Hardbone fish marking method based on multi-element composite marker

Abstract

The invention relates to the technical field of fish marking and tracking, and discloses a scleroderma fish marking method based on multi-element composite marking. The method specifically comprises the following steps: marking the sclerobone fish for two or more times by adopting a plurality of marking elements, and introducing the marking elements into the sclerobone fish body, so as to form a composite element fingerprint spectrum formed by a plurality of marking peaks in the otolith of the sclerobone fish. And then, scanning along the growth axis of the otolith through electron probe microscopic analysis (EPMA), analyzing the position, intensity and width characteristics of a mark peak, and realizing high-resolution identification and tracking of an individual or a group. According to the method, the stable and recognizable compound element fingerprints can be formed in the otolith by reasonably designing element types, concentration gradients and processing time, and the method is suitable for multiple scenes such as fish population dynamic monitoring, fishery management and species protection.

Description

Technical Field

[0001] This invention relates to fish tagging and tracking technology, and more particularly to a tagging method for bony fish based on multi-element composite tagging. Background Technology

[0002] Fish tagging and tracking are of great significance in aquaculture, ecological research, resource management, and species conservation. To achieve accurate identification of individual fish or fish populations, existing tagging methods must balance success rate, distinguishability, cost, and ease of operation. However, traditional tagging techniques often focus on external visibility or survival rate, making it difficult to generate unique identifiers for individuals or populations. Furthermore, their identification accuracy is limited in complex environments, thus reducing their effectiveness in aquaculture management, ecological monitoring, and conservation applications.

[0003] In recent years, otolith labeling technology has provided a new approach to labeling technology by forming characteristic fingerprints through the biomineralization of elements in water. However, existing multi-element composite labeling technologies still have significant shortcomings: (1) Limited information capacity of the label: it is mostly concentrated on a single element (such as strontium) and does not fully utilize the synergistic potential of multiple elements; (2) The formation pattern of markers is unclear: There is a lack of systematic research on the deposition patterns of multi-element combinations under different methods (immersion, feeding), concentrations and time conditions; (3) Insufficient recognition accuracy: The dynamic control and standardization methods of composite markers are not yet perfect, making it difficult to form a map with high discrimination and strong traceability.

[0004] In addition, existing research focuses on the "single-element single-labeling" method, which usually achieves a single-peak signal in otoliths by adjusting the concentration of a single element, but lacks applied research on multi-element joint labeling or multiple time series labeling, resulting in limited otolith labeling information and low traceability.

[0005] Therefore, there is an urgent need for an otolith labeling method that can achieve multi-element collaborative labeling and time-layered deposition, so as to improve information capacity and recognition accuracy while maintaining ease of operation and controllable cost, and provide a more reliable technical means for fish farming, ecological research and resource management. Summary of the Invention

[0006] To address the problems of existing otolith tagging methods, which are mostly single-element single-time tagging, have limited information capacity, low tag map discrimination, and limited application scenarios, this invention provides a tagging and tracking method for bony fish based on multi-element composite tagging.

[0007] This invention achieves synergistic and layered deposition of multiple elements in otoliths by introducing various chemical elements and performing multiple labeling processes at different time stages, thereby obtaining a composite labeling spectrum with stable structure, controllable signal, and high discrimination. The technical solution of this invention is as follows: Two or more marker elements were used to mark the otoliths of bony fish two or more times, and the marker elements were introduced onto the otoliths of bony fish.

[0008] Preferably, the marking element is any two or three of strontium, barium, and zinc.

[0009] Preferably, when zinc is used as a marker element, it is labeled by feeding, and the concentration of zinc ions in the feed is 5000 mg / kg to 6500 mg / kg; when strontium or barium is used as a marker element, it is labeled by immersion, and the concentration of strontium ions in the water is 2.0 mg / L to 210 mg / L, and the concentration of barium ions is 100 μg / L to 160 μg / L.

[0010] Preferably, the amount of feed is 1% to 2.5% of the fish's body weight.

[0011] Preferably, the time interval between each marking is 20 to 90 days. During the marking interval or the recovery and feeding phase, clean water without the addition of strontium and barium is used for cultivation, and feed with a zinc content of less than 50 mg / kg (i.e. no additional zinc is added) is given.

[0012] The application of the multi-element composite marker-based marker method for marking bony fish described in this invention in determining the origin of fish or the batch to which the fish belong.

