1 Introduction

Gene transfer techniques have revolutionized the field of aquaculture, providing powerful tools for genetic modification and enhancement of various aquatic species. These techniques, including CRISPR/Cas9, TALENs, and Zink finger nucleases, have been widely applied to fish species to improve traits such as disease resistance, growth, and reproduction (Gutási et al., 2023). The application of these methods in shrimp, however, is still in its nascent stages, with significant potential for development. The ability to modify the genome of shrimp can lead to substantial improvements in aquaculture productivity and sustainability (Zenger et al., 2019; Jacinda and Yustiati, 2021).

Shrimp aquaculture is a critical component of the global seafood industry, contributing significantly to food security and economic development. However, the industry faces challenges such as disease outbreaks and environmental sustainability. Gene transfer techniques offer a promising solution to these issues by enabling the development of disease-resistant shrimp strains and enhancing growth rates (Sun et al., 2005; Jacinda and Yustiati, 2021). For instance, the use of transfection methods has shown high efficiency in gene transfer, with significant improvements in hatching rates and gene expression in shrimp (Sun et al., 2005). Additionally, the identification of horizontally transferred genes in shrimp genomes highlights the potential for natural genetic enhancements that can be harnessed through gene transfer technologies (Yuan et al., 2013).

This study aims to provide a comprehensive overview of the current state of gene transfer techniques in shrimp, their applications, and potential benefits. The objectives are to review the various gene transfer methods used in shrimp, including microinjection, electroporation, and transfection, discuss the efficiency and outcomes of these methods in terms of gene expression and phenotypic changes, explore the potential applications of gene transfer in improving shrimp aquaculture, focusing on disease resistance, growth enhancement, and environmental sustainability, and address the ethical and environmental considerations associated with the use of genetically modified organisms (GMOs) in aquaculture.

2 Background on Shrimp Genetics

2.1 Genetic structure and diversity in shrimp

Shrimp species exhibit significant genetic diversity, which is crucial for their adaptability and survival in various environments. For instance, the Pacific white shrimp (Litopenaeus vannamei) has been extensively studied for its genetic diversity using techniques such as microsatellite markers. These markers have revealed high levels of heterozygosity and numerous alleles specific to different populations, indicating a rich genetic variation within the species (Wolfus et al., 1997). Additionally, the development of high-throughput SNP genotyping methods has further enhanced our understanding of genetic relationships and diversity in shrimp populations, facilitating more efficient breeding programs (Yu et al., 2020).

2.2 Challenges in shrimp genetic manipulation

Despite the advances in genetic studies, manipulating shrimp genetics presents several challenges. One major issue is the efficient delivery and stable integration of foreign genes into shrimp genomes. Traditional methods like microinjection and electroporation have shown limited success, with low hatching rates and gene expression levels (Sun et al., 2005). Moreover, the presence of mitochondrial pseudogenes, which can be mistaken for functional genes, complicates genetic analyses and manipulations (Williams and Knowlton, 2001). The development of more effective gene transfer techniques, such as the use of nuclear localization signals (NLS) to enhance nuclear import of vector DNA, has shown promise but still requires further optimization (Arenal et al., 2004).

2.3 Historical overview of gene transfer in shrimp

The history of gene transfer in shrimp has seen gradual improvements in techniques and outcomes. Early methods focused on microinjection and electroporation, which had limited success due to low efficiency and high mortality rates. The introduction of transfection reagents significantly improved gene transfer efficiency, with studies reporting up to 60% hatching rates and 72% gene transfer efficiency when using DNA/jetPEI complexes (Sun et al., 2005). More recently, the development of a VP28-pseudotyped baculovirus expression system has achieved near 100% infection efficiency in specific tissues, marking a significant advancement in the field (Wu et al., 2021). These historical developments highlight the ongoing efforts to refine gene transfer techniques to enhance their applicability and success in shrimp genetic manipulation.

