A method for accurately locating spawning grounds of schizothorax

Abstract

The present invention provides a method for accurately locating the spawning grounds of schizothoracine fish, comprising: step 1, determining the study area; step 2, determining the sexual maturity stage based on the egg diameter and the stage of gonad development, and then determining whether spawning has occurred based on whether the eggs are emptied and whether the parent fish's anal fin is broken, thereby determining the spawning time; step 3, based on the study area determined in step 1, identifying the river section of the spawning ground of the genus Gymnosoma schizothoracine fish during the spawning period of the genus Gymnosoma schizothoracine fish determined in step 2; step 4, based on the spawning ground section determined in step 3, accurately locating the spawning ground: determining the specific time of spawning within the spawning period obtained in step 2; after determining the specific spawning time, accurately locating the spawning ground of the genus Gymnosoma schizothoracine fish based on the bottom soil differences of the river section of the spawning ground of the genus Gymnosoma schizothoracine fish. The present invention accurately locates the spawning grounds of schizothoracine fish, fills a gap, and provides scientific support for the protection of fish habitats in the source river of the Yangtze River.

Description

Technical Field

[0001] The present invention relates to the field of schizothorax spawning ground positioning, and in particular to a method for accurately positioning a schizothorax spawning ground. Background Art

[0002] Fish spawning grounds are places where fish lay eggs, hatch sticky fertilized eggs, and where newborn fry of some species complete their initial growth. They are crucial for maintaining the continuation of fish populations and fish diversity, and are an important part of fish ecology research.

[0003] The Qinghai-Tibet Plateau, known as Asia's Water Tower and the source of many major rivers, is also a sensitive region to climate change, which could adversely impact the quality of schizothorax spawning grounds. Due to the unique plateau environment, the fish population of the Qinghai-Tibet Plateau is unique, with schizothorax predominating among the cyprinid fish. These specialized schizothorax species that inhabit the headwater rivers of the Plateau are globally endemic and a vital biological resource. China accounts for over 80% of the world's schizothorax species, comprising 70 recognized species and nine subspecies, belonging to 11 recognized genera. Currently, research on the location of schizothorax spawning grounds is limited, with only three species of natural spawning grounds discovered. Schizothorax typically spawn in rivers, laying sinking eggs. They typically dig nests during spawning, but burying eggs in gravel after spawning has not been reported. The Qinghai Lake naked carp of the genus Gymnocypris digs a pit to spawn, but does not bury the eggs. Researcher Chen Yifeng's team found that the Selin Co naked carp spawns in its spawning nest, with fertilized eggs visible to the naked eye, but no burying of the eggs was observed. The Sichuan schizothorax of the genus Schizothorax can be seen spawning in sand pits 30-50 cm in diameter, but does not bury the eggs. Researcher Yang Deguo's team found that male Schizothorax breviscarus buries exposed eggs in depressions with sand in their mouths. For exposed eggs on flat surfaces, they use body movements to guide water flow, flushing the eggs downstream into crevices in the gravel. Burying eggs with sand is unlikely to achieve a true burial; subtle hydraulic forces can expose them, and fertilized eggs primarily develop in rock crevices. The spawning grounds of these four schizothorax species can be located by directly visually detecting fertilized eggs. Currently, the lack of basic research on spawning grounds has severely hindered in-depth study of the hydrological requirements of spawning grounds and the impacts of climate change. Research on the habitat location of schizothorax, a Yangtze River source fish, is particularly lacking. The Yangtze River Protection Law explicitly mandates fish habitat surveys in key areas, and the Qinghai-Tibet Plateau Protection Law also explicitly mandates river water ecological protection. Locating schizothorax spawning grounds is a crucial foundation for studying the ecohydrological requirements of major fish spawning events on the Qinghai-Tibet Plateau and their responses to climate change. Therefore, conducting schizothorax spawning ground location research on the Qinghai-Tibet Plateau is urgently needed.

