumulative (gray bars) number of annual publications on zebrafish studies related to animal venom or toxin, spanning the years from 1955 to 2023 on a global scale.

Upon closer scrutiny, an analysis of the top 10 countries exhibiting the highest volume of research documents was conducted (Table 1). The United States secures the foremost position with a cumulative count of 240 publications, constituting 34.1% of the total. Subsequently, China (n = 164; 23.3%) and Germany (n = 65; 9.2%) follow in succession. This ranking is attributed to the prolific output of research emanating from the United States, showcasing its substantial contribution to the scholarly landscape.

Table 1.

Top 10 Countries with the Highest Publication Output in Studies Utilizing Zebrafish as Model Organism for Animal Venom and Toxin Research

Rank	Country	No. of articles (%)
1	United States	240 (34.1)
2	China	164 (23.3)
3	Germany	65 (9.2)
4	Brazil	47 (6.6)
5	United Kingdom	45 (6.4)
6	Canada	28 (3.9)
7	India	26 (3.6)
8	Japan	24 (3.4)
8	Australia	24 (3.4)
10	Italy	22 (3.1)

From the pool of articles acquired through Scopus, not every article was incorporated into this comprehensive review. The meticulous selection process adhered strictly to the predefined inclusion and exclusion criteria elucidated in the methodology section. This selective approach was undertaken to ensure a focused analysis that adheres to the established criteria, preventing an exhaustive review of the entire set of 703 studies obtained from Scopus. In addition, this targeted selection process was employed to ensure a focused examination of articles that best contribute to the objectives and robustness of this review.

Numerous studies have reported the use of zebrafish in assessing the impact of animal venoms and toxins. These studies covered a wide array of venomous sources, including snakes,^37–40 jellyfish,^41–43 cuttlefish,⁴⁴ anemone,^45,46 spider,^47–49 and cone snail^50–55 (Table 2). Researchers have exposed zebrafish embryos, larvae, and adults to various types of venoms or purified toxins, monitoring physiological changes, and recording the observable outcomes.

Table 2.

Applications of Zebrafish as Model Organism in Animal Venom and Toxin Studies

No.	Source of venom or toxin	Species	Venom concentrations	Stage of zebrafish	Exposure time	Effects	References
1	Snake	Bothrops alternatus	20 μL of 0.13 mg/mL	Adult	3 h	Necrosis, inflammation, and weight gain	³⁷
		Lachesis muta muta	60 or 80 μg/80 μL	Larvae	15, 30, 40, and 60 min	Heart rate alterations, circulation modifications, thrombus, and pericardial damages	³⁹
		Montivipera bornmuelleri	1, 1/10, 1/100, and 1/1000 mg/mL	Larvae and embryo	24, 48, and 60 h	Heartbeat rate reduction and hemorrhage	⁴⁰
2	Jellyfish	Chrysaora quinquecirrha	2, 20, 200, 400, and 500 μg/g	Juvenile	24 h	Eye hemorrhage and gill hyperplasia	⁴¹
2	Jellyfish	Nemopilema nomurai	10, 30, 100, 300, and 500 μg/g	Adult	24 h	Hemorrhage and inflammation in cardiopulmonary regions	^42,43
3	Cuttlefish	Sepia prashadi	0.1, 1, 5, 10, and 50 μM	Egg and embryo	24, 48, 72, and 96 h	Premature hatching, aberrant somite migration, heart arrhythmia, and otolith deformation	⁴⁴
4	Anemone	Zoanthus natalensis	250 μM	Embryo and larvae	2 and 4 days	Neuronal loss and altered locomotor behavior	⁴⁵
4	Anemone	Nematostella vectensis	0.5 mg/mL	Larvae	1 h	Bent and contracted tails	⁴⁶
5	Spider	Loxosceles intermedia	20 μL of 250, 500, 1000, and 2000 ng	Adult	7 days	No pathological effects	⁴⁹
		Dolomedes sulfurous	From 11.4 to 71.4 μg/g body weight	Adult	5 min, 20 min, and 24 h	Severe neurotoxic effects and death	^47,48
		Dolomedes mizhoanus	From 11.4 to 71.4 μg/g body weight	Adult	5 min, 20 min, and 24 h	Severe neurotoxic effects and death	^47,48
6	Cone snail	Conus catus	2.75–55 ng/mL	Adult	0–500 min	Decreased swimming movement and rigid paralysis	⁵⁰
		Conus virgo	50, 75, 100, and 125 μg/kg body weight	Adult	24 h	Paralysis and erratic swimming	⁵³
		Conus tulipa	0.001–100 ng/μL	Larvae and adult	5, 10, and 15 min	No alteration in escape response	⁵²
		Conus episcopatus	10 μM	Larvae and adult	1 and 3 h	Early hypoactivity and increased movement	⁵⁵
		Conus inscriptus	100, 200, 400, 600, 800, and 1000 μg/mL	Embryo and larvae	72 h	Deformities and death in larvae	⁵⁴
		Conus geographus	65 ng/g	Adult	110 min	Reduced blood glucose	⁵¹