[0013] Beneficial effects of the present invention (1) Improvement of the labeling method: A dual-channel independent labeling system based on the difference in element absorption was constructed. This invention addresses the differences in the solubility characteristics of different labeling elements in water and the absorption mechanisms of fish, and constructs a dual-channel introduction scheme that does not interfere with each other. Taking advantage of the high solubility of Sr and Ba in water and their rapid absorption through the gills, they are allocated to the water soaking channel to establish a high-intensity time anchor point. Taking advantage of the good stability of Zn in feed and its easy accumulation through the intestines, it is allocated to the feed feeding channel to establish a stable identity imprint. This "separate path" strategy not only effectively avoids the technical defects of poor solubility stability of Zn in water labeling and easy damage to fish gills, but more importantly, it realizes the physical diversion of labeling elements by utilizing different physiological absorption pathways of fish. This independent absorption mechanism allows the elements introduced by the dual channels to operate in parallel and without interference during physiological metabolism and transport, thereby fundamentally avoiding the absorption competition that may occur under a single pathway and ensuring the integrity and independence of the signal when multiple elements are compositely labeled.

[0014] (2) Improved spectral structure: This invention achieves "spatiotemporal physical isolation" and high-resolution recognition of labeled signals. Addressing the issue that traditional continuous labeling easily leads to signal tailing and overlap, thus affecting recognition accuracy, this invention introduces a standardized "metabolic cleaning" mechanism. A specific cleaning interval (e.g., 20-30 days) is set between different labeling operations. The fish's own metabolic function removes residual labeled elements from the blood, artificially constructing a pure, low-background deposition band on the otolith structure. This interval plays a "signal isolation" role at the microscale, spatially decoupling adjacent labeled events and transforming the originally continuous biological deposition process into a discrete spectral structure of "signal unit—background isolation band—signal unit". Regardless of whether adjacent signals are short-term or long-term labeled, this physical isolation effectively eliminates crosstalk and ambiguity at signal boundaries, greatly reducing background noise interference with feature extraction and significantly improving the signal-to-noise ratio and recognition accuracy of complex multidimensional coding.

[0015] (3) Dimensional expansion of coding capacity: A multi-dimensional coding system of "element composition + temporal position + signal width" was constructed. This invention breaks through the limitation of traditional markers that can only identify "presence / absence". By utilizing the differences in physical characteristics at the microscale, a high-capacity information coding system was constructed. This invention constructs a three-dimensional coding strategy, namely, synchronously controlling the element type of the marker (distinguishing signal source), temporal position (distinguishing sequence), and signal width (distinguishing duration). These three dimensions are independent of each other and organically combined, together constituting the composite fingerprint feature of this invention. This multi-dimensional combination strategy is similar to writing a complex "barcode" on the otolith microstructure. By arranging and combining different signal widths, and with different element types and time intervals, the amount of information that a single fish can carry increases exponentially without increasing the cost of additional elements. This not only greatly improves the coding capacity of the marker system, but also more effectively meets the needs of refined tracing of multiple batches, multiple sizes, and different sources of released groups in the same water area. Attached Figure Description

[0016] Figure 1 This is the composite element fingerprint spectrum formed in the otoliths of fish fry in the control group of Example 1.

[0017] Figure 2 This is the composite element fingerprint spectrum formed in the otoliths of fish fry in treatment group A in Example 1.

[0018] Figure 3 This is a composite element fingerprint spectrum formed in the otoliths of fish fry in treatment group B of Example 1.

[0019] Figure 4 This is the composite element fingerprint spectrum formed in the otoliths of fish fry by treatment group C in Example 1.

[0020] Figure 5 The labeling strategy provided in Example 2 is used to form a composite element fingerprint spectrum in the otoliths of fish fry.

[0021] Figure 6 The labeling strategy provided in Example 3 is used to form a composite element fingerprint spectrum in the otoliths of fish fry.

[0022] Figure 7 The composite element fingerprint spectrum formed in the otoliths of fish fry using the labeling strategy 1 provided in Example 4.

[0023] Figure 8 The composite element fingerprint spectrum formed in the otoliths of fish fry using the labeling strategy 2 provided in Example 4. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] All experimental subjects used in all embodiments were juvenile round-mouthed copper fish ( Coreius guichenoti The average body length is 6.0±0.5cm, and the animals are in good health.

