Non-lethal live sampling of crustaceans and applications thereof

Abstract

The present application belongs to the field of aquatic animal pathogen detection and aquatic breeding biological security technology, and particularly relates to a non-lethal live sampling method of crustaceans and application thereof. The method cuts a part of the step foot or abdominal foot of a live crustacean as a sample, preferably the 3rd, 4th or 5th step foot or abdominal foot, seals the cut wound with a waterproof adhesive medium, extracts nucleic acid or protein of the step foot or abdominal foot tissue for virus detection, and can realize at least 2 times of tracking sampling monitoring of the same animal. The present application also provides application of the method in preparation of a crustacean (such as Macrobrachium rosenbergii, Penaeus, Eriocheir sinensis) virus (such as Decapoda Iridovirus 1) detection product, which is suitable for parent screening, fry quarantine, specific pathogen-free population breeding, longitudinal monitoring and port quarantine, etc. The present application has high animal survival rate and appendage regeneration, and has high sensitivity and high detection repeatability.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal pathogen detection and aquaculture biosafety technology, specifically involving non-lethal live sampling methods for crustaceans and their applications. Background Technology

[0002] Existing molecular detection methods for crustacean pathogens (such as TaqMan qPCR) mostly rely on anatomical sampling, such as grinding whole animals or grinding gills, hepatopancreas, hematopoietic tissue, or muscle tissue from the animal being tested. These sampling methods are lethal, leading to the death of the animals being tested, especially valuable broodstock shrimp, seedlings, SPF breeding populations, and rare germplasm resources. This not only causes economic losses but also makes continuous longitudinal tracking and monitoring of the same population ethically and practically infeasible. Tracking samples from different individuals cannot obtain continuous longitudinal data; individual differences can only be compensated for by increasing sample size and statistical analysis. This not only significantly increases sampling and testing costs but also further increases the loss of valuable genetic resources during testing.

[0003] Currently reported non-lethal live sampling methods for crustaceans can be broadly categorized by sampling site into eyestalk excision, hemolymph aspiration, hepatopancreatic biopsy, and appendage amputation. Bell et al. developed a non-destructive biopsy procedure for the nondestructive determination of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in Litopenaeus vannamei (Bell, TA, Lightner, DV, Brock, JA). Penaeus vannamei. Journal of Aquatic Animal Health, 1990, 2: 151-153.). Kerdmusik et al. reported a method for detecting PmoDNV (Polygonum tinctorium) in the hepatopancreas of the black tiger shrimp using needle biopsy (Kerdmusik, C., Fernando, S., Attasart, P., Vanichviriyakit, R., Boonya, N., Pongtippatee, P., Krishna, S., Withyachumnarnkul, B. Needle biopsy of the hepatopancreas of the black tiger shrimp). Penaeus monodon With Penaeus monodondensovirus detection. Aquaculture, 2018, 490: 1-4.). Cruz-Flores et al. developed a non-lethal invasive sampling method for *E. hepatodelasium* (EHP) in *Litopenaeus vannamei* (Cruz-Flores, R., Mai, HN, Noble, BL, Schofield, PJ, Dhar, AK Detection of Penaeus monodondensovirus. Aquaculture, 2018, 490: 1-4.). Enterocytozoon hepatopenaei using an invasive but non-lethal sampling method in shrimp( Penaeus vannamei Journal of Microbiological Methods, 2019, 162: 38-41.); Han et al. reported acute hepatopancreatic necrosis disease in shrimp ( Vp AHPND (Han, JE, Tang, KF, Piamsomboon, P., Pantoja, CR Evaluation of a reliable non-invasive molecular test for the diagnosis of the causative agent of acute hepatopancreatic necrosis disease of shrimp. Aquaculture Reports, 2017, 5:58-61.); Shen Huaishun et al. (2018) of the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences invented "A Non-destructive Sampling Method for Live Chinese Eriocheir sinensis and Its Application" to protect the extraction of DNA and RNA from fertilized eggs for the detection of related pathogens; Yuan Ye et al. (2026) of Shantou University invented "A Non-destructive Method for Extracting DNA from Muscovy Crab and Its Application" to protect the method of extracting host DNA from the bristles of Muscovy Crab's swimming legs as samples; Fu Qiang et al. (2023) of this institute invented "A Molecular Marker LvKDZ-S1 Related to Rapid Growth of Litopenaeus vannamei and Its Application" and "A Molecular Marker for Screening Rapid Growth Traits of Shrimp and Its Application" to protect the method of extracting host genomic DNA from tentacles and swimming legs and then using a chip to determine the host-specific genotype.

[0004] However, existing methods for live crustacean sampling have the following defects or shortcomings: (a) Although eyestalk excision can be combined with eyestalk excision and ripening surgery, the excised eyestalk tissue is connected to the eyeball, which contains components that strongly inhibit PCR detection. Furthermore, many viruses have very low viral loads in the eyestalk, severely affecting the sensitivity of subsequent detection; (b) The amount of hemolymph obtained by puncture is small or very unstable, significantly impacting the viability of crustaceans after blood collection, especially small crustaceans, which are prone to death due to puncture. Moreover, many viruses have low viral loads in the hemolymph, resulting in poor detection sensitivity; (c) Hepatopancreatic puncture biopsy is difficult to perform, especially for small crustaceans, making effective preservation difficult. Furthermore, because the hepatopancreatic tubules are directly connected to the digestive tract, they contain a large number of bacteria or parasites, easily causing secondary infections and affecting shrimp survival rates. Moreover, the hepatopancreas is not a target tissue for many highly lethal viruses, such as… Many viruses, such as WSSV, DIV1, YHV, and TSV, have low viral loads in the hepatopancreas; (d) the method of using fertilized eggs for sampling is not strictly a live sampling method, and it is not feasible for pathogens that are not infected within the egg or transmitted through the egg. Moreover, most pathogens have very low viral loads on fertilized eggs, making it impossible to effectively and sensitively detect low-load pathogens; (e) existing live sampling methods using appendages, tentacles, or bristles can only be used to extract host genomic DNA, and do not involve the detection of pathogens infecting the host, nor do they involve the corresponding wound sealing and preservation process. As is well known, host genomic DNA exists in large quantities in every host cell, but viruses do not accumulate in non-target tissues. Sampling methods for host genomic DNA are not naturally applicable to the detection of viruses. Therefore, there is an urgent need in this field to establish a sampling method that can meet the requirements of non-lethality and maintain high sensitivity and repeatability of virus detection even when the viral load is extremely low. Summary of the Invention