Snake venom

The assessment of snake venom impact on various life stages of organisms provides valuable insights into its physiological effects. In this context, zebrafish have emerged as important models for understanding venom-induced changes. Adult, larval, and embryonic zebrafish have been utilized to examine the consequences of snake venom exposure. This approach sheds light on both developmental and physiological alterations caused by venom components.

In a study by Carvalho et al.,³⁷ adult zebrafish injected with Bothrops alternatus venom showed necrosis and edema in the chest area, as well as increased body weight. The venom-injected fish exhibited various histological abnormalities in the gills, including hypertrophy, hyperplasia, epithelial rupture, and leukocyte infiltration. In addition, the livers displayed derangement of hepatic cords, vacuolization, and degeneration upon exposure to venom.

Similarly, the kidneys showed hyaline degeneration, hypertrophy, glomerular degeneration, and hyperemia. Moreover, the intestines exhibited detachment, hypertrophy, sloughing, degeneration, and leukocyte infiltration. These venom-associated tissue alterations observed by Carvalho et al.³⁷ in the zebrafish resemble those previously seen in humans and other animals exposed to specific snake venoms, further highlighting the relevance of this model for understanding the impact of venom-induced physiological changes.

Zanotty et al.³⁹ presented the envenomation effects of Lachesis muta muta snake venom on zebrafish larvae. The study found that Mutacytin-1 (MC-1), a C-Type lectin-like protein from the venom, caused a significant decrease in the cardiac frequency in the zebrafish larvae. In addition, MC-1 fraction induced thrombus formation and apoptosis in the cardiac myocytes of the larvae. The cardiac tissue exhibited significant alterations, including cell edema and damage, as observed through histology and fluorescence microscopy.³⁹ These findings underscore the intricate physiological impact of snake venom components on delicate organisms such as zebrafish larvae.

A study by Sahyoun et al.⁴⁰ assessed the effects of Montivipera bornmuelleri, a venomous snake of the Viperidae family from the Middle East, on zebrafish embryos. Analyses of its venom revealed >60 protein compounds, including serine proteases, phospholipase A2, metalloprotease III, and L-amino acid oxidase.⁵⁶ Sahyoun et al.⁴⁰ also reported that exposure to M. bornmuelleri venom led to dose-dependent mortality and developmental defects. Cardiotoxicity of M. bornmuelleri venom manifested as a transient decrease in heartbeat rate and pericardial hemorrhage. Overall, these findings demonstrate the developmental and cardiotoxic impacts of M. bornmuelleri venom on zebrafish embryos, implicating potential cardiovascular targeting by venom components.

Although understanding the intricate physiological impacts of snake venom on zebrafish models sheds light on its effects, novel approaches are being explored to address the challenges posed by snakebite envenomation. The validated treatment for severe snakebite envenomation involves passive immunization with animal-derived antisera administered intravenously, but this treatment is often unavailable due to cost, species identification challenges, and limited availability of the antisera.^57,58 To address this issue, Anderson et al.³⁸ investigated alternative options and discovered that human tryptase β can effectively degrade and detoxify venoms from various snake species.