3 Gene Transfer Techniques in Shrimp

3.1 Overview of common gene transfer methods

Gene transfer techniques are essential tools in shrimp genetic research, enabling the study and manipulation of genes to understand their functions and improve shrimp breeding programs. The most common methods include microinjection, electroporation, lipofection, and viral vectors.

3.1.1 Microinjection

Microinjection is a direct and reliable method for gene transfer, widely used in various aquatic species. This technique involves injecting nucleic acids, gene constructs, or other substances directly into shrimp embryos or cells using fine needles. It has been extensively used in fish and other aquatic organisms due to its precision and effectiveness (Abdelrahman et al., 2021; Lane et al., 2021; Gultom, 2023). Despite requiring sophisticated equipment and skilled personnel, microinjection remains a preferred method due to its high success rate and the ability to deliver precise amounts of genetic material (Takahashi et al., 2015; Gu et al., 2018).

3.1.2 Electroporation

Electroporation involves applying an electrical field to cells to increase the permeability of the cell membrane, allowing nucleic acids to enter the cells. This method is less invasive than microinjection and can be used to transfect a large number of cells simultaneously. It has been successfully applied in various species, including mice, where it has shown effectiveness in delivering CRISPR/Cas9 components (Takahashi et al., 2015). Electroporation is advantageous for its simplicity and ability to handle large-scale gene transfer experiments.

3.1.3 Lipofection

Lipofection uses lipid-based reagents to form complexes with nucleic acids, facilitating their entry into cells. This method is less commonly used in shrimp but has potential due to its non-invasive nature and ease of use. Lipofection is particularly useful for in vitro applications and can be optimized for different cell types and conditions.

3.1.4 Viral Vectors

Viral vectors are engineered viruses that can deliver genetic material into host cells. This method is highly efficient and can achieve stable gene expression. Viral vectors have been used in various model organisms and hold promise for shrimp gene transfer due to their ability to infect a wide range of cell types and integrate genetic material into the host genome.

3.2 Comparative analysis of techniques

Each gene transfer technique has its advantages and limitations. Microinjection is highly precise but labor-intensive and requires specialized equipment (Abdelrahman et al., 2021; Lane et al., 2021; Gultom, 2023). Electroporation is less invasive and suitable for large-scale applications but may have lower efficiency in some cases (Takahashi et al., 2015). Lipofection is easy to use and non-invasive but may not be as effective in vivo. Viral vectors offer high efficiency and stable gene expression but pose biosafety concerns and require careful handling.

3.3 Efficiency and success rates in shrimp

The efficiency and success rates of gene transfer techniques in shrimp vary depending on the method used and the specific experimental conditions. Microinjection has shown high success rates in various aquatic species, including shrimp, due to its precision and control over the amount of genetic material delivered (Abdelrahman et al., 2021; Lane et al., 2021; Gultom, 2023). Electroporation and lipofection offer alternative approaches with varying degrees of success, depending on the optimization of protocols and reagents used. Viral vectors, while promising, require further research to ensure their safety and effectiveness in shrimp.

3.4 Case study: successful gene transfer using CRISPR-Cas9 in shrimp

A notable example of successful gene transfer in shrimp involves the use of CRISPR-Cas9 technology. This method has revolutionized genetic engineering by allowing precise and targeted modifications of the genome. In a study involving zebrafish, automated microinjection of CRISPR/Cas9 components demonstrated high efficiency and success rates, highlighting the potential for similar applications in shrimp (Cordero-Maldonado et al., 2019). The use of CRISPR-Cas9 in shrimp can facilitate the study of gene functions and the development of genetically improved shrimp strains, contributing to advancements in aquaculture and biotechnology.

4 Cellular Applications of Gene Transfer

4.1 Gene editing for disease resistance

Gene editing technologies, such as CRISPR/Cas9, have been instrumental in enhancing disease resistance in shrimp. By integrating antimicrobial peptide genes (AMGs) through genome editing, researchers have successfully modulated the innate immune system of aquatic animals, leading to improved survival rates and immune responses against pathogens. This approach has shown promise in reducing bacterial colony-forming units and increasing lysozyme activity, which are critical for disease resistance. Additionally, the expression of immune-related genes such as IL, IKβ, TGFβ, C3b, and TLR is significantly enhanced, contributing to a robust immune response (Wang and Cheng, 2023).