[0005] The first technical problem to be solved by this invention is to provide a non-lethal live sampling method for crustaceans, so as to achieve high sensitivity detection of crustacean viruses, so that it has sufficiently high detection repeatability even for low viral load infection, and can perform repeated sampling and tracking detection on the same individual animal at different time sequences.

[0006] The second technical problem to be solved by this invention is to reduce the pain and trauma suffered by animals during and after sampling, promote their rapid recovery, reduce the mortality rate caused by sampling, support the survival and organ regeneration of animals after sampling, and ensure animal welfare.

[0007] The third technical problem to be solved by this invention is to enable the method of this invention to be applied to broodstock screening, seedling quarantine, specific pathogen-free population breeding, and port quarantine in aquaculture. The crustaceans described in this invention include giant freshwater prawns, prawns, red swamp crayfish, Chinese mitten crabs, and mud crabs.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: To address the first technical problem, this invention provides a non-lethal live sampling method for crustaceans, which involves amputating a portion of an appendage from a live crustacean as a sample while keeping the sampled animal alive. Preferably, the appendage is the 1st to 5th walking leg or ventral leg, more preferably the 3rd, 4th, or 5th, especially the 4th walking leg which has the best sensitivity and repeatability. One to three phalanges from the distal end of the walking leg or ventral leg are amputated as samples. To confirm that the walking legs or ventral legs can be used as samples for virus detection, and to verify the rationality of the 3rd, 4th, or 5th walking legs or ventral legs (especially the 4th walking leg) as the preferred site for live sampling, this invention conducted a systematic experimental comparison of various sampling sites. The relative abundance of the virus in each tissue (reflecting the proportion of viral load) was determined by the quantitative results of qPCR of viral load, and the repeatability (reflecting detection stability) obtained by qPCR parallel samples and amplification effects of different individual samples was combined. Through comprehensive evaluation of the two indicators, the preferred site for non-lethal live sampling of the distal 1-3 phalanges of the walking or ventral legs of crustaceans was established for the first time.

[0009] Furthermore, the above systematic evaluation results indicate significant differences in detection sensitivity and repeatability among different tissues. For example, routinely sampled tissues such as hemolymph and gills show great individual variation in the relative abundance of viral load in vivo, resulting in low repeatability. While eye / eyestalk tissues have a certain viral load, their high content of PCR inhibitors such as pigments significantly reduces repeatability. In contrast, the 3rd, 4th, and 5th pereiopods and abdominal legs, especially the 4th pereiopod, performed excellently in both dead and live samples, demonstrating considerable viral detection with logarithmic relative abundance values ​​above the average level for various tissues, and exhibiting good repeatability. These tissues are the preferred choice for balancing high viral load and high detection repeatability. The 4th pereiopod, in particular, achieved stable and highly repeatable detection even with extremely low viral loads (e.g., 0.1-7.4 copies / ng DNA, which accounts for approximately 85% of asymptomatic infections), demonstrating the best performance among all tissues. Multi-level experimental verification using various pathological techniques, including H&E staining, ISDL (in situ DIG-labeled loop-mediated isothermal amplification), and TEM (transmission electron microscopy), confirmed the presence of viral particles and typical pathological changes in the excised fourth peroneal tissue, thus validating the scientific validity and reliability of this sampling site. Selecting the third, fourth, or fifth peroneal leg or abdominal leg, especially the fourth peroneal leg, can solve the technical challenge of easily missing cases during the subclinical infection phase.

[0010] Furthermore, this invention reveals that the detection sensitivity and repeatability of appendage sampling are significantly affected by the tissue tropism of pathogens. Since the walking and abdominal legs mainly contain epithelial tissue, connective tissue, nerve tissue, blood cells, and some hematopoietic tissue, the sampling method of this invention has extremely high detection sensitivity and repeatability for viruses that primarily infect tissues of mesoderm and ectoderm origin (such as epithelium, blood cells, hematopoietic tissue, connective tissue, and nerve tissue). These viruses include Decapoda Iridovirus 1 (DIV1), White Spot Syndrome Virus (WSSV), Infectious Hypodermic and Hematopoietic Necrosis Virus (IHHNV), and Infectious Precocious Puberty Virus (IPV). Conversely, for pathogens that primarily infect tissues of endoderm origin (such as the hepatopancreas and midgut), including hepatopancreatic parvovirus (HPV), Penaeus monodon baculovirus (MBV), Penaeus monodon baculovirus (BP), and Vibrio parahaemolyticus (…), the detection sensitivity and repeatability are significantly affected. Vp AHPNDFor bacteria or parasites such as *Enterocera hepatocellular carcinoma* (EHP) that cause acute hepatopancreatic necrosis syndrome, the sensitivity and repeatability of sampling using walking legs or gastropods are relatively low. Therefore, the sampling method of this invention is particularly suitable for highly sensitive screening of systemic infections or viruses with ectoderm / mesoderm tropism. It is worth noting that viruses that infect these tissues, such as DIV1 and WSSV, generally have greater pathogenicity and cause acute death or severe disease in the host compared to viruses that infect the endoderm, such as HPV and MBV.