In this study, zebrafish embryos were used as a biological toxicity assay to demonstrate that recombinant human tryptase β can significantly reduce venom toxicity and render most snake venoms nonlethal. This approach could serve as a first aid treatment for snakebites, minimizing tissue damage and venom spread before further medical intervention.

Jellyfish venom

Several studies have shed light on the intricate and often detrimental interactions between jellyfish venom and zebrafish, providing insights into the physiological responses and potential impacts on these aquatic organisms. Becerra-Amezcua et al.⁴¹ reported the impact of crude venom from the jellyfish Chrysaora quinquecirrha on zebrafish. The most significant effect observed in zebrafish exposed to venom was hemorrhaging in the eyes after intraperitoneal and subcutaneous injections.

Subcutaneous injection also led to convulsions, paralysis in the fins, and reduced swimming capacity, resulting in fish mortality. The psychological impact of venom was mainly evident in disturbances of circulation, hyperplasia, hypertrophy of gill epithelium, and erythrocyte morphological changes. Furthermore, comparing hemolytic activity on fish and human erythrocytes, fish erythrocytes proved more sensitive to the venom than human erythrocytes, showing hemolytic activity at a lower venom concentration.⁴¹

Meanwhile, Mohan Prakash et al.⁴² and Prakash et al.⁴³ delved into the toxic effects of Nemopilema nomurai jellyfish venom on zebrafish. High doses of venom resulted in reduced survival rates and increased body weights, accompanied by evident physiological changes. Notably, in the neural system, an increase in brain weight was observed, coupled with alterations in several enzyme activities, giving rise to discernible behavioral abnormalities and instances of brain hemorrhage. Likewise, the respiratory system exhibited disarrayed gills and heightened enzyme activities. The cardiac system also displayed augmented heart weights, elevated enzyme activities, and signs of inflammation. In addition, the venom inflicted damage upon intestinal, renal, and hepatic tissues, provoking cellular degeneration and necrosis.^42,43

These investigations into the effects of jellyfish venom on zebrafish underline the vulnerability of zebrafish to venom-induced changes across various organ systems. Importantly, the parallels drawn between zebrafish responses and potential human reactions to venom exposure offer a platform for anticipating and addressing human health risks associated with jellyfish venom. By highlighting the shared physiological reactions, these findings help to lay the groundwork for the development of timely interventions and therapeutic strategies that can effectively counteract and alleviate the potential adverse effects of jellyfish stings on human well-being.

Cuttlefish toxin

Despite the limited number of studies available, research investigating the effects of cuttlefish toxins on zebrafish has proven both valuable and representative of human physiological responses. A study by Karthik et al.⁴⁴ examined the impact of Sepia prashadi cuttlefish toxin on zebrafish embryos, revealing noteworthy insights into its influence on development and health. A low molecular weight toxin extracted and purified from the posterior salivary gland of S. prashadi prompted premature hatching of the eggs and led to lethal effects at higher concentrations.

Various developmental abnormalities, such as tail deformation, spine bend, yolk sac diffuse, and malformations in eye, head, and somite differentiation, were observed in the embryos exposed to the toxin. In addition, the toxin caused deformities in otoliths, inhibited morphogenetic movements, and induced heart arrhythmia at later developmental stages (72 and 96 hpf). Pectoral and caudal fin development were also arrested at 96 hpf, affecting locomotive ability.⁴⁴ With the scarcity of studies focused on cuttlefish toxin, a compelling opportunity arises to delve deeper into the mechanisms underlying its impact on humans by employing zebrafish as model organisms.

Anemone toxin

Toxins derived from marine organisms have been acknowledged as a promising and emerging source of peptide-based therapeutic agents.^59,60 Among these, peptide toxins from sea anemones have garnered attention as potential therapeutic leads and pharmacological tools. A study by Liao et al.⁴⁵ employed transcriptomic and proteomic analyses to identify six distinct groups of expressed peptide toxins in the Zoanthus natalensis anemone.

In vivo experiments confirmed the neuroprotective effects of ZoaKuz1, a peptide derived from Z. natalensis toxin, wherein it significantly mitigated the reduction of dopaminergic neurons in the zebrafish model of Parkinson's disease. Furthermore, ZoaKuz1 effectively counteracted the alterations in swimming behavior caused by 6-hydroxydopamine (6-OHDA) in zebrafish larvae. In summary, Liao et al.⁴⁵ demonstrate the potential of ZoaKuz1 as a neuroprotective agent against 6-OHDA-induced dopaminergic neuron loss in zebrafish larvae.