4.2 Enhancing growth and reproduction

Gene transfer techniques have also been employed to enhance growth and reproduction in shrimp. For instance, dietary supplementation with specific nutrients, such as hydrolyzed yeast and Bacillus licheniformis, has been shown to improve feed efficiency and protein efficiency ratios in shrimp. These dietary interventions, facilitated by gene transfer, lead to better growth performance and reproductive outcomes. Moreover, the upregulation of genes related to growth and immune responses, such as CAT, GPX, SOD, Pen-3a, and PPO, further supports the enhanced growth and reproductive capabilities of shrimp (Chen et al., 2020).

4.3 Case study: application of gene transfer in improving immune responses

A notable case study involves the use of Sargassum polycystum and nucleotides in the diet of juvenile whiteleg shrimp (Litopenaeus vannamei) to improve their immune response and cold tolerance. The study demonstrated that shrimp fed with a diet supplemented with both Sargassum polycystum and nucleotides exhibited significant improvements in survival, growth, and feed utilization indices (Figure 1). Additionally, the nonspecific immune responses, such as phagocytosis, lysozyme, phenoloxidase, and superoxide dismutase (SOD) activity, were markedly enhanced. The upregulation of immune-related genes, including cMnSOD, Penaeidin4, and heat shock protein70 (HSP70), further underscores the effectiveness of gene transfer in bolstering the immune system of shrimp (Abdel-Rahim et al., 2021).

4.4 Exploring cellular pathways using transgenic shrimp

Transgenic shrimp serve as valuable models for exploring cellular pathways and understanding the molecular mechanisms underlying various physiological processes. For example, the use of Sargassum horneri extracts in shrimp diets has been shown to stimulate innate immunity and enhance growth performance. The study revealed significant modulation of immune-related genes, such as prophenoloxidase I, prophenoloxidase II, peroxinectin, α2-macroglobulin, clotting protein, lysozyme, superoxide dismutase, and glutathione peroxidase, in shrimp fed with Sargassum horneri extracts (Lee et al., 2020). These findings highlight the potential of transgenic shrimp in elucidating the roles of specific genes and pathways in immune responses and growth regulation.

5 Advances in Gene Transfer Technology

5.1 Innovations in delivery systems

Recent advancements in gene transfer technology have significantly improved the efficiency and specificity of gene delivery systems in shrimp. One notable innovation is the development of a VP28-pseudotyped baculovirus expression system, which has demonstrated high infection efficiency in adult shrimp tissues such as gill, heart, and intestine, while showing tissue-specific tropism (Wu et al., 2021). Additionally, the improvement of lentivirus-mediated gene transfer systems, incorporating envelope proteins VP19 and VP28 from the white spot syndrome virus (WSSV), has enhanced the infectivity and tropism of lentiviruses in shrimp cells, achieving higher infection efficiencies compared to traditional methods (Chen et al., 2018). Furthermore, the use of biodegradable nanocarriers resembling extracellular vesicles has emerged as a promising approach for efficient gene delivery, achieving near 100% efficiency in various cell types, including primary cells (Tarakanchikova et al., 2019) (Figure 2). These innovations highlight the potential of advanced delivery systems to revolutionize gene transfer in shrimp.

5.2 Case study: development of non-viral gene transfer techniques

Non-viral gene transfer techniques have also seen significant progress. For instance, the use of DNA nuclear targeting sequences, such as 3NFs, has been shown to enhance the nuclear import of plasmid DNA, thereby improving gene transfer efficiency in both in vitro and in vivo settings (Guen et al., 2021). This method leverages the interaction with the transcription factor NF-κB to facilitate the translocation of nucleic acids into the nuclear compartment of target cells. Another notable development is the application of carbon dot-polyethyleneimine (CDP) nanocomposites, which have proven effective in delivering functional DNA in both plant and animal cells. This system not only facilitates plasmid transport into cells but also protects DNA from degradation, offering a highly efficient and versatile tool for gene transfer (Wang et al., 2020). These case studies underscore the potential of non-viral techniques to provide safer and more efficient alternatives for gene transfer in shrimp.