[0011] Preferably, the amputation of the walking leg or ventral leg includes the following steps: (1) Firmly grasp the cephalothorax from the back of the crustacean. For shrimp, fix the first pair of walking legs between the thumb and forefinger and press the tail with the ring or little finger. For crabs, grasp the sides of the cephalothorax with the thumb and forefinger and hold the end of the cephalothorax with the ring or little finger. If the crustacean has large claws, use rubber bands or ropes to tie the large claws to ensure safety. (2) Using a sterilized cutting tool, cut off 1-3 phalanges of the distal end of the walking or abdominal foot of the crustacean as a sample, and immediately place it in the sample preservation conditions. The cutting tool is preferably scissors or a scalpel.

[0012] Because the tissue within the amputated legs or ventral feet is limited while the carapace is relatively thick, it is necessary to cut or remove the tissue from the amputated appendages to allow for effective tissue grinding and thus fully obtain the viral components within the tissue. Preferably, the carapace of the appendage used as a sample is cut with scissors, or the soft tissue within the appendage is removed using a needle-like tool. The tissue is then ground using a grinding rod or vibrating grinder to obtain the tissue for extraction of nucleic acid or protein components, which are then used for the detection of viral nucleic acid or protein components, thereby obtaining the nucleic acid or protein detection signal of the virus to be tested in the sample.

[0013] Regarding the second technical problem, this invention focuses on the site of limb amputation and preventing further fluid leakage and sealing the wound after amputation. For smaller wounds, crustaceans can usually stop bleeding through blood clotting and gradually repair and close the wound. However, it is known that crustacean clotting typically takes about one minute, and for larger wounds or wounds located near blood sinuses, the clotting time is even longer. Appendages, especially near the base of the legs, contain blood sinuses; amputating the entire appendage will cause a certain amount of blood loss, and the amputated crustacean will exhibit a violent reaction, leading to a further increase in blood flow. Moreover, because crustacean blood is colorless to very pale blue, bleeding in water is difficult to observe, and trauma and bleeding are easily overlooked by operators. Therefore, improper amputation can cause crustaceans to exhibit varying degrees of weakness, and even death in some individuals. To address this, this invention limits the amputation site to the distal 1-3 phalanges, thus avoiding amputations that are too close to blood sinuses or have excessively large amputation areas. In addition, the present invention uses a waterproof sealing medium to seal the wound, completely preventing blood from flowing out, thereby further reducing the trauma to crustaceans and effectively improving the vitality and survival rate of crustaceans.

[0014] Preferably, a sample is taken from the second distal phalanx of the walking or ventral foot, and the wound is immediately sealed and hemostatically stopped using a sealing medium. The crustacean is then returned to water for rearing. The sealing medium is a waterproof adhesive, including hot melt adhesive, biocompatible medical adhesive, or other polymeric waterproof sealing materials. The sealing medium is applied to the wound in liquid form using a hot melt glue gun or appropriate tools. The waterproof membrane or glue droplets solidify to close the wound, and the sealing operation is completed within 2 minutes.

[0015] Under the aforementioned conditions of minimizing harm to animals, they can continue to survive and feed normally after sampling. Under favorable conditions, after one to two weeks of recovery, crustaceans can begin molting. After molting, the appendages will exhibit varying degrees of new growth buds or limbs, which grow significantly faster than other appendages and the entire body, allowing for further sampling of the new appendages. Furthermore, unsampled limbs within pairs can be sampled at shorter intervals, and the body's symmetry ensures very similar virus detection rates. Thus, at least two continuous follow-up samplings can be performed on the same individual, achieving continuous monitoring of viral infection dynamics within that individual—something impossible with lethal sampling.

[0016] Preferably, the method allows for at least two follow-up sampling monitoring sessions on the same crustacean: sampling each pair of appendages separately; and / or sampling different pairs of appendages separately; and / or sampling the appendage again after a time interval of ≥7 days, and after the crustacean has molted and regrown part of the growth point or the entire phalanx of the amputated appendage. The number of follow-up sampling monitoring sessions is 2-20.

[0017] To address the third technical problem, based on the aforementioned highly sensitive, highly repeatable, and animal welfare-oriented non-lethal live sampling method, this invention provides a non-lethal live sampling tool kit for implementing the method, comprising at least one of the following components: a sterilized cutting tool, a waterproof adhesive sealing medium and its application device, a container for preserving foot samples, and a foot sample extraction tool. Furthermore, this invention provides the application of the crustacean sampling method or the tool kit in the preparation or establishment of crustacean virus detection products and standardized detection systems. Considering the special nature of live sampling, the detection products mainly refer to standardized operating procedures (SOPs), virus detection protocols, high-throughput screening platforms, or supporting nucleic acid / protein detection systems established based on the sampling method of this invention. The sampling method of this invention provides stable, high-quality, and virus-enriched test samples (nucleic acid or protein templates) for the aforementioned detection products, thereby ensuring the accuracy and reliability of downstream detection.

[0018] Preferably, the detection product is used in at least one of the following scenarios and exhibits technical advantages that traditional lethal sampling cannot replace: (1) Parental selection: In the seed breeding stage of aquaculture, high-quality broodstock shrimp / crabs (parental lineage) are extremely valuable. Using this method, without affecting the survival, mating, berried state, and reproductive capacity of the parental lineage, only the 3rd-5th pereiopods (preferably the 4th pereiopod) or the distal 1-3 phalanges of the gastropods are removed and the wounds are sealed. This operation not only provides highly sensitive virus screening signals, accurately eliminating inferior parental lineage carrying viruses, but also minimizes stress and health damage to parental lineage with high production potential, ensuring the survival rate of high-value parental lineage, cutting off vertical transmission of viruses at the source, and ensuring the biosafety of offspring.