Meanwhile, a separate investigation by Sachkova et al.⁴⁶ centered on toxins from Nematostella vectensis, using zebrafish larvae and Cherry shrimps (Neocaridina davidi) as test subjects. The outcomes revealed that N. vectensis toxins proved lethal to zebrafish larvae whereas leaving shrimps unaffected, suggesting that they might have evolved in response to selective pressure from natural fish predators.

These studies underscore the inherent potential of investigating anemone toxins through the utilization of zebrafish as a model organism. The distinctive characteristics of these toxins, as illustrated by both the neuroprotective attributes of Z. natalensis toxin-derived peptides and the predator-specific impacts of N. vectensis toxins, emphasize the multifaceted avenues of research that await exploration.

Spider venom

Despite its potential significance, the study of spider venom remains limited. Loxoscelism, triggered by the bite of spiders belonging to the Loxosceles genus, is a notable public health concern, particularly in the Americas. The venom of Loxosceles intermedia (brown spider) poses a threat, with phospholipase D enzyme toxins being the primary cause of loxoscelism.⁶¹ To better understand the pathophysiology, a zebrafish model was utilized to study the hemolytic properties and organ damage induced by L. intermedia venom.⁴⁹

Hemolytic assays employing zebrafish and human erythrocytes revealed that the venom caused dose-dependent lysis of human cells but not zebrafish cells. Despite its vascular effects, the venom did not impact zebrafish survival or fin regeneration. Histological analysis also indicated no organ abnormalities post-venom injection. Behavioral assessments showed minimal differences, suggesting limited stress-induced responses. Overall, this investigation sheds light on L. intermedia venom effects and zebrafish as a model for studying loxoscelism and regeneration processes.⁴⁹

Meanwhile, a series of studies reported that the venom of Dolomedes sulfurous and Dolomedes mizhoanus spiders showed neurotoxicity affecting voltage-activated sodium, potassium, and calcium channels.^47,48 Upon intrathoracic injection, these venoms caused severe neurological disturbances in zebrafish, leading to altered swimming behavior, disorientation, and rapid spinning in small circles. Most fish exhibiting spinning symptoms quickly sank to the bottom and died within 20 min, although some recovered and swam normally after 24 h.^47,48 Although studies on spider venom effects in zebrafish are currently limited, the diverse range of outcomes showcased by Dolomedes spider venoms, and the insight gained from L. intermedia venom effects on zebrafish emphasize the potential of this field for future exploration.

Cone snail toxin

The use of zebrafish as a model organism also offers a unique perspective in understanding the effects of cone snail toxins. With an abundance of research focused on the impact of these toxins on zebrafish, it becomes imperative to explore the significance of such studies and the insights they provide into the broader field of toxinology.

The behavioral effects of cone snail Conus catus conotoxins were assessed in adult zebrafish through in vivo assays.⁵⁰ Venom fractions of C. catus were administered through intramuscular injections to adult zebrafish, causing an initial decrease in movement and rapid breathing, followed by a burst of hyperactive swimming, particularly at higher doses. This hyperactivity was succeeded by spastic paralysis characterized by stiff and fibrillating fins. The study revealed that neurotoxic/paralytic conotoxins were the predominant components of C. catus venom.⁵⁰

Similarly, a study by Ganesan et al.⁵³ reported the impact of crude venom from Conus virgo on adult zebrafish, focusing on their behavioral responses, locomotory activities, and paralytic effects. Control zebrafish displayed normal swimming behavior with rapid tail movements. However, when exposed to C. virgo venom, the fish exhibited delayed tail movements and a significant decrease in travel distance, dependent on the venom dosage.