[0026] Acclimation treatment: Before the experiment, all juvenile fish were temporarily held in the laboratory recirculating aquatic system for 7 days and fed with basic commercial feed (Zn content <50mg / kg). Feeding was stopped 24 hours before the experiment.

[0027] Water quality conditions: The experimental water was fully aerated for 2 days, and the natural background values ​​of Sr, Ba, and Zn in the water were approximately 0.1 mg / L, 40 μg / L, and 20 μg / L, respectively. During the experiment, dissolved oxygen was ≥6.0 mg / L, water temperature was controlled at 20±1℃, and pH was 7.15±0.1.

[0028] Reagent Notes: Unless otherwise specified, the concentrations mentioned in this embodiment refer to the ion concentrations of the target elements. Strontium chloride hexahydrate (SrCl2·6H2O), barium chloride dihydrate (BaCl2·2H2O), and zinc sulfate heptahydrate (ZnSO4·7H2O) were used as labeling sources.

[0029] Example 1 Concentration threshold screening and biosafety verification of Sr-Ba composite immersion labeling Experimental objective: To establish the range of "minimum identification concentration" and "maximum safe concentration" for effective labeling of Sr and Ba elements, and to define the optimal safe labeling window.

[0030] Experimental Procedure: Four immersion treatment groups with different concentration gradients and one control group were set up, with a treatment time of 48 hours for each group, during which no feeding was performed. First, Group A (environmental disturbance simulation group) was established, with the Sr concentration in the water adjusted to 0.5 mg / L (slightly higher than the background value) and the Ba concentration to 50 μg / L, aiming to simulate the natural high background fluctuations that may occur in outdoor water bodies and verify whether low concentration addition would be misjudged. Group B (low concentration effective threshold group) was established, with the Sr concentration in the water adjusted to 2.0 mg / L (approximately 20 times the background value) and the Ba concentration to 100 μg / L, to explore the minimum chemical addition amount for forming a recognizable marker. Group C (high concentration verification group) was established, with the Sr concentration in the water adjusted to 210 mg / L and the Ba concentration to 160 μg / L, to verify the marker clarity and biosafety at extremely high concentrations. Finally, Group D (lethal verification group) was established, with the Sr concentration in the water set to 200 mg / L and the Ba concentration increased to 800 μg / L, to determine the biological lethal boundary of the composite labeling solution. The control group was kept in the basic aquaculture water without the addition of any exogenous marker elements to establish biological survival and chemical background baselines. All groups were transferred to the basic water for a 20-day recovery period after soaking to observe long-term effects.

[0031] Experimental results: First, refer to the test results of the control group ( Figure 1 In aquaculture waters without added exogenous marker elements, the Sr / Ca ratio (×10³) and Ba / Ca ratio (×10³) of otoliths from *Cyprinus circinus* were significantly different. 5 All of them exhibited natural background fluctuations. This result establishes the natural background baseline of otolith elements, and any effective artificially labeled signal must be significantly higher than this background fluctuation range.

[0032] For the environmental disturbance simulation group (Group A, Sr ion concentration in water 0.5 mg / L, Ba ion concentration 50 μg / L), the detection results showed that ( Figure 2 Although the Sr / Ca ratio and Ba / Ca ratio showed slight fluctuations, they were not significantly different from the background values. This indicates that at extremely low concentrations, exogenous elements cannot overcome the interference of the environmental background, which can easily lead to misjudgment or missed judgment of the labeled signal. Therefore, this concentration ratio was determined to be an invalid labeled concentration.

[0033] For the low concentration effective threshold group (Group B, Sr ion concentration in water 2.0 mg / L, Ba ion concentration 100 μg / L), the detection results showed ( Figure 3 The otolith Sr / Ca ratio showed a clear and independent peak, demonstrating distinct identifiability; although the Ba / Ca ratio peak was slightly above the upper limit of background noise, the signal strength was sufficient for effective identification. These results confirm that this concentration ratio represents the minimum effective threshold for forming identifiable markers.

[0034] For the high-concentration validation group (Group C, Sr ion concentration of 210 mg / L and Ba ion concentration of 160 μg / L in water), the detection results showed excellent labeling effect. Figure 4 The Sr / Ca ratio exhibited an extremely high signal-to-noise ratio; the Ba / Ca ratio waveform was stable and clearly distinguishable from background noise. Furthermore, the fish in this experimental group survived well, demonstrating that no acute lethal effect occurred under conditions where Sr concentrations were as high as 210 mg / L.