[0004] The genus Schizothorax contains nine species of schizothoracines. With the exception of the plateau schizothoracine, the remaining eight species (soft-spined schizothoracine, Lhasa schizothoracine, hot schizothoracine, Jialing schizothoracine, Dadu schizothoracine, Yellow River schizothoracine, anterior-belly schizothoracine, and small-headed schizothoracine) are endemic to my country. Among them, the small-headed schizothoracine, also known as the small-headed plateau schizothoracine, is the most highly evolved species of schizothoracine and has the highest altitude distribution among the cyprinids. Its distribution is narrow, confined to high-altitude rivers and lakes north and south of the Tanggula Mountains. It is widely distributed in the Yangtze River source region, possessing extremely high scientific and ecological value. Among existing methods for locating fish spawning grounds, patent publication number CN108932377B describes a method and system for accurately locating fish spawning grounds. This method targets drifting eggs and fry, using a hydrodynamic model to analyze the turbulent flow fields required for spawning drifting eggs. However, it is not suitable for schizothoracic fish that lay sinking eggs. Patent publication number CN113111956B describes a method for accurately locating fish spawning grounds. This method uses a hydrodynamic analysis grammar to determine the suitability of spawning ground hydrological factors, and is also targeted at fish that lay drifting eggs. Patent publication number CN115413608B describes a system and method for locating spawning grounds for fish that lay drifting eggs. This system involves identifying spawning grounds for drifting eggs, incorporating artificial digital roe technology based on digital simulation. All three methods are applicable to fish that lay drifting eggs, and are indirect methods, not direct methods for identifying the locations where broodstock mate or fertilized eggs first appear. Furthermore, locating the spawning grounds of schizothorax cannot be done by simply visually spotting fertilized eggs; new methods must be developed. Given the exceptional scientific and ecological value of schizothorax, achieving precise location of spawning grounds is crucial for protecting the Qinghai-Tibet Plateau, advancing the ecology of schizothorax on the Qinghai-Tibet Plateau, and addressing the requirements of the Yangtze River Protection Law and the Qinghai-Tibet Plateau Protection Law. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a precise positioning method for the spawning grounds of schizothorax fish, which can accurately locate the spawning grounds of naked schizothorax, which is beneficial to the protection of the spawning grounds of this type of fish and the subsequent research on the ecological and hydrological needs of the spawning grounds, and can also provide a reference for the positioning of the spawning grounds of other schizothorax fish.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for accurately locating a spawning ground of a schizothorax fish comprises the following steps:

[0008] Step 1: Determine the research area

[0009] The study area was a 10-km river section, 2.5 km upstream and 7.5 km downstream of the wintering grounds of the genus Schizophora.

[0010] Step 2: Determine the spawning period of the genus Schizontidae

[0011] The gonad development cycle of Gymnosoma spp. was determined by dissecting the fish in the wintering grounds. The natural reproduction time was determined by observing the presence of eggs in the abdominal cavity of mature female Gymnosoma spp. and whether the anal fin was worn. The time when male and female fish left the wintering grounds and were both sexually mature but not spawning was the pre-spawning time T0. The time when female fish were found to have spawned was T1. The spawning period was T S Located between T0 and T1, sexually mature female fish have a secondary sexual characteristic, the bead star. The sexually mature female fish and the time of sexual maturity can be identified by the bead star on the anal fin;

[0012] Step 3: Based on the research area determined in step 1, identify the river section where the spawning ground of the genus Schizonotrichia is located during the spawning period of the genus Schizonotrichia determined in step 2. Specifically, the spawning river section is determined based on two events: parent fish chasing each other and finding newborn fry.

[0013] Step 4. Based on the spawning grounds determined in step 3, the spawning grounds are precisely located within the river section. The specific time of spawning is determined within the spawning period obtained in step 2. After the specific spawning time is determined, the spawning grounds of the genus Nasturtium are precisely located based on the bottom differences of the river section where the spawning grounds of the genus Nasturtium are located.

[0014] Furthermore, in step 2, it was determined that the sexual maturity period of the genus Gymnosoma is between June 1 and June 15.

[0015] Furthermore, the step three specifically includes: the method for determining the river section of the spawning ground adopts the broodstock method and the early resource method. The broodstock method is to simultaneously discover V-stage broodstock of the genus Nasciurus that are mature but have not yet spawned, and the number of broodstock in the habitat reaches a certain scale and the broodstock are chasing each other in this area, and the egg-carrying condition of the broodstock is checked by squeezing the eggs and observing whether the anal fin is broken, and monitoring whether the broodstock have spawned; the early resource method is to collect possible fish eggs and newborn fry through a handheld D-type net and a sinking egg collection net, and determine the section location. The spawning ground is located in the upstream area near the section where the early resources are collected.

[0016] Furthermore, the step four specifically includes: after determining that breeding behavior has occurred, immediately searching for the spawning ground in the spawning river section, the search range includes the parent fish activity area and the non-parent fish activity area, if many clean blue-black pebble patches appear in the searched spawning river section, and the surface substrate of the blue-black patches is composed of pebbles and gravels of varying sizes, with coarse and fine substrates distributed, but mostly fine gravels, then the area discovered at this time is determined to be the spawning ground.