Paralysis was observed in toxin-administered fish, manifesting as lethargic movements, floating on the water surface, settling at the bottom of the tank, and erratic swimming behavior. These effects were associated with spasmodic and less frequent opercular respiratory movements. The percentage of affected fish and the duration of recovery increased significantly with higher toxin doses. Furthermore, histological analysis revealed minimal nerve fiber distortion and slightly elongated neurons in the brain sections of toxin-exposed fish compared with the normal architecture observed in control fish.⁵³

Concurrently, in a study by Dutt et al.,⁵² the effects of Conus tulipa toxin on adult and larval zebrafish were reported. Toxin from different duct sections was administered to adult zebrafish through intramuscular injection, leading to a reduction in swimming ability. Toxin from the proximal section of the duct showed the most potent effect, causing an 80% reduction in swim distance. However, when the dissected toxin was added to the water column for larval zebrafish, there was no observable change in fish behavior or escape response.

Furthermore, the study explored the impact of specific peptides (conantokin, conopressin, and ρ-TIA) on larval zebrafish escape responses. Among them, ρ-TIA exhibited a dose-dependent effect on reducing the escape response in larval zebrafish. In conclusion, Dutt et al.⁵² highlight the effects of C. tulipa toxin and specific peptides on zebrafish behavior, shedding light on potential mechanisms of prey capture by C. tulipa.

Meanwhile, in a recent report by Bosse et al.,⁵⁵ a zebrafish larvae behavioral assay was established to screen for bioactive compounds in conotoxins. In this assay, light conditions were used to trigger behavioral shifts in larvae with locomotion changes serving as readouts. By testing a library of 89 synthetic conotoxins, Bosse et al.⁵⁵ found that 36 conotoxins elicited significant behavior alterations, with CNF-Ep1, a conorfamide from Conus episcopatus, showing pronounced effects.

Interestingly, CNF-Ep1 demonstrated persistent effects even after treatment cessation. Adult zebrafish also exhibited reduced locomotion upon CNF-Ep1 injection. Therefore, this study highlights conotoxins diverse impact on zebrafish behavior, providing insights into potential therapeutic applications.⁵⁵

In a similar manner, the investigation conducted by Jain et al.⁵⁴ focuses on Conus inscriptus, a vermivorous cone snail abundant in Indian coastal waters. Zebrafish embryos were subjected to the toxin, with initial hours revealing no effects due to its impermeability. However, after hatching, deformities emerged at higher venom concentrations, resulting in complete mortality. Furthermore, to test anticonvulsant properties, exposure to conotoxins resulted in decreased mobility in chemically induced epileptic zebrafish larvae. Thus, this research contributes significantly to the comprehension of C. inscriptus venom and its potential utility as an anticonvulsant.

Lastly, to investigate the ability of the conotoxin in lowering blood glucose, Conus geographus cone snail insulin-like toxins were tested in a zebrafish model of diabetes induced by streptozotocin (STZ).⁵¹ After STZ treatment, the zebrafish exhibited significantly elevated blood glucose levels, which were then effectively lowered upon administration of insulin-like conotoxin. These results indicate that C. geographus toxin can bind to and activate the fish insulin receptor, showcasing their role in inducing insulin shock in fish prey.

Taken together, the experiments utilizing zebrafish have yielded invaluable insights into the mechanisms of cone snail toxins on living organisms. Through careful observation of changes in behavior, morphology, and physiological parameters, researchers have unraveled potential pathways that underlie venom-induced tissue damage, neurological dysfunction, and other adverse effects.

Potential Approaches for Zebrafish-Based Venom and Toxin Assay

The intersection of advanced biotechnologies and zebrafish research has opened new alternatives for investigating the field of animal venoms and toxins. Two prominent methodologies, CRISPR/Cas9 gene editing and optogenetics, along with high-throughput screening methods and the construction of a library for transgenic lines, have emerged as powerful tools in this exploration. These techniques may provide not only insights into the mechanisms underlying the effects of venoms but also novel approaches for studying their impact on organisms.