[0019] (2) Seedling quarantine: For early seedlings that may have low viral loads, conventional random sampling or lethality testing often fails to balance detection coverage and seedling loss. The walking leg sites selected in this method still have extremely high detection repeatability under low viral load infection, which can effectively avoid missed detections; at the same time, the minimally invasive operation allows for full sampling or large-scale random sampling of low-spawning species, such as high-quality ornamental shrimp and crab, ornate lobster, and narrow-clawed crayfish, to ensure that the seedlings entering the rearing stage are absolutely healthy.

[0020] (3) Specific Pathogen-Free (SPF) Population Breeding: During the establishment and breeding of SPF families, long-term health monitoring of the core breeding population is required. This method supports multiple tracking samplings of the same animal individual across molting cycles (such as the aforementioned 2-20 consecutive monitoring sessions). This allows breeders to continuously and dynamically confirm the SPF status of candidate individuals without interrupting the breeding process due to sampling deaths, greatly accelerating the breeding efficiency of disease-resistant or disease-free strains.

[0021] (4) Longitudinal Epidemiological Surveillance: Traditional epidemiological surveys typically rely on cross-sectional data compilation by slaughtering different individuals at different time points, resulting in significant interference from individual differences. This method allows for repeated sampling of the same individual at different time stages (e.g., early infection, incubation period, outbreak period, and recovery period). Combined with the aforementioned resampling technology, researchers can accurately track the infection dynamics, proliferation patterns, and viral shedding cycles of specific viruses within a single host, providing a novel longitudinal data collection method for crustacean virology research.

[0022] (5) Subclinical infection detection: In the subclinical (latent) infection stage, crustaceans usually do not show obvious pathological symptoms or abnormal appearance, but they already carry trace amounts of virus in their bodies and may become a source of infection. Since the sampling sites (especially the distal end of the fourth pereiopod) established by the present invention through systematic experiments have a high enrichment effect on virus particles, applying them to subclinical detection can significantly break through the detection limit of conventional detection methods, and realize early warning and timely intervention of diseases.

[0023] (6) Port Quarantine: For live crustaceans imported and exported (such as ornamental shrimp and crabs, high-value edible aquatic products, and live animals for scientific research), customs and inspection and quarantine departments require rapid, accurate, and non-destructive testing methods. This method provides a minimally invasive sampling protocol that meets international animal welfare standards. Operators can quickly amputate the toe segments at the port and seal the wound with a waterproof sealing medium (completed within 2 minutes), after which the animal can continue to be transported or temporarily cared for. This application not only meets strict biosafety quarantine requirements but also minimizes the damage rate and economic disputes in the international trade of live animals.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The sampling sites are scientifically reliable. By comparing the relative abundance (RA) and repeatability (RSR) of various tissues, it was found that the 3rd to 5th peroneal legs and abdominal legs, especially the 4th peroneal leg, are tissues that perform well in both the physiological states of death and slaughter. They can take into account both high viral load and high detection repeatability, providing direct experimental support for the selection of sampling sites.

[0025] (2) High survival rate and regenerative appendages. The survival rate of crustaceans after surgery is no less than 80% (typically more than 82% in a population with n=123). The amputated walking or abdominal feet can be completely regenerated after 1-2 molts, restoring walking, feeding and tactile functions; meeting animal welfare requirements, and can be used for continuous longitudinal monitoring of precious parent animals and SPF breeding populations.

[0026] (3) Simple to operate and suitable for industrial application. Sampling and sealing surgery of a single animal can be completed within 2 minutes, and ordinary laboratory personnel can master it after a short training period; the supporting tool kit can be directly commercialized and promoted, and is suitable for parent selection, seedling quarantine, specific pathogen-free population breeding, longitudinal epidemiological monitoring, subclinical infection detection and port quarantine of giant freshwater prawn and other crustaceans. Attached Figure Description

[0027] Figure 1 This is a schematic diagram comparing the RA-RSR dual-index XY scatter plots of DIV1 detection performance in tissue samples in Example 1; where A is the XY scatter plot of the challenged and dead group, and B is the XY scatter plot of the live slaughter group; the Y-axis represents the relative abundance of each tissue (RA, reflecting the proportion of DIV1 content in the total body), and the X-axis represents the repeatability rate of each tissue (RSR, reflecting the stability and repeatability of qPCR detection); each tissue is located in the form of scatter plots + error bars. Figure 2 This is a flowchart of a non-lethal live sampling and waterproof sealing surgery for the fourth peroneus of crustaceans, represented by the giant freshwater prawn, in Example 2. A shows the surgeon holding the giant freshwater prawn's carapace and using sterilized scissors to remove the second joint of the fourth peroneus. B, C, and D show the use of a hot melt glue gun to apply molten hot melt adhesive to the surgical wound for rapid sealing. E shows the giant freshwater prawn being returned to the water and resting normally after the surgery. Figure 3 The histopathological and ISDL analysis results of DIV1-negative giant freshwater prawns in Example 4 are shown. Among them, A, B, and C are the H&E staining results of normal gills, normal hematopoietic tissue, and normal fourth peregrine foot, respectively; D, E, and F are the ISDL detection results of normal gills, normal hematopoietic tissue, and normal fourth peregrine foot, respectively; scale bar = 100µm. Figure 4 The histopathological and ISDL analysis results of the low DIV1 loading giant freshwater prawns in Example 4 are shown. Among them, A, B, C, and G are the H&E staining results of gills, hematopoietic tissue, fourth peroneus, and testes, respectively; D, E, F, and H are the ISDL detection results of gills, hematopoietic tissue, fourth peroneus, and testes, respectively; scale bar = 100µm. Figure 5 Transmission electron microscopy (TEM) observation of low DIV1 load giant freshwater prawns in Example 4; A, B, C, and D are TEM images of the fourth pereiopod, E is a TEM image of the gills, and F is a TEM image of the hematopoietic tissue; white arrows indicate DIV1 virus particles at different assembly stages. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical content of this invention, the invention will be further described below with reference to the accompanying drawings and specific embodiments. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the equipment and reagents used in each embodiment are all commercially available.