[0017] Furthermore, the time for locating the spawning ground in step 4 is 1-7 days after the occurrence of the breeding behavior is determined.

[0018] Furthermore, it also includes step five, identification of the species of fertilized eggs: after determining the spawning ground, the bottom sediment is removed to collect the fertilized eggs, which are brought back to the laboratory for incubation. After hatching, the morphology of the fry and molecular biology methods are used to identify whether they are fish of the genus Gymnosoma.

[0019] Furthermore, the artificial incubation method for the fertilized eggs in step 5 is indoor basin incubation, with a daily rhythmic water temperature of 8-22°C. The incubation water source is well water with a natural water temperature of 8-9.5°C. The incubation water temperature is achieved by mixing with hot water, and the room temperature is 13-14°C. During incubation, the dissolved oxygen is appropriate, the eggs are incubated in the dark or low light, and 2 / 3 of the water is changed daily.

[0020] Furthermore, in step 1, the upstream 2.5 km section represents a single-beam river type, which is shallow and narrow, with a river width of 20-50 m; the downstream 7.5 km section represents a braided river system, which is a multi-branched river with diverse habitats and a river width of 600-1000 m.

[0021] The positive effects of the present invention are:

[0022] (1) The spawning grounds of the schizothorax were accurately located, filling a gap in the knowledge and providing scientific support for the protection of the fish habitat in the Yangtze River source. It can also provide a reference for the accurate location of the spawning grounds of other fish of the same genus, the schizothorax.

[0023] (2) By using the characteristics and patterns of surface sediment color and particle size changes, we accurately identified the high-quality spawning nests and spawning grounds of the genus Nasciurus, thereby improving the accuracy of spawning ground identification; we innovated the spawning nest sediment evolution method, thus avoiding the drawbacks of the indirect method and direct egg-finding method that cannot locate the spawning grounds of the genus Nasciurus.

[0024] (3) The spawning nest bottom evolution method is effective and can be carried out on the river bank without wading, thus avoiding the potential damage to fertilized eggs caused by large-scale wading operations to search for eggs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the color and particle size changes of the surface substrate before spawning (A) and after spawning (B) in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0027] Dangqu, the southern source of the Yangtze River, is located in the heart of the Qinghai-Tibet Plateau. Its fish diversity is an important part of the fish diversity of the entire Yangtze River. Fish of the genus Schizophrenia are key fish in the southern source of the Yangtze River and are also indicator species of whether the aquatic ecosystem is healthy. The fish lays eggs in the river, the spawning substrate is gravel, and the spawning ground has the phenomenon of buried eggs and hatching. Based on the above biological and behavioral characteristics, the goal of the present invention is to determine the sexual maturity period according to the size of the egg diameter and the stages of gonadal development, and then determine whether spawning behavior has occurred based on whether the eggs are emptied and whether the anal fin of the parent fish is broken, so as to determine the spawning time. The time period between the maturity time and the spawning time is the spawning period. The spawning river section is determined by two events: the parent fish chasing each other and the discovery of newborn fry. Once spawning is determined during the spawning period, a precise positioning study of the spawning ground is immediately carried out in the spawning river section. Visual observation of the changes in the substrate color and particle size composition of the spawning nest is the key to the precise positioning method.

[0028] The spawning nest substrate evolution method is based on the unique biological behavior of broodstock of the genus Gymnosoma, which buries and incubates their eggs. This behavior protects the fertilized eggs of their offspring from UV damage on the Qinghai-Tibet Plateau. This is key to identifying the microhabitat of a single spawning nest (10-20 cm in diameter). The digging of gravel to form the nest before spawning and the burying of the eggs in gravel after spawning result in two stages of agitation of the gravel substrate. This removes impurities such as silt from the spawning area, making its color distinct from that of non-spawning areas. This is the core principle for accurately locating spawning sites. Following minor floods in March and April each year, the riverbed is mostly covered in a yellowish-brown gravel substrate. The substrate is uniformly sized, consisting of large-grained gravels, with a maximum diameter of 4-5 cm. Because naked schizont fish dig pebbles to form spawning nests for laying eggs, and then use the excavated pebbles to push and bury the eggs, the mud and sand covering the surface of the pebbles that have been stirred twice will be cleaned up, forming a color contrast between the spawning nest and other unused substrates. Therefore, many clean blue-black pebble patches appear in the spawning grounds, while the non-spawning areas still have a yellowish-brown substrate. The surface substrate composition of the blue-black patches is also different from the original yellowish-brown substrate composition. The surface substrate composition of the blue-black patches is composed of pebbles and gravels of varying sizes, with coarse and fine substrates distributed, but fine gravels are the majority. It is found that the areas with abnormal habitat color and benthic particle size are spawning grounds ( Figure 1 Because the spawning and pre-hatching period of naked-tailed fish occurs during the dry season before flooding, the spawning grounds are shallow and clear, and the spawning nests are visible to the naked eye, so spawning grounds can be identified visually.