CRISPR/Cas9 gene editing

CRISPR/Cas9 technology has revolutionized genetic manipulation and has been seamlessly integrated into zebrafish research.²³ This breakthrough technique allows researchers to precisely modify zebrafish genomes, creating tailored models that can mimic specific conditions for studying diseases and developmental biology.^62,63 The advent of CRISPR/Cas9 genome editing technology in zebrafish marked a transformative milestone in 2013, utilizing an expression vector for Cas9 mRNA and a customizable single guide RNA to induce somatic indel mutations effectively.²³

The CRISPR/Cas9 technology in zebrafish further evolve by demonstrating exceptional efficiency in large fragment deletions, simultaneous multi-site edits, and tissue-specific knockouts.^64–66 Notably, Wu et al.⁶⁷ introduced a system ensuring consistent null phenotypes in G0 zebrafish through yolk injection of CRISPR/Cas9 ribonucleoprotein complexes. Meanwhile, expanding the CRISPR toolkit, ErCas12a, a member of the Cas12a family, also exhibited successful indel induction in zebrafish embryos.⁶⁸

In addition, Sun et al.⁶⁹ systematically knocked out all genes on chromosome 1 of zebrafish, resulting in 1029 mutated genes and providing insights into gene functions related to human diseases. These advancements underscore CRISPR/Cas9 as a pivotal tool for precise genetic manipulation in zebrafish, promising broad applications in venom and toxin studies. By knocking out or modifying specific genes, scientists can investigate the molecular mechanisms underlying the effects of venoms and toxins.

For instance, delving into the physiological responses elicited by venom, known to be controlled by specific ion channels,^70–72 can be achieved by utilizing gene editing methods that precisely target and examine these ion channels. This technology not only may accelerate the discovery of venom targets but also provide insights into potential therapeutic interventions.

Optogenetics

Optogenetics, a sophisticated approach that employs light to control cellular activities, has found its way into the field of zebrafish-based research.^73,74 By utilizing optogenetic tools, researchers can manipulate neural circuits and cellular responses in real time, which may help to shed light on the intricate interplay between venom components and the nervous system.

This technique might also enable the dissection of complex venom-induced behaviors and physiological changes, offering a deeper understanding of venom toxicity and its impact on the host organism. Importantly, optogenetics, a technique that involves modulating targeted neural pathways,^74,75 can also offer a distinctive platform for evaluating the efficacy of antivenom or therapeutic interventions directed toward these pathways.

High-throughput screening methods

High-throughput screening methods have considerably expedited research using zebrafish models.^76–79 Automated imaging systems^80,81 and microfluidic platforms^82,83 may allow for rapid assessment of venom-induced phenotypic changes in zebrafish embryos or larvae. These platforms can be used to simultaneously screen multiple venom samples or compounds, facilitating the identification of novel venom components, toxicity profiles, and potential therapeutic agents. High-throughput approaches also enable researchers to efficiently explore large venom libraries and unravel the complex interactions between venoms and host systems, paving the way for the discovery of new bioactive molecules and the development of innovative treatments.

Transgenic lines of zebrafish for hepatotoxicity assay

Liver transgenic lines are indispensable for toxicity testing in zebrafish.⁸⁴ The liver, a key organ in vertebrates, plays a vital role in metabolism and detoxification.^13,29 Transgenic lines expressing specific markers in the liver allow real-time in vivo monitoring of hepatotoxicity. This approach provides dynamic insights into the effects of toxic compounds, surpassing traditional in vitro methods. Therefore, liver transgenic lines enhance the precision, efficiency, and comprehensiveness of toxicity assessments in zebrafish, crucial for advancing preclinical safety evaluations.

An investigative approach has been established to label keratin,⁸⁵ notch signaling component,⁸⁶ and hepatic stellate cells,⁸⁷ facilitating comprehensive examinations of hepatocellular development in zebrafish. Moreover, to expedite the evaluation of drug-induced hepatotoxicity in zebrafish, a frequently employed approach involves the utilization of transgenic lines expressing liver fatty acid-binding protein or Wnt signaling fused with fluorescent proteins.^84,88–91

In addition, through a combination of transcriptomic and transgenic approaches, Poon et al.⁹² identified markedly upregulated genes as biomarkers indicative of drug-induced liver injury (DILI) in zebrafish. Poon et al.⁹² also successfully constructed three distinct transgenic lines expressing fluorescent protein reporter that manifested a dose- and time-dependent induction in relation to DILI drugs.