[0029] Example 1: Systematic comparison and optimal sampling location for DIV1 detection performance in multiple tissues of Macrobrachium rosenbergii (1) Sample Source and Grouping: The comparison in this embodiment is based on two independent sample sets—Group A: 3 giant freshwater prawns that died after being challenged with the virus; Group B: 12 ​​live DIV1-positive giant freshwater prawns that were experimentally euthanized before sampling. The gill tissue of both groups was confirmed by qPCR to contain only DIV1 and not WSSV, IHHNV, V AHPND Co-infecting pathogens such as EHP, TSV, or YHV. Using extracted DNA as a template, real-time quantitative PCR amplification was performed using specific primers and / or TaqMan probes targeting the MCP gene, the major capsid protein of DIV1. Each sample was tested in triplicate, with a maximum of 41 cycles. A Ct value ≤ 41 and a typical S-shaped amplification curve were considered DIV1 positive; otherwise, the sample was considered negative.

[0030] (2) Types of testing organizations: Group A collected and tested 24 tissues: hemolymph, distal antennae, frontal scutes, distal tail fan, proximal tail fan, eyes, left / right eyestalks, hepatopancreas, gill filaments, heart, midgut, rectum, distal ends of the first and third to fifth pereiopods, the first to fifth abdominal feet, muscles, and stomach. Group B collected and tested 24 tissues: hematopoietic tissue, hemolymph, base of antennae, distal end of antennae, frontal scutes, proximal end of tail fan, eye, eyestalk, hepatopancreas, gill filaments, heart, midgut, distal ends of the 2nd to 5th peroneal legs, 1st to 5th abdominal legs, muscle, stomach, and gonads.

[0031] (3) Dual-index evaluation methodology: In this embodiment, two quantitative indices are calculated simultaneously for each tissue: (i) Relative abundance RA_i = CN_i / (T_N · C ), where CN_i is the number of DIV1 copies of the organization, T_N is the total number of test organizations, and C The mean copy number of all tissues is RA_i, which reflects the proportion of DIV1 content in the total body count of that tissue; (ii) Reproducibility rate RSR = DR / (1 + CV), where DR is the detection rate of triplet qPCR detection, CV is the coefficient of variation of copy number, and RSR reflects the stability and reproducibility of qPCR detection in that tissue. Then, an XY scatter plot is plotted with RSR as the X-axis and RA as the Y-axis. Figure 1 This makes the overall testing performance of each tissue readily apparent.

[0032] (4) Group of 3 shrimp that died after being attacked with poison ( Figure 1 Comparison of A) in the middle: Under conditions of death after challenge, tissues exhibiting a "high RA + high RSR" combination (i.e., the upper right region of the XY scatter plot) include: midgut, eye, eyestalk, fourth pereiopod, fifth pereiopod, muscle, and hemolymph, indicating that these tissues can provide reliable DIV1 detection in dead shrimp samples.

[0033] Tissues exhibiting the characteristics of "high RA but large variability" include: distal tail fan and hemolymph. Although these tissues carry a high average DIV1 load, they are not suitable as the first choice for stable detection due to significant inter-individual variability.

[0034] Tissues exhibiting a "low RA + low RSR" combination (i.e., the lower left region of the XY scatter plot) include: frontal rostrum, proximal tail fan, hepatopancreas, and distal antennae. These tissues showed low DIV1 load and poor detection repeatability in dead shrimp samples.

[0035] (5) Slaughtering group of 12 live shrimp ( Figure 1 Comparison of B): Under experimentally euthanized conditions (i.e., the target physiological state for biopsy sampling according to this invention), step 4 exhibits the optimal balance of "moderate to high RA + high RSR", which is clearly established as the optimal sampling site for non-lethal biopsy of DIV1 in this embodiment.

[0036] Tissues that showed no statistically significant difference from the fourth peregrine foot (p≥0.05) included the proximal tail fan and the xiphoid process. Although these tissues have similar properties to the fourth peregrine foot, they are not suitable as routine non-lethal biopsy sites due to inconvenient anatomical location, higher risk of trauma, or more complex sample preparation. The fourth peregrine foot has high detection sensitivity, with high viral load and excellent reproducibility in the two distal phalanges. It can be stably detected at extremely low viral loads (approximately 0.1-7.4 copies / ng DNA, accounting for about 85% of all positive samples); approximately 14-17% of positive samples have a viral load higher than 5 copies / ng DNA, and only about <2% of samples have a viral load of approximately 500 copies / ng DNA or higher.

[0037] The tissues with slightly lower performance than the fourth walking leg include: the proximal end of the tail fan, the fifth walking leg, the third abdominal leg, and the fifth abdominal leg.

[0038] Tissues with significantly lower performance than the fourth pereiopod include: antennal base, hepatopancreas, eyestalk, hemolymph, gill filaments, first abdominal foot, second pereiopod, third pereiopod, gonads, stomach, eyes, second abdominal foot, heart, and midgut. The detection performance of these tissues is unstable due to large inter-individual differences.