[0029] Furthermore, the optimal time to pinpoint spawning grounds is 1-7 days after spawning, before flooding occurs. During this time, fertilized eggs have not yet hatched and are present in the nest. Otherwise, once the fertilized eggs hatch, the spawning grounds will no longer contain fertilized eggs, lacking direct evidence of their location. Overall, determining the spawning period, identifying the spawning river section, and visually observing changes in habitat substrate color and surface particle size are crucial steps in accurately locating spawning grounds for Gnatia fish on the Qinghai-Tibet Plateau. Previous studies have lacked precise methods for pinpointing spawning grounds for Gnatia fish.

[0030] The object of the present invention is to provide a method for accurately locating the spawning ground of schizothoracine fish, by determining the spawning period, determining the location time. The spawning river section is determined by the presence of parent fish or the location of fry. After the time and space are determined, the spawning nest and the fertilized eggs inside the spawning nest are determined by a key spawning nest identification method, and the fertilized eggs are finally hatched and the type of fry is determined to be a schizothoracine fish. The specific embodiment of the spawning ground accurate location of schizothoracine fish in the Yangtze River source is described in detail below.

[0031] Example 1

[0032] Precise positioning of a 300㎡ spawning ground

[0033] (1) Determine the research area

[0034] The study area was the wintering grounds of a population of schizontids (Schizontidae) found in the southern headwaters of the Yangtze River and a 7.5 km section downstream. This section represents a braided river system with multiple branching channels, diverse habitats, and a river width of 600-1000 m.

[0035] (2) Determine the spawning period

[0036] Based on the research area determined in step (1), the gonadal development cycle of the small-headed naked schizont was observed by dissecting the small-headed naked schizont, observing whether spawning occurred, observing whether the anal fin was worn, and whether the fry were collected, so as to determine the natural reproduction time. The time when male and female fish leave the wintering grounds and are sexually mature but have not spawned is the pre-spawning time T0, the time when female fish are found to have spawned is T1, and the spawning period T SLocated between T0 and T1. In terms of egg diameter, in April, the gonads were at stage IV development, with an egg diameter of 2.37±0.15mm. On May 13, the eggs were still at stage IV, with an egg diameter of 2.4±0.18mm. In early June, the gonads matured, with the maximum egg diameter of 2.4±0.1mm. On June 30, the gonads were found to be empty and spawning had occurred. On August 6, the eggs were found to be at stages II-III, with an egg diameter of 0.5mm. In November, the female fish's gonads developed again to stage IV, and the IV gonads lasted until May 2020; on May 31, the gonads were found to have entered stage V. In terms of secondary sexual characteristics, the body surface of sexually mature male and female fish is covered with pearl stars. The male fish has obvious pearl stars on each fin and the entire body surface; the female fish has relatively fewer pearl stars on the body, with the most obvious pearl star on the anal fin. The last two branched fin rays of the male fish have become spines, and the fin ray membrane is arc-shaped and concave. Females are significantly larger than males, reaching twice their total length and ten times their weight. However, there are also large males and females of equal size. During capture, females shed their eggs. Gentle pressure on the male's abdomen triggers the ejection of liquefied semen, followed by an open mouth and violent tail movements. By June 18th, 80% of mature females had ovarian emptying, confirming the spawning period as May 31st to June 18th.

[0037] (3) Identify the river sections where spawning sites are located

[0038] Based on the research area determined in step (1), the identification of the river sections where the spawning grounds are located was carried out during the spawning period of the genus Gnatia in step (2). The broodstock method was used to determine the approximate range of the spawning grounds. The areas where the breeding groups of the summer breeding populations are located and where they like and chase each other are the river sections where the spawning grounds are located. At the same time, it was found that 150 mature but unspawned stage V broodstock of Gnatia smallheaded, including females and males, were cruising in the 7.5 km upstream river section and had a high degree of loyalty to the deep water area. Females were significantly larger than males, and on average, five males chased one female, confirming that this river section was the spawning ground section.

