CN113796356B - Method for detecting and evaluating quality of Cereus lucidus imagoes - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
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Abstract
The invention discloses a quality detection and evaluation method for Cereus lucidus imagoes, and belongs to the field of agricultural pest biological control. The invention firstly detects 7 quality indexes of species morphological characteristics, species molecular characteristics, appearance, product quantity, female bee rate, parasitic rate and death rate of the glazed small bees at different temperatures, and then evaluates the quality indexes according to established quality standards. The method can improve the commodity quality of the glazed wasp imagoes and strengthen the product quality management, and ensures the quality safety of agricultural products and the safety of ecological environment; the quality-qualified amelogia lucida is screened by the detection method and released to the field, so that the population quantity of the diaphorina citri can be effectively controlled, and the commercialization of the natural enemy product of the amelogia lucida is promoted.
Description
Technical Field
The invention belongs to the field of agricultural pest biological control, and particularly relates to a quality detection and evaluation method for Cereus lucidus imagoes.
Background
Bee with glaze (2)Tamarixia radiata) Is diaphorina citri (Diaphorina citri Kuwayama) The dominant parasitic wasp of (2) parasitizing 4~5-old diaphorina citri nymph. One small belly-brightening amelogia can kill more than 500 nymphs of diaphorina citri by feeding and parasitizing. In 1998, the release of amelogus lucida was introduced in florida, which was spread throughout all the citrus gardens of florida by 2006, in addition to which the amelogus lucida was successfully introduced in california, colombia guarumu, france, siniwann, taiwan, philippines and african-hair quest, etc.; it is distributed in Brazil, argentina, venezuela, mexico, etc. Currently, lucida glaucomatodes has been produced industrially in brazil, mexico, california, texas, and florida, and in china, the institute of sciences in guangdong province, animal research, produced in large scale in 2020.
However, the parasitic rates of the lucida neriidae produced in different areas are obviously different. In Florida, USA, the parasitic rate of the small glazed bee is relatively low in spring and summer, the highest in autumn, and the average parasitic rate is more than 60%; in texas, a large number of wasps releasing bright belly glaze can reduce the population quantity of diaphorina citri by 91.2%; in Brazil, the parasitic rate is 43.3% -88.8%; in the west of mexico, the average parasitic rate reaches 60.58%. This may be influenced by environmental factors such as temperature, humidity, etc. of the release site, and also may be related to the quality of the lucent-belly glazed wasps. Especially, the large fluctuation of the parasitic rate in the same release area is probably directly related to the quality of parasitic wasps. Therefore, the quality detection of the Cervus elaphus Linnaeus adults and the establishment of the Cervus elaphus Linnaeus quality evaluation standard are of great significance to the production and application of the Cervus elaphus Linnaeus adults.
Disclosure of Invention
In order to improve the commodity quality of the small glazed bee products, strengthen the product quality management and guarantee the quality safety of agricultural products and the ecological environment safety, the invention provides a method for detecting and evaluating the quality of the small glazed bee products, which can be used for identifying the small glazed bee products and detecting the quality of the small glazed bee products, and refers to other parasitic bee quality evaluation systems.
The invention aims to provide a method for detecting and evaluating quality of amethyst wasps, which comprises the steps of detecting 7 quality indexes including species morphological characteristics, species molecular characteristics, appearance, product quantity, female bee rate, parasitic rate and death rate of amethyst wasps stored at different temperatures, and evaluating according to quality standards:
in a preferred embodiment, the quality detection of the species morphological characteristics is to identify the male bees and the female bees of the glazed wasps, and the identification contents comprise body length, body color, antennae, handle sections, bar sections and abdomen.
In another preferred example, the quality detection of the molecular characteristics of the species is to perform molecular identification of the species by using a conserved region and internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) of cytochrome oxidase I (cytochrome oxidase subunit I, COI).
In another preferred example, the quality of the appearance is tested by recording the number of female bright belly glazed bees with dark body color and incomplete limb and residual wing, and counting the ratio of the number to the total number.
In another preferred example, the quality of the product quantity is detected by manually counting, and recording the ratio of the actual quantity of the small glazed bees in each bottle to the number of the packaging bottle identifiers (200 heads/bottle).
In another preferred example, the quality of the female bee rate is detected by manually counting, and the ratio of the female lucida ames in each bottle to the total number in each bottle is recorded.
In another preferred example, the quality detection of the parasitism rate is to adopt a 60-mesh insect-proof bag of 15 cm × 15 cm, transfer 4-year-old terminal diaphorina citri nymphs and female bees of the small glazed bee (the head ratio is 10: 1), and remove the small glazed bee after 24 h; and 7-9 d later, checking and counting the number of the parasitized diaphorina citri, and counting the parasitizing rate.
In another preferred example, the quality test of the death rate of the leucorrhea bees at different temperatures is to store the leucorrhea bees at 15 ℃,25 ℃ and 30 ℃ by using an artificial climate box for 10 h, record the death number of the leucorrhea bees and count the death rate.
Compared with the prior art, the invention has the following beneficial effects: the detection and evaluation method can improve the quality of the product of the amethystoides aurantiaca imagoes, strengthen the quality management of the product, and ensure the quality safety of agricultural products and the safety of ecological environment; the quality-qualified amelogia lucida is screened by the detection method and released to the field, so that the population quantity of the diaphorina citri can be effectively controlled, and the commercialization of the natural enemy product of the amelogia lucida is promoted.
Drawings
Fig. 1 is the morphological characteristics of female bees of glazed wasps.
Fig. 2 is the morphological characteristics of the drone of the glazed belly wasp.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The amelogia lucida used in the following examples is a product produced on a large scale by the animal institute of academy of sciences of Guangdong province.