In summary, liver transgenic lines in zebrafish significantly improve precision in toxicity assessments and provide a comprehensive understanding of hepatocellular development. These lines, expressing key markers and signaling components, expedite drug-induced hepatotoxicity evaluation and contribute to the identification of biomarkers for liver injury, enhancing the reliability of preclinical safety evaluations.

Future Perspectives

In the field of zebrafish-based venom and toxin research, several promising directions are expected to expand our understanding of venomous effects and their implications. Future investigations could delve deeper into the molecular mechanisms underlying venom-induced injuries in zebrafish by scrutinizing the alteration of targeted gene expression and elucidating the affected signaling pathways. Pioneering techniques, such as single-cell transcriptome analysis^93,94 and CRISPR/Cas9-mediated genetic manipulations,^23,63 could offer unprecedented insights into these mechanisms, unraveling the intricate tapestry of venom-induced responses.

The translational potential of zebrafish research could also be harnessed to develop targeted therapies for venom-induced injuries. Employing zebrafish models to rigorously test the efficacy of potential antivenom, neutralizing agents, or treatments against animal venoms and toxins could serve as a pivotal bridge between basic research and medical applications, potentially saving lives and mitigating the consequences of envenomation. Lastly, in the pursuit of a comprehensive understanding of venomous effects, integrating zebrafish studies with findings from other animal models also emerges as a promising approach.

By juxtaposing zebrafish data with observations from mice, rats, and cell cultures, researchers can validate the consistency of venom-induced effects across distinct organisms. This cross-model validation not only enhances the robustness of findings but also widens the perspective on the potential relevance of zebrafish-based results. Moreover, comparative studies across different species might unveil species-specific responses to venoms and toxins, aiding in predicting potential human reactions and informing clinical interventions.

Conclusion

The study of animal venoms and toxins, driven by their diverse applications and potential benefits, has captivated the scientific community for centuries. As interest in their medical and biotechnological potentials gains momentum, the need for precise, holistic, and ethical testing methods assumes paramount importance. The urgency lies in addressing crucial research gaps related to venom-induced effects on physiology and molecular mechanisms. Zebrafish, as a potent model organism, is pivotal in this endeavor due to its genetic similarity to humans, rapid development, and transparency.

Rapid and time-sensitive studies on zebrafish can bridge the gaps of other animal models in advancing toxicology and accelerating the development of medical and biotechnological applications of animal venom and toxin. Through the investigation of a spectrum of animal venom sources, including snakes, jellyfish, cuttlefish, anemones, spiders, and cone snails, zebrafish-centered studies have unveiled intricate physiological responses and toxic effects. To propel this domain forward, cutting-edge techniques, such as CRISPR/Cas9 gene editing, optogenetics, library of transgenic lines, and high-throughput screening methods, offer promising avenues for further exploration.

Future studies could focus on elucidating the precise molecular mechanisms underlying venom-induced injuries in zebrafish. In addition, the translational potential of zebrafish research could be harnessed to rigorously test the efficacy of novel treatments against animal venoms and toxins. Lastly, comparative studies across different species, including zebrafish, mice, rats, and cell cultures, may unveil species-specific responses to venoms and toxins, informing clinical interventions and enhancing our understanding of venomous effects.

Footnotes

Acknowledgments

The authors acknowledge the valuable insights and discussion from colleagues in the Faculty of Biology, Universitas Gadjah Mada that have enriched this review article.

Authors' Contributions

Conceptualization and formal analysis by F.S., Y.A.P., and T.R.N. Writing—original draft by F.S., N.I.S., D.S.Y., E.A.W., D.S.P., and W.A.P. Formal analysis and data curation by F.S., D.S.P., and W.A.P. Writing—review and editing by F.S., N.I.S., D.S.Y., E.A.W., A.P., A.R.P.R., Y.A.P., and T.R.N. Investigation by F.S., N.I.S., and D.S.Y. Funding acquisition by F.S., N.I.S., D.S.Y., and E.A.G. Supervision by Y.A.P. and T.R.N.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was funded by grants from Doctoral Competency Improvement Program Universitas Gadjah Mada Number 7743/UN1.P.II/Dit-Lit/PT.01.03/2023.

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