[0039] (4) Accurate positioning of spawning grounds

[0040] Based on the spawning ground determined in step (3), the spawning ground is accurately located in the river section. The specific time of spawning is determined within the spawning period obtained in step (1). After determining the specific spawning time, based on the spawning nest bottom evolution method, the spawning ground of the genus Gymnosoma is accurately located according to the bottom differences of the spawning nests in the spawning ground of the small-headed Gymnosoma schizont. From June 1 to 18, 2021, the ovaries of female Gymnosoma schizont are monitored daily to see whether they are empty. No spawning occurred on June 17, and 30% of the female fish ovaries were empty on June 18. The anal fins of the female fish with empty ovaries were severely damaged, and it was determined that spawning occurred. Immediately search and locate the spawning ground in the spawning river section. The search range includes the parent fish activity area and the non-parent fish activity area. By finding the different points of bottom difference, the spawning ground is accurately located. 2.5 km downstream, a mixture of khaki and bluish-black riverbed was found, with bluish-black circular patches evenly distributed among the khaki riverbed. The bluish-black patches were distributed with coarse and fine sediments, with fine gravels being the majority. The coarse and fine distribution of these particles was significantly different from the large-grained gravels of 4-5 cm uniformly distributed in the surrounding khaki areas. The spawning area is 300 m 2 .

[0041] (5) Identification of fertilized egg species

[0042] After identifying the distinctively colored substrate, the bluish-black substrate was removed and the fertilized eggs were collected. The fertilization rate was calculated. The minimum fertilization rate, calculated as the percentage of white eggs removed, was 98%. The collected fertilized eggs were brought back to the laboratory for incubation. The fertilized eggs were artificially incubated indoors in a basin-type incubator, with a daily water temperature of 8-20°C. The incubation water source was well water, naturally at 8°C, which was maintained by hot water blending. The room temperature was 13°C, with an appropriate dissolved oxygen level. Incubation was conducted in the dark or in low light, with two-thirds of the water changed daily. Morphological identification after hatching revealed that the species lacked whiskers and had small spots on its back. Given that only the small-headed schizothorax species, Gymnosoma microcephala, reproduces naturally in Dangqu, while other schizothorax species, including juveniles of the family Cobitidae, possess three pairs of whiskers, the fertilized eggs were identified as Gymnosoma microcephala. Dead eggs were sorted daily during the incubation process until all larvae hatched. The hatch rate of the fertilized eggs was calculated to be 82%.

[0043] Area 300m 2 There are about 900 nests in the spawning ground, with a fertilization rate greater than 98% and a high hatching rate of 82%. This not only indicates that the spawning ground is of high quality, but also proves that our method can achieve the precise positioning of high-quality spawning grounds.

[0044] Example 2

[0045] Precise positioning of the 240㎡ spawning ground

[0046] (1) Determine the research area

[0047] The study area was the wintering grounds of a population of schizontids (Schizontidae) found in the southern source of the Yangtze River and a 2.5 km section upstream. This 2.5 km section represents a single-beam river, with a shallow and narrow channel and a width of 20-50 m.

[0048] (2) Determine the spawning period

[0049] Based on the research area determined in step (1), the natural reproduction time of the fish was determined by dissecting the fish in the wintering grounds to observe the gonadal development cycle, whether spawning occurred, whether the anal fin was worn, and whether the fry were collected. The time when male and female fish leave the wintering grounds and are sexually mature but have not spawned is the pre-spawning time T0, the time when female fish are found to have spawned is T1, and the spawning period is T S Located between T0 and T1. On June 2nd, female gonads were observed to have entered stage V. Regarding secondary sexual characteristics, sexually mature males and females are covered in asterisms. Males display distinct asterisms on all fins and throughout their bodies; females have relatively few asterisms, most prominent on the anal fin. The last two branched fin rays of males have transformed into spines, and the ray membranes are curved and concave. Females are significantly larger than males, reaching twice their total length and ten times their weight. However, there are also large males and females of equal size. During capture, females shed eggs, with slight pressure on the male's abdomen triggering the ejection of liquefied semen, followed by a mouth-opening movement and violent tail swaying. By June 14th, 50% of mature females were found to have ovarian emptying, confirming the spawning period as June 2nd to 14th.

[0050] (3) Identify the river sections where spawning sites are located

[0051] Based on the research area determined in step (1), the identification of the river sections where the spawning grounds are located was carried out during the spawning period of the genus Gnaphalidae determined in step (2). The broodstock method was used to determine the approximate range of the spawning grounds. The areas where the breeding groups of summer breeding groups breed and where they like and chase each other were the river sections where the spawning grounds were located. At the same time, it was found that 300 mature but unspawned stage V broodstock of Gnaphalidae, including females and males, were cruising in the 2.5 km upstream river section and had a high degree of loyalty to the deep water areas where they swam. Females were significantly larger than males, and on average, six males chased one female, confirming that this river section was the spawning ground section.