5.3 Future prospects in shrimp gene transfer research

The future of gene transfer research in shrimp holds promising prospects, driven by continuous advancements in both viral and non-viral delivery systems. The development of more sophisticated and targeted delivery methods, such as the use of adeno-associated virus (AAV) vectors, which offer high gene transfer efficiency and low immunogenicity, could further enhance the precision and effectiveness of gene transfer in shrimp (Kimura et al., 2019). Additionally, the integration of modern gene technologies, such as genome editing and cell-specific promoters, with these advanced delivery systems could pave the way for more precise genetic modifications and improved disease resistance in shrimp. As research progresses, the focus will likely shift towards optimizing these techniques for large-scale applications in aquaculture, addressing environmental and health concerns associated with genetically modified organisms (GMOs) (Jacinda and Yustiati, 2021). Overall, the continued innovation in gene transfer technology promises to unlock new possibilities for enhancing shrimp culture and ensuring sustainable aquaculture practices.

6 Integration with Breeding Programs

6.1 Role of gene transfer in shrimp breeding

Gene transfer techniques, particularly those involving genome editing tools like CRISPR/Cas9, have revolutionized shrimp breeding by enabling precise modifications to the shrimp genome. These techniques allow for the introduction of desirable traits such as disease resistance, improved growth rates, and enhanced feed efficiency, which are critical for the sustainability and profitability of shrimp aquaculture (Kishimoto et al., 2018; Sun and Zhu, 2019; Dai et al., 2020). The ability to directly edit genes in shrimp accelerates the breeding process, reducing the time required to develop new breeds with specific traits compared to traditional breeding methods (Kishimoto et al., 2018; Dhugga, 2022).

6.2 Strategies for combining traditional breeding with gene editing

Combining traditional breeding methods with modern gene editing techniques can enhance the efficiency and effectiveness of shrimp breeding programs. Traditional methods, such as selective breeding and marker-assisted selection (MAS), can be used to identify and propagate desirable traits within a population. These methods can be complemented by genome editing to introduce or enhance specific genetic traits more rapidly and accurately (Yu et al., 2020; Ampofo et al., 2023). For instance, genomic selection (GS) and single-step genomic best linear unbiased prediction (ssGBLUP) have been shown to improve the accuracy of breeding value predictions, thereby enhancing the selection process for traits like feed efficiency and growth (Dai et al., 2020; Ampofo et al., 2023). Additionally, high-throughput SNP genotyping methods can facilitate the integration of genomic data into breeding programs, enabling more precise selection and breeding strategies (Yu et al., 2020).

6.3 Case study: breeding shrimp for specific traits using gene transfer

A notable example of the successful integration of gene transfer techniques in aquaculture is the breeding of the Pacific white shrimp (Litopenaeus vannamei) for improved feed efficiency and growth traits. In a study evaluating genomic information to enhance genetic evaluation, methods such as genomic best linear unbiased prediction (GBLUP) and single-step GBLUP (ssGBLUP) were employed to predict breeding values for feed efficiency ratio (FER) and residual feed intake (RFI). The study demonstrated that genomic-based methods significantly increased the accuracy of breeding value predictions compared to traditional pedigree-based methods, highlighting the potential of gene transfer techniques to accelerate the breeding of shrimp with desirable traits (Dai et al., 2020). Furthermore, the development of high-throughput SNP genotyping approaches has provided valuable tools for genetic studies and the application of molecular breeding methods, such as MAS and GS, in shrimp breeding programs (Yu et al., 2020). These advancements underscore the transformative impact of gene transfer and genome editing technologies in the field of shrimp aquaculture.