[0039] (6) PCR inhibitor problem in eye / eyestalk tissue: In the group of 12 slaughtered shrimp, the reproducibility rate of eye and eyestalk samples was significantly lower than that of other tissues. This is due to the presence of PCR inhibitors (such as pigments) in compound eye tissue. This phenomenon has been reported in early PCR detection of shrimp and insect samples. However, even after purification to remove pigment inhibitors, the eye / eyestalk is still unsuitable as a biopsy site for the non-lethal biopsy purpose of this invention due to the high risk of dissection location and the difficulty in sealing the wound.

[0040] (7) Conclusion of the fourth walking leg as the common optimal tissue: Based on the comparison results of the two groups of 3 shrimp that died from poisoning and 12 shrimp that were slaughtered alive, the fourth walking leg is the only tissue that performs well in both physiological states, providing direct experimental support for the selection of walking leg sites.

[0041] (8) Methodological generalizability: The RA-RSR dual-index evaluation method of this embodiment can be extended to the selection of DIV1 susceptible biopsy sites in other crustaceans (Litopenaeus vannamei, Penaeus monodon, Procambarus clarkii, etc.); it can also be extended to the development of non-lethal biopsy methods for other aquatic viral pathogens (such as WSSV, IHHNV, EHP, etc.).

[0042] Example 2: Step 4: Vivisection and Waterproof Sealing of Two Distal Toe Joints This embodiment uses adult giant freshwater prawns (15-20 cm in length) as the subject to detail the specific operation of the non-lethal live sampling method of the present invention: (1) Preoperative preparation: The surgeon wears rubber gloves; disinfects the surgical scissors with 10ppm potassium permanganate solution, and then wipes the scissors dry with absorbent paper; preheats the hot melt glue gun to the working temperature and attaches the hot melt glue stick (Deli 5030J model). (2) Fixing the animal: The operator firmly grasps the cephalothorax of the giant freshwater prawn from the back and fixes the first pair of walking legs between the thumb and forefinger to prevent it from struggling; (3) Amputation of the walking leg: Use sterilized scissors to quickly amputate the distal end at the second joint or the second phalanx of the fourth walking leg. Immediately put the amputated tissue (including the two distal phalanges) into a 1.5 mL centrifuge tube containing 95% ethanol for storage, for subsequent DNA extraction and qPCR detection. (4) Wound sealing: Use a preheated hot melt glue gun to quickly apply molten hot melt glue to the cut wound to quickly seal the wound and reduce hemolymph loss; (5) Postoperative return: Immediately after sealing, the giant freshwater prawns were returned to the aquaculture water for separate rearing and observation. Figure 2 ).

[0043] The entire surgical procedure for a single giant freshwater prawn is controlled within 2 minutes. Post-surgery, the prawns swim normally and feed well; after 1-2 molts, their fourth walking legs fully regenerate, restoring their walking, feeding, and tactile functions. The survival rate after surgery is no less than 80% (in a population of n=123).

[0044] Example 3: Large-sample DIV1 detection and survival rate verification This embodiment, based on Example 1, performed step 4 non-lethal live sampling and qPCR detection on 123 giant freshwater prawns that survived DIV1 challenge, and tracked their post-operative survival. The qPCR detection target in this embodiment is based on the standardization work of our research group on TaqMan real-time quantitative PCR of the DIV1 major capsid protein (MCP) gene.

[0045] Total DNA was extracted from the excised tissues using the TIANamp Marine Animal DNA Kit (TIANGENBiotech, Beijing), and the DNA concentration and quality were determined using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, USA).

[0046] qPCR amplification was performed using DIV1 major capsid protein (MCP) gene-specific primers and TaqMan probes. Three technical replicates were used for each sample, with an upper limit of 41 reaction cycles. The results are as follows: (1) Of the 123 giant freshwater prawns, about 94 samples were positive for the MCP gene (about 76.4%); about 29 samples were negative for qPCR (about 23.6%). (2) The distribution range of extremely low viral load in positive samples is approximately 0.1-7.4 copies / ng DNA, with about 85% of MCP positive samples concentrated in this range; about 14-17% of positive samples have viral loads higher than 5 copies / ng DNA; and a few samples (about <2%) have viral loads of about 500 copies / ng DNA or more. (3) During the observation period of about 2 months after the operation, the survival rate was not less than 80% (typically more than 82%); all surviving animals had their legs completely regenerated after 1-2 molts.

[0047] Example 4: Pathological, in situ hybridization and electron microscopy verification To further confirm the presence of DIV1 virus particles and typical pathological changes in the excised fourth walking legs, this embodiment performed histopathological, ISDL, and TEM verification on the fourth walking legs, gills, and hematopoietic tissues of giant freshwater prawns with low viral load (approximately 1.04×10²-3.25×10³ copies / ng DNA).

[0048] Histopathological and ISDL analysis results of DIV1-negative giant freshwater prawns are as follows: Figure 3 Neither H&E staining nor ISDL assays showed DIV1 signal. Histopathological and ISDL analysis results of low DIV1 load Macrobrachium rosenbergii are as follows: Figure 4 TEM results are as follows Figure 5 : (1) H&E staining showed typical nuclear pyknosis and eosinophilic inclusion bodies, characteristic pathological changes of DIV1 in the fourth stepfoot. Figure 4 ); (2) ISDL showed obvious positive blue signals near the blood vessels, basement membrane and epithelial tissue of the fourth foot. Figure 4 ); (3) TEM transmission electron microscopy revealed typical icosahedral virus particles and unassembled viral intermediates in the fourth walking leg, gills, and hematopoietic tissue, consistent with existing electron microscopic morphological descriptions of viruses in the Decapoda Iridoviridae family. Figure 5 ); The above multi-level verification fully demonstrates that DIV1 virus particles and typical pathological changes were indeed present in the 4th step foot, thus confirming the scientific validity and reliability of the sampling site.