[0052] (4) Accurate positioning of spawning grounds

[0053] Based on the spawning ground determined in step (3), the spawning ground is accurately located in the river section. The specific time of spawning is determined within the spawning period obtained in step (1). After determining the specific spawning time, based on the spawning ground bottom evolution method, the spawning ground of the genus Gymnosoma is accurately located according to the bottom differences of the spawning grounds in the spawning ground of the small-headed Gymnosoma schizophora. From June 2 to 14, 2022, the ovaries of female Gymnosoma schizophora are monitored daily to see whether they are empty. No spawning occurred on June 13, and 50% of the female fish ovaries were empty on June 14. The anal fins of the female fish with empty ovaries were severely damaged, which confirmed that spawning occurred. Immediately search and locate the spawning ground in the spawning river section. The search range includes the parent fish activity area and the non-parent fish activity area. By finding the different points of bottom difference, the spawning ground is accurately located. 2.5 km upstream, a mixture of khaki and bluish-black riverbed was found, with bluish-black circular patches evenly distributed among the khaki riverbed. The bluish-black patches were distributed with coarse and fine sediments, with fine gravels being the majority. The coarse and fine distribution of these particles was significantly different from the large-grained gravels of 4-5 cm uniformly distributed in the surrounding khaki areas. The spawning area is 240 m 2 .

[0054] (5) Identification of fertilized egg species

[0055] After identifying the distinctively colored substrate, the bluish-black substrate was removed and the fertilized eggs were collected. The fertilization rate was calculated. The minimum fertilization rate, calculated as the percentage of white eggs removed, was 99%. The collected fertilized eggs were brought back to the laboratory for incubation. The fertilized eggs were artificially incubated indoors in a basin-type incubator, with a daily water temperature of 9.5-22°C. The incubation water source was well water with a natural temperature of 9.5°C, which was maintained by hot water blending. The room temperature was 14°C, with an appropriate dissolved oxygen level. Incubation was conducted in the dark or in low light, with two-thirds of the water changed daily. Morphological identification after hatching revealed that the species lacked whiskers and had small spots on its back. Given that only the small-headed schizothorax, Gymnosoma microcephala, reproduces naturally in Dangqu, while other schizothorax species, including juveniles of the family Cobitidae, possess three pairs of whiskers, the fertilized eggs were identified as juveniles of the small-headed Gymnosoma microcephala. Dead eggs were sorted daily during the incubation process until all larvae hatched. The calculated hatch rate for the fertilized eggs was 85%.

[0056] Area 240m 2 There are about 744 nests in the spawning ground, the fertilization rate in the nests is greater than 99%, and the hatching rate reaches a high value of 85%. On the one hand, it shows that the spawning ground is of high quality. On the other hand, it also proves that our method can achieve the precise positioning of high-quality spawning grounds.

[0057] Example 3

[0058] Precise positioning of the 450㎡ spawning ground

[0059] (1) Determine the research area

[0060] The study area is the wintering ground of the breeding population of small-headed schizont found in the southern source of the Yangtze River and 5 km downstream.

[0061] (2) Determine the spawning period

[0062] Based on the research area determined in step (1), the natural reproduction time of the fish was determined by dissecting the fish in the wintering grounds to observe the gonadal development cycle, whether spawning occurred, whether the anal fin was worn, and whether the fry were collected. The time when male and female fish leave the wintering grounds and are sexually mature but have not spawned is the pre-spawning time T0, the time when female fish are found to have spawned is T1, and the spawning period is T S Located between T0 and T1. On June 3rd, gonadal development into stage V was observed. Regarding secondary sexual characteristics, sexually mature male and female fish are covered in asteroses. Males display distinct asteroses on all fins and throughout their bodies, while females have fewer asteroses, most prominent on the anal fin. The last two branched fin rays in males have transformed into spines, and the ray membranes are curved and concave. Females are significantly larger than males, reaching twice their total length and ten times their weight. However, there are also large males and females of equal size. During capture, females shed eggs, with slight pressure on the male's abdomen triggering the ejection of liquefied semen, followed by a mouth-opening movement and violent tail swaying. By June 12th, 30% of mature females had ovarian emptying, confirming the spawning period as June 3rd to 12th.