[0035] In contrast, the lethality verification group (Group D, with a water concentration of 200 mg / L Sr ions and 800 μg / L Ba ions) had a cumulative mortality rate of over 90% within 48 hours, indicating that a Ba concentration of 800 μg / L reached the biological lethal boundary.

[0036] Based on the results of the above groups, this invention has determined the optimal safe and effective window for Sr-Ba composite labeling: the effective labeling concentration of Sr should be higher than 2.0 mg / L, and can be safely increased to above 210 mg / L to enhance signal intensity; the effective labeling concentration of Ba should be higher than 100 μg / L, but must be strictly controlled below 800 μg / L to balance labeling clarity and biosafety.

[0037] Example 2 Dual-channel independent labeling verification of Sr and Zn Experimental objective: To verify the solution to the problem of difficult Zn water labeling using a dual-channel mode of "Sr soaking in water + Zn feeding with feed" and to confirm that the two do not interfere with each other.

[0038] Experimental procedure: The procedure of "long-term feeding of Zn + short-term soaking of Sr" was adopted.

[0039] Zn feeding procedure: Prepare a labeled feed containing 6000 mg / kg of Zn.

[0040] Ten juvenile fish were selected and fed continuously for 14 days in the basic water body at a daily feed amount of 2% of their body weight. On the 7th day of the feeding cycle, the experimental fish were removed from the rearing tank and transferred to labeled water containing 30 mg / L of Sr ions for 24 hours of immersion treatment. Feeding was suspended during the immersion period. After the immersion, the fish were returned to their original rearing tank and fed Zn-containing feed until the 14th day. After the labeling was completed, they were transferred back to the basic water body for a 20-day recovery period.

[0041] Experimental results: such as Figure 5As shown, Zn exhibits a significant high content during otolith labeling, effectively overcoming the difficulty of Zn deposition in single-body water labeling. Simultaneously, Sr presents an extremely high and sharp peak. The clear coexistence of both elements in the spectrum, without mutual obscuring, conclusively demonstrates that this dual-channel mode can achieve non-interference between the two labeled elements, perfectly fulfilling the experimental expectations for constructing composite labels.

[0042] Example 3 Time-sharing isolation marker verification of Ba and Zn Experimental objective: To verify the construction of a clear physical isolation map by using trace amounts of Ba and feeding Zn, combined with the "cleaning interval" design described in this invention.

[0043] Experimental procedure: First, the experimental juvenile fish were transferred to a labeled water containing 160 μg / L of Ba ions and soaked for 48 hours. After labeling, the fish were transferred back to the basic water and fed ordinary feed to resume breeding for 20 days. This cleaning interval allowed the fish to metabolize the residual Ba in their blood, thus forming a low-background physical isolation zone on the otoliths. Subsequently, the fish were fed labeled feed containing 6000 mg / kg of Zn ions for 10 consecutive days. After labeling, the fish were transferred back to the basic water to resume breeding for another 20 days.

[0044] Experimental results: The results show that the sharp characteristic peak formed by Ba and the high-content broad plateau formed by Zn are completely separated by a significant low background region, presenting a clear "front peak - middle valley - back plateau" time sequence structure. Figure 6 This phenomenon definitively proves that introducing a recovery period between two markings allows the fish to effectively metabolize and clear the residue of the previous marking, thereby forming a pure physical isolation zone on the otoliths. This not only fundamentally eliminates the signal overlap and interference that may occur between different marking elements, but also achieves precise physical spatial separation of the marking signals at the microscopic scale, perfectly verifying the effectiveness of the time-sharing isolation strategy.

[0045] Example 4 Verification based on "immersion duration modulation" multidimensional width coding Experimental objective: To verify whether the "physical width" of characteristic peaks on otoliths can be modulated by changing the duration of immersion labeling (24h vs 168h), thereby establishing the feasibility of "width" as a third-dimensional encoding information.