7 Future Directions and Research Opportunities

7.1 Emerging technologies in gene transfer

The development of new gene transfer technologies holds significant promise for advancing shrimp aquaculture. For instance, the VP28-pseudotyped baculovirus expression system has demonstrated high efficiency in gene transfer and expression in adult shrimp tissues, although it shows tissue-specific and cell-specific tropism (Wu et al., 2021). This system could be further optimized and expanded to other tissues and developmental stages. Additionally, the transfection method has been identified as the most effective gene transfer technique for shrimp, characterized by high hatching rates and low toxicity (Jacinda and Yustiati, 2021). Future research should focus on refining these methods and exploring novel gene transfer technologies to enhance their efficiency and applicability.

7.2 Potential for multi-omics approaches

Integrating multi-omics approaches, such as genomics, transcriptomics, proteomics, and metabolomics, can provide comprehensive insights into the genetic and molecular mechanisms underlying shrimp biology and production traits (Guppy et al., 2018). The application of single-cell omics technologies can further enhance our understanding by providing high-resolution data on cellular phenotypes and gene regulatory networks (Blencowe et al., 2019; Efremova and Teichmann, 2020). Future research should aim to develop and apply multi-omics techniques to address key challenges in shrimp aquaculture, such as disease resistance, growth rates, and reproductive success.

7.3 Collaboration between researchers and industry

Collaboration between academic researchers and the aquaculture industry is crucial for translating scientific discoveries into practical applications. The integration of genomic resources and omics data into breeding programs and disease management strategies can significantly benefit the industry (Guppy et al., 2018). Establishing partnerships and collaborative projects can facilitate the development of cost-effective genotyping tools and selective breeding programs, ultimately leading to improved shrimp production and sustainability.

7.4 Identifying gaps and setting research priorities

Despite the progress made in gene transfer and omics research, several gaps remain that need to be addressed. For example, the full potential of omics resources has not yet been realized in the industry, and there is a need for better integration of these resources (Guppy et al., 2018). Additionally, the challenges associated with single-cell omics data modeling and the dynamic nature of gene regulatory networks require further investigation (Blencowe et al., 2019). Identifying these gaps and setting research priorities will be essential for advancing the field and achieving practical outcomes. Future research should focus on overcoming these challenges and developing robust methodologies for gene transfer and multi-omics integration in shrimp aquaculture.

8 Concluding Remarks

The research on gene transfer techniques in shrimp has made significant strides, particularly in the application of microinjection, electroporation, and transfection methods. Among these, transfection has emerged as the most efficient technique, demonstrating higher hatching rates and gene expression levels compared to microinjection and electroporation. For instance, transfection methods have shown hatching rates of 50%-60% and gene expression levels of 40%-60%, significantly outperforming the other methods. Additionally, the use of transfection reagents like jetPEI has been particularly effective when applied at the prejelly layer stage of shrimp zygotes, achieving a gene transfer efficiency of 72%.

The advancements in gene transfer techniques hold promising implications for the future of shrimp genetic research. The ability to efficiently introduce and express foreign genes in shrimp can lead to significant improvements in disease resistance, growth rates, and overall aquaculture productivity. The development of genetic resources such as transcriptomics, genomics, DNA markers, and linkage maps will further enhance selective breeding programs, enabling the cultivation of shrimp with desirable economic traits. Moreover, the integration of next-generation sequencing (NGS) techniques will continue to revolutionize the genomics of commercially important aquaculture species, providing deeper insights into their biological, reproductive, and physiological functions at the molecular level.

Gene transfer techniques have proven to be a powerful tool in the field of shrimp aquaculture, offering a means to address critical challenges such as disease outbreaks and the need for improved performance traits. While transfection has shown the most promise due to its high efficiency and low toxicity, it is essential to continue exploring and refining all available methods to maximize their potential applications. The ongoing development and exploitation of genomic resources will be crucial in fully realizing the benefits of gene transfer techniques, ultimately leading to more sustainable and productive shrimp farming practices. As the field progresses, it will be important to address the environmental and health concerns associated with genetically modified organisms (GMOs) to ensure the responsible and ethical application of these technologies.

Acknowledgments

The authors extend sincere thank to two anonymous peer reviewers for their feedback on the manuscript of this study.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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