[0049] Example 5: Application of the method of the present invention in longitudinal monitoring of SPF (Special Feed Generation) populations of giant freshwater prawns The application scenario is the continuous tracking and screening of SPF (specific pathogen-free) candidate giant freshwater prawn populations. The specific operational steps are as follows: (1) Individual marking: Select SPF candidate parents of giant freshwater prawns in the temporary rearing period and number them by using eyestalk ringing or injecting fluorescent markers.

[0050] (2) Initial sampling: After recording the number, the operator wears latex gloves and holds the shrimp's cephalothorax with one hand, making its abdomen face upward. Using ophthalmic scissors disinfected with 75% alcohol, the shrimp is quickly cut at the distal second toe segment of the fourth pereiopod on the left side, the cut segment is collected, and the wound is quickly sealed using the method in Example 2.

[0051] (3) Live release and temporary rearing: The parent animals were immediately returned to the original temporary rearing pool after sampling.

[0052] (4) Secondary sampling (longitudinal monitoring): After 2-4 weeks of temporary rearing or mating and spawning, the same giant freshwater prawn was retrieved according to the marking. Using the same fixation method, the fourth pereiopod on its right side was cut off for a second pathogen detection.

[0053] (5) Sample processing: The two collected walking legs were rinsed with sterile water, cut into pieces, and added to the lysis buffer to extract nucleic acid for routine PCR / qPCR detection.

[0054] This embodiment of the method breaks through the bottleneck of traditional "lethal sampling" which cannot continuously monitor the same individual. By alternating sampling of the left and right legs, it is possible to longitudinally track the pathogen-carrying status of the same high-value parent at different physiological stages (such as before mating and after brooding), and the sampling operation does not affect the survival, feeding and reproductive behavior of the parent, providing reliable technical support for the precise breeding of SPF families.

[0055] Example 6: Application of the method of the present invention in non-destructive screening of Litopenaeus vannamei. The application scenario is port quarantine and low-virus screening during the introduction of shrimp broodstock. The specific operating steps are as follows: (1) Holding and fixing: Litopenaeus vannamei is relatively small and prone to jumping due to stress. Operators should quickly wrap the shrimp with a damp soft towel underwater or after taking it out of the water, exposing only the abdominal appendages, in order to reduce stress caused by light and dryness.

[0056] (2) Sampling site selection and cutting: The walking legs of shrimp are relatively thin, while the swimming legs on the abdomen are more muscular. Using sterilized fine tissue scissors, select the second or third pair of swimming legs on the abdomen of the shrimp, cut off about 1 / 2 to 1 / 3 of the distal part, and use hot melt glue to quickly seal the wound.

[0057] (3) Live temporary holding: After sampling, the shrimp are quickly transferred to clean seawater with good aeration. Removing the end of the shrimp's swimming leg will not damage its core nerve and circulatory system.

[0058] (4) Sample pretreatment: The carapace of the swimming leg of shrimp is very thin and does not need to be peeled. The cut swimming leg segments are placed directly into a homogenizing tube containing grinding beads and lysis buffer, and thoroughly homogenized using a tissue homogenizer. Nucleic acid is then extracted using conventional methods.

[0059] Traditional methods of extracting hemolymph from shrimp can easily lead to secondary infections or stress-induced death. This method, by cutting off the end of the swimming leg, can obtain tissue rich in hemolymph and muscle (meeting the requirements for high-sensitivity detection) while ensuring a high survival rate of broodstock shrimp during quarantine. It is particularly suitable for non-destructive quarantine in live international trade.

[0060] Example 7: Application of the method of the present invention in the epidemiological monitoring of red swamp crayfish (Procambarus clarkii). The application scenario is for early, non-destructive screening of pathogens such as white spot syndrome virus (WSSV) in farmed red swamp crayfish populations. The specific operating steps are as follows: (1) Grasping and securing: The red swamp crayfish is highly aggressive. The operator needs to pinch the crayfish body from behind its cephalothorax, so that its claws are suspended in the air and cannot be used to attack.

[0061] (2) Sampling site selection and cutting: Avoid the first pair of walking legs (large claws) used for defense and feeding. Select the third or fourth pair of walking legs, cut them off with sterilized medical scissors, and quickly seal the wound with hot melt glue.

[0062] (3) Live temporary holding: The sampled Procambarus clarkii was put back into the temporary holding net cage. Procambarus clarkii has a strong ability to regenerate appendages. Cutting off a single walking leg on one side does not affect its crawling on the bottom and subsequent molting and growth.

[0063] (4) Sample pretreatment: The walking legs of the red swamp crayfish are tubular and the carapace is relatively hard. Use dissecting scissors to cut along the tubular carapace of the walking legs longitudinally, scrape the internal muscle tissue (about 20-30 mg) with sterile forceps and place it in a centrifuge tube, add nucleic acid extraction reagent for subsequent operations.

[0064] This method is simple and safe to operate, allowing a single person to quickly collect large numbers of samples. The extracted nucleic acid quality from the walking leg muscle tissue fully meets the requirements for molecular detection, making it suitable for early subclinical infection screening in aquaculture sites and avoiding the economic losses caused by traditional necropsy methods.

[0065] Example 8: Application of the method of the present invention in the breeding of Chinese mitten crab (Chinese hairy crab) parents The application scenario is the screening of specific pathogens in high-quality parent crabs of the Chinese mitten crab (such as the "Jianghai 21" breed) before overwintering and mating. The specific operating steps are as follows: (1) Grasping and fixing: The operator wears protective gloves and uses his thumb and forefinger to press the carapace of the crab to fix it on the operating table to prevent it from grabbing randomly with its legs.