[0046] Experimental Procedure: Two full-process treatment groups were set up, both following the labeling procedure of "Sr / Ba immersion—30-day interval—Zn feeding". Only the immersion time was changed to verify the width coding. For the short code group, the experimental juvenile fish were transferred to labeled water with Sr ion concentration of 30 mg / L and Ba ion concentration of 160 μg / L and immersed for 24 hours (labeling strategy 1). After the immersion, the fish were transferred back to the basic water and fed ordinary feed for 30 days to recover. Then, they were fed labeled feed containing Zn ion concentration of 6000 mg / kg for 10 consecutive days. For the long code group, the labeling concentration in the water was kept unchanged, and the immersion time was extended to 168 hours (7 days, labeling strategy 2). After the labeling, the fish were transferred back to the basic water and recovered for 30 days. The same Zn feed feeding operation was then performed to construct composite labeled samples with different time-series widths.

[0047] Experimental results: First, the detection spectrum of short code groups ( Figure 7 The results show that the Sr / Ca and Ba / Ca ratios exhibit a typical "narrow and sharp characteristic peak," occupying an extremely narrow physical width on the horizontal axis, indicating a transient high-intensity signal injection. Subsequently, the Zn signal exhibits a wider band, and the results clearly demonstrate the signal combination pattern of "narrow Sr / Ba peak—time interval—wideband Zn."

[0048] Secondly, the detection spectrum of long code groups ( Figure 8 The results show that the characteristic peak shape of the Sr / Ca and Ba / Ca ratios has changed significantly, from a "narrow and sharp characteristic peak" of short code groups to a wide band. At the same time, the Zn / Ca marker band on the right side of the spectrum still appears stably and maintains a clear physical distance from the Sr / Ba marker band.

[0049] This result strongly confirms the feasibility of using the "physical width" of the marker peak as encoding information, thus providing a solid experimental basis for constructing a multidimensional composite marker coding system that includes "element type, signal strength, and signal width".

[0050] Comprehensive analysis suggests that by regulating the exposure concentrations of Sr and Ba and the timing of Zn intake, a structurally stable, multi-element composite marker system with distinct signal stratification can be formed in otoliths. This method achieves synergistic utilization of water absorption and feeding absorption pathways, constructing a stable marker structure with temporal sequence characteristics and elemental hierarchical differences, suitable for individual fish identification and fish stocking tracking studies.

[0051] It should be noted that the peak position, peak intensity, and peak width of each marker peak in otoliths exhibit some variability among different individuals. This variability mainly stems from two aspects: natural physiological factors such as the individual's own growth rate, otolith deposition rate, and differences in physiological metabolism; and external factors such as the temporary holding environment, water quality conditions, and treatment methods. The differences in peak intensity formed by different marker elements in otoliths may also be related to the element's absorption efficiency, deposition rate, and metabolic characteristics.

[0052] It is worth emphasizing that the differences between these individuals and elements are controllable in the design of this method. They can be adjusted by reasonably selecting element combinations, concentration gradients, processing durations and intervals, thereby ensuring that the resulting composite element fingerprints are stable and identifiable.

[0053] In summary, this invention, by rationally setting the types of marker elements, concentration gradients, and time series, can form a structurally stable composite element fingerprint spectrum with distinct signal stratification in otoliths. This method can provide a high-resolution identification tool for individual fish identification, group tracking, and fish stocking management, and has broad application prospects.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit its protection scope; equivalent modifications and improvements made by those skilled in the art without departing from the concept of the present invention should all fall within the protection scope of this application.

Claims

1. A method for marking and tracking a hard bone fish based on a multi-element composite marker, characterized in that: Introducing the marking elements into the otoliths of the bony fish by marking the bony fish twice or more times with two or more marking elements.

2. A method for marking hard bone fishes based on multi-element composite markers according to claim 1, characterized in that: The marking elements are any two or three of strontium, barium and zinc.

3. A method of marking a hardy fish based on a multi-element composite marker according to claim 2, characterized in that: When zinc is used as the marking element, the marking is performed by feeding, and the concentration of zinc ions in the feed is 5000 mg / kg to 6500 mg / kg; when strontium or barium is used as the marking element, the marking is performed by soaking, and the concentration of strontium ions in the water is 2.0 mg / L to 210 mg / L, and the concentration of barium ions is 100 μg / L to 160 μg.

4. The method according to claim 3, wherein: The feeding amount of the feed is 1% to 2.5% of the weight of the fish.

5. The method according to any one of claims 1 to 4, wherein the method is based on multi-element composite markers. The time interval between each marking is 20 to 90 days, and during the interval, the bony fish is cultured in clean water without additional strontium and barium, and is fed with feed with a zinc content of less than 50 mg / kg.

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