[0063] (3) Identify the river sections where spawning sites are located

[0064] Based on the research area determined in step (1), the identification of the river sections where the spawning grounds are located was carried out during the spawning period of the genus Nasturtium determined in step (2). The broodstock method was used to determine the approximate range of the spawning grounds. The areas where the breeding groups of the summer breeding populations are located and where they like to chase each other are the river sections where the spawning grounds are located. At the same time, it was found that 500 mature broodstock of the genus Nasturtium in stage V, including females and males, which had not yet spawned, were swimming in the 2.5 km downstream river section and had a high degree of loyalty to the deep water areas where they swam. Females were significantly larger than males, and on average, seven males chased one female, confirming that this river section was the spawning ground section.

[0065] (4) Accurate positioning of spawning grounds

[0066] Based on the spawning ground determined in step (3), the spawning ground is precisely located in the river section. The specific time of spawning is determined within the spawning period obtained in step (1). After determining the specific spawning time, the spawning ground of the genus Gymnosoma is precisely located based on the spawning ground bottom evolution method and the bottom differences of the spawning grounds in the spawning ground of the small-headed Gymnosoma schizont. From June 3 to 12, 2023, the ovaries of the female Gymnosoma schizont are monitored daily to see whether they are ovarian empty. No spawning occurred on June 11, and 30% of the female fish ovaries were emptied on June 12. The anal fins of the female fish with ovaries emptied were severely damaged, which confirmed that spawning occurred. Immediately search and locate the spawning ground in the spawning river section. The search range includes the parent fish activity area and the non-parent fish activity area. By finding the different points of bottom difference, the spawning ground is precisely located. In the 5km downstream river section, a mixture of khaki and blue-black riverbed bottom was found, and blue-black circular patches were evenly distributed between the khaki riverbed. The dark blue patches are distributed with coarse and fine substrates, with fine gravels being the majority. The distribution of coarse and fine grains is significantly different from the surrounding yellowish-brown areas with uniform 4-5cm large-grain gravels. The spawning area is 450m 2 .

[0067] (5) Identification of fertilized egg species

[0068] After identifying the distinctively colored substrate, the bluish-black substrate was removed and the fertilized eggs were collected. The fertilization rate was calculated, with the minimum fertilization rate being the percentage of white eggs removed, which was 97%. The collected fertilized eggs were brought back to the laboratory for incubation. The fertilized eggs were artificially incubated indoors in a basin-type incubator, with a daily water temperature of 9.5-20°C. The incubation water source was well water, naturally at 9.5°C, which was then heated by hot water blending. The room temperature was 13°C, with an appropriate dissolved oxygen level. Incubation was conducted in the dark or in low light, with two-thirds of the water changed daily. Morphological identification after hatching revealed that the species lacked whiskers and had small spots on its back. Given that only one species of schizothoracic fish, the Gymnosoma genus, reproduces naturally in Dangqu, while most other species, including Cobitidae, possess three pairs of whiskers, the fertilized eggs were identified as juvenile Gymnosoma larvae. Dead eggs were sorted daily during the incubation process until all larvae hatched. The calculated hatch rate for the fertilized eggs was 81%.

[0069] Area 450m 2 There are about 1,000 nests in the spawning ground, the fertilization rate in the nests is greater than 97%, and the hatching rate reaches a high value of 81%. On the one hand, it shows that the spawning ground is of high quality. On the other hand, it also proves that our method can achieve the precise positioning of high-quality spawning grounds.

[0070] The present invention is to identify and locate the spawning grounds of the world's highest-altitude Cyprinidae fish, the genus Gymnosoma, at the southern source of the Yangtze River at an altitude of over 4,800 meters, from the three aspects of spawning time, spawning space, and precise identification mechanism. The gonadal development cycle of the fish was determined, and the spawning period was clarified. The river section of the spawning ground was determined by tracking the parents and discovering the newborn fry, and finally the high-quality spawning ground was accurately located by the color change of the spawning ground substrate. The buried egg incubation is a significant feature of its spawning ground structure. The present invention has achieved the precise positioning of the spawning grounds of Gymnosoma on the Qinghai-Tibet Plateau, laying a solid foundation for the protection of the spawning grounds of this fish and the subsequent research on the ecological and hydrological needs. The breakthrough in the precise positioning of the spawning grounds of Gymnosoma, the Cyprinidae fish with the highest altitude distribution and the highest evolutionary level of the schizothorax fish in the world, has important academic value and lays a solid foundation for the subsequent research on the ecological and hydrological needs of the spawning grounds, as well as the research on plateau adaptation and climate change response. This achievement is unique in China and is in a leading position both domestically and internationally. Therefore, the method for accurately locating the spawning grounds of schizothorax fish proposed in the present invention is scientific, innovative, and original. It is a pioneering work in the field of fish habitat research and patents. The invention can also provide a reference for research and inventions in the same field at home and abroad.