Example 1 species morphological characterization
The quality of the glazed wasp product is evaluated, and morphological identification is firstly carried out to ensure that species morphological characteristics are matched with detection results. Herein, 5 bottles of lucida wasps were randomly selected, and 10 of them (5 female and 5 male) were taken from each bottle for species morphological identification. The identification results are as follows:
(1) Female bees (fig. 1):
the body length is 0.92 to 1.04 mm, most of the body is black brown, the compound eye and single eye are red brown, the head is wider than the body, 2 teeth and 1 cutting tooth of the upper jaw, and 1 section of the lower jaw whisker and the lower lip whisker are respectively arranged. An antenna 8 section (antenna type 1,1,3,3) with a small ring section of 1; the length of the handle section is 3.7 times of the width; the peduncle is equal to or slightly longer than the No. 1 cable knot; 1~3 has a rope length ratio of 11; the end of each rod section 3 is provided with a needle-shaped protrusion, and each rod section is provided with 7-11 thorn-shaped sensors. The base section of the antenna is dark brown, the handle section is light colored, the back edge is dark colored, and the rest sections are dark yellow.
The medial aspect of the middle lobe of the middle sternum has 2 pairs of hair, the lateral margin of the inferior midline of the scutellum has 2 pairs of hair, and the discoid sensor is close to the front 1 pair of hair; the ruffles of the chest and abdomen are incomplete and have irregular net-shaped lines. The wings are transparent, and the length of the front wing is 2.3 times of the width; the end part of the inferior limbus vein is a fracture mark, the inferior limbus vein is provided with 1 bristle, and the lower part of the end part is provided with 2~3 bristles; the base of the front edge chamber is provided with 1~2 fine hairs, and the end part is provided with 3~4 fine hairs; the border vein has 7~9 setae. Tarsal nodes 4-4-4, yellowish, dark brown at the base and tarsal terminals.
The belly web is yellow, the back plate of 1~4 section or 1~5 section is mostly yellow in the center of the back, and is in a large yellow spot. The 3 rd oviposition valve is dark brown, the oviposition device extends out from the 2 nd abdominal joint of the abdomen and slightly protrudes out of the tail end of the abdomen, the oviposition device is 1.6 times of the length of the midfoot section, and the 3 rd oviposition valve is 0.2 times of the length of the oviposition device.
(2) Drone (fig. 2):
the body color and the shape are similar to those of female worms, and the body length is 0.84 to 1.05 mm. Antenna 9 section (antenna 1,1,4,3); a 1 small oblong sensing area is arranged at the position of the handle section base close to 2/5~1/2; 4 cable joints, each joint is provided with 1 row of 6~7 long setae; the setae of the 1 st row of the bar section 1 are slightly shorter, and the tail end of the bar section is provided with a needle-shaped protrusion. Most of the center of the back plate of the 1~2 section of the abdomen is dark yellow, the edge is dark brown, and the back plate is small dark yellow spots; the web 1~3, the belly, is mostly dark yellow, with the remaining webs blackish brown. The male external genitalia is long, extending from the base of the 4 th abdominal segment of the abdomen, and the tip of the finger is dark brown.
Example 2 species molecular characterization
Species molecular identification was performed using conserved regions of cytochrome oxidase subunit I (COI) and internal transcribed spacers 1,2 (ITS-1, ITS-2).
Randomly sampling and selecting 5 bottles of small glazed bees, extracting total DNA from 1 head of each bottle, selecting a Tiangen biological blood/cell/tissue genome DNA extraction kit (DP 304) as the kit, and strictly following the kit instructions for experimental steps. The upstream and downstream primers are shown in Table 1.
A50 muL system, 2 muL DNA template, 0.5 muM each of primers (Table 1), 25 muM LMix (2X SanTaq PCR Mix premix containing blue dye, biological B532061) were used for PCR reaction, and were made up with sterile water. Reaction conditions are as follows: pre-denaturation 94 ℃ (5 min) → denaturation 94 ℃ (30 s) → annealing 50-60 ℃ (30 s) → extension 72 ℃ (30 ~ 60s) → extension 72 ℃ (10 min) → preservation 4 ℃ (infinity), wherein the denaturation of step 2 to the extension of step 4 are repeated for 35 cycles.
Molecular identification results of the glazed wasps show that the intra-species similarity of target genes COI, ITS-1 and ITS-2 is higher in NCBI (National Center for Biotechnology Information Search database) database (Table 2), the intra-species similarity of COI is 98.5% -100.0%, and the inter-species similarity is 94.0% at most between T.radiata and T.dryi; the ITS-1 intraspecies similarity is as low as 97.9%, while the T. Drukyulensis similarity with the generic T.drukyulensis is only 82.0%; the ITS-2 internal phase similarity is 99.0% -100.0%, and the similarity with T.drukyulensis is less than 82.7%. Thus, the standard specifies that COI, ITS-1 and ITS-2 have a similarity of greater than 97%, and that species is considered to be a lucent-belly bumblebee.
Example 3 female bee body color and disability detection
Observing whether the female lucent abdomen glaze wasp is dark in body color or whether the female lucent abdomen glaze wasp is damaged by wings or not by using a stereoscopic microscope. Randomly selecting 10 bottles of small glazed bees, taking 1 sample of every two bottles, testing 200 female small glazed bees of every sample, respectively recording the body color darkness and the number of female bees with residual wing and residual limb, respectively counting the ratio of the body color darkness and the number of the residual limb and residual wing individuals to the total number (the body color darkness rate and the residual limb and residual wing rate), and detecting for 5 times, wherein the detection results are shown in table 3.
As can be seen from table 3, in the appearance test of the female bees of the lucent-abdomen wasps 5 times, the body color shading rate of the lucent-abdomen wasps is up to