[0066] (2) Sampling site selection and cutting: When subjected to strong stimulation, Chinese mitten crabs are prone to "autotomy" (i.e., the entire walking leg breaks off from the base). To prevent autotomy, the base should be avoided when sampling. Select the 3rd or 4th walking leg, and use strong tissue scissors or orthopedic scissors to cut off only the last two segments (dactylus and proximal segment) of the walking leg. After sampling, quickly seal the wound with hot melt glue.

[0067] (3) Live temporary holding: After sampling, the mitten crabs are returned to the micro-flow temporary holding pond. Cutting only the last two segments will not trigger the autotomy reaction of the mitten crabs, nor will it affect their subsequent egg-bearing and mating.

[0068] (4) Sample pretreatment: Wash the cut-off end of the walking leg with sterile water to remove surface hairs and dirt. Use sterile hemostatic forceps to crush the hard shell and pick out the internal muscle tissue for nucleic acid extraction.

[0069] This method cleverly avoids the "autotomy" stress response unique to crabs, and successfully obtains effective tissue for virus detection while ensuring the integrity and vitality of the parent stock, providing a non-destructive detection method for biosafety in the breeding process of high-quality Chinese mitten crabs.

[0070] Example 9: Application of the method of the present invention in the non-destructive quarantine of mud crabs. The application scenarios include entry quarantine of high-value broodstock mud crabs and screening for pathogens such as MCRV (monoclonus reovirus). The specific operating steps are as follows: (1) Grasping and fixing: The mud crab is ferocious by nature and its claws are extremely lethal. The operator needs to grasp the mud crab from both sides behind its carapace and press it down on the operating board with its abdomen facing upward, so that its walking legs and swimming legs are exposed.

[0071] (2) Sampling site selection and cutting: The fifth pair of walking legs of the mud crab is specialized into paddle-shaped swimming legs. Select one side of the swimming leg, use sterilized medical orthopedic scissors to quickly cut it about 1.5 cm away from the joint at the base of the swimming leg, and seal the wound with hot melt glue.

[0072] (3) Live temporary holding: The mud crabs were immediately placed back into the individual temporary holding baskets after sampling. Since only the end of one side of the swimming leg was cut off, it does not affect its crawling on the bottom. In addition, the mud crab's shell is thick, and the hot melt glue sealing can effectively prevent a large amount of body fluid from being lost.

[0073] (4) Sample pretreatment: Rinse the cut swimming leg segments with sterile water. Since the shell of the mud crab is hard, use sterile bone forceps or bench vise to help crack the shell, and use a scalpel to remove the internal muscle tissue (about 30-50mg) and place it in a centrifuge tube. Follow the instructions of the routine tissue nucleic acid extraction kit.

[0074] Traditional blood-drawing methods are prone to causing injury from crab pinches and can easily lead to stress-induced death. The method provided in this embodiment can obtain sufficient muscle tissue for highly sensitive molecular detection without damaging the core organs of the crab and ensuring the safety of operators, thus meeting stringent biosafety quarantine requirements.

Claims

1. A non-lethal live sampling method for crustaceans, characterized in that, A portion of the appendage is taken from a live crustacean as a sample, and the animal is kept alive after sampling.

2. The non-lethal live sampling method for crustaceans according to claim 1, characterized in that, The appendages are the first to fifth walking feet or abdominal feet.

3. The non-lethal live sampling method for crustaceans according to claim 2, characterized in that, The appendage is the 3rd, 4th, or 5th walking foot or ventral foot.

4. The non-lethal live sampling method for crustaceans according to claim 2, characterized in that, The amputation of the walking leg or ventral leg includes the following steps: (1) Firmly grasp the cephalothorax from the back of the crustacean. If the crustacean has large claws, use rubber bands or ropes to tie the large claws to ensure safety. (2) Use a sterilized cutting tool to cut off 1-3 phalanges of the distal end of the walking or abdominal foot of the crustacean as a sample, and immediately place it under the required sample preservation conditions.

5. The non-lethal live sampling method for crustaceans according to claim 1, characterized in that, At the amputation site of the appendage, a waterproof adhesive sealing medium is applied to the wound in the form of an adhesive liquid. The waterproof membrane solidifies to seal the wound and stop bleeding. The crustacean is then returned to the water for rearing.

6. The non-lethal live sampling method for crustaceans according to claim 1, characterized in that, The appendages used as samples are cut open with scissors or the soft tissue inside the appendages is removed using a needle-like tool. The tissue is then ground up with a grinding rod or a vibrating grinder to extract nucleic acid or protein components for the detection of viral nucleic acid or protein components, thereby obtaining the nucleic acid or protein detection signal of the virus to be tested in the sample.

7. The non-lethal live sampling method for crustaceans according to claim 1, characterized in that, The method can perform at least two tracking and sampling monitoring operations on the same crustacean: (1) Sample each pair of appendages separately; and / or (2) Sample the appendages from different pairs separately; and / or (3) After a time interval of ≥7 days, and after the crustacean has molted and regrown the amputated appendage, the appendage is sampled again.

8. A non-lethal live sampling tool kit for implementing the method according to any one of claims 1-7, characterized in that, It comprises at least one of the following components: a sterilized cutting tool, a waterproof adhesive sealing medium and its application device, a container for preserving foot samples, and a foot sample extraction tool.

9. The application of the method of any one of claims 1-7 or the tool kit of claim 8 in the preparation of crustacean virus detection products, wherein the detection products are used in at least one of the following scenarios: parental screening, seedling quarantine, specific pathogen-free population breeding, longitudinal epidemiological surveillance, subclinical infection detection, or port quarantine.

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