[0071] The present invention accurately locates the spawning grounds of schizothorax fish. Buried egg hatching is the main structural feature of its spawning grounds, and the fertilization rate and hatching rate of fertilized eggs in the spawning grounds are both high, proving that the located spawning grounds are of extremely high quality.

[0072] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for accurately locating the spawning grounds of schizothorax fish, characterized in that: The steps include: Step 1: Determine the research area The study area was a 10-km river section, 2.5 km upstream and 7.5 km downstream of the wintering grounds of the genus Schizophora. Step 2: Determine the spawning period of the genus Schizontidae The gonad development cycle of Gymnosoma spp. was determined by dissecting the fish in the wintering grounds. The natural reproduction time was determined by observing the presence of eggs in the abdominal cavity of mature female Gymnosoma spp. and whether the anal fin was worn. The time when male and female fish left the wintering grounds and were both sexually mature but not spawning was the pre-spawning time T0. The time when female fish were found to have spawned was T1. The spawning period was T S Located between T0 and T1, sexually mature female fish have a secondary sexual characteristic, the bead star. The sexually mature female fish and the time of sexual maturity can be identified by the bead star on the anal fin; Step 3: Based on the research area determined in step 1, identify the river section where the spawning ground of the genus Schizonotrichia is located during the spawning period of the genus Schizonotrichia determined in step 2. Specifically, the spawning river section is determined based on two events: parent fish chasing each other and finding newborn fry. Step 4: Based on the spawning grounds determined in step 3, the spawning grounds are precisely located within the river section. The specific time of spawning is determined within the spawning period determined in step 2. After the specific spawning time is determined, the spawning grounds of the genus Gymnosoma are precisely located based on the bottom sediment differences of the river section where the spawning grounds of the Gymnosoma genus are located. The step four specifically includes: after determining that breeding behavior has occurred, immediately searching for the spawning ground in the spawning river section, the search range includes the parent fish activity area and the non-parent fish activity area, if many clean blue-black pebble patches appear in the searched spawning river section, and the surface substrate of the blue-black patches is composed of pebbles and gravels of varying sizes, with coarse and fine substrates distributed, but mostly fine gravels, then the area discovered at this time is determined to be the spawning ground.

2. The method for accurately locating the spawning grounds of schizothorax according to claim 1, characterized in that: In step 2, it was determined that the sexual maturity period of the genus Gymnosoma was between June 1 and June 15.

3. The method for accurately locating the spawning grounds of schizothorax according to claim 1, wherein: The step three specifically includes: the spawning ground river section is determined by the broodstock method and the early resource method. The broodstock method is to simultaneously discover mature but unspawned V-stage broodstock of the genus Gymnosoma, and the number of broodstock in the habitat reaches a certain scale and the broodstock are chasing each other in this area. The broodstock are checked for egg-carrying status by squeezing eggs and observing whether the anal fin is broken to monitor whether the broodstock have spawned. The early resource method is to collect possible fish eggs and newborn fry through a handheld D-type net and a sinking egg collection net to determine the section location. The spawning ground is located in the upstream area near the section where the early resources are collected.

4. The method for accurately locating the spawning grounds of schizothorax according to claim 3, characterized in that: The time for locating the spawning ground in step 4 is 1-7 days after the occurrence of the breeding behavior.

5. The method for accurately locating the spawning grounds of schizothorax according to claim 1, wherein: It also includes step five, identification of the species of fertilized eggs: after determining the spawning ground, the bottom sediment is removed to collect the fertilized eggs, which are then brought back to the laboratory for incubation. After hatching, the morphology of the fry and molecular biology methods are used to identify whether they are fish of the genus Gymnosoma.

6. The method for accurately locating the spawning grounds of schizothorax according to claim 5, characterized in that: The artificial incubation method of the fertilized eggs in step five is indoor basin incubation. The incubation water temperature is a daily rhythm water temperature of 8-22°C. The incubation water source is well water with a natural water temperature of 8-9.5°C. The incubation water temperature is achieved by mixing with hot water. The room temperature is 13-14°C. The dissolved oxygen is appropriate during incubation. Incubation is carried out in the dark or low light environment, and 2 / 3 of the water is changed every day.

7. The method for accurately locating the spawning grounds of schizothorax according to claim 1, characterized in that: In step 1, the upstream 2.5 km section represents a single-branch river type, with a shallow and narrow river channel and a river width of 20-50 m; the downstream 7.5 km section represents a braided river system, with multiple branched river channels, diverse habitats, and a river width of 600-1000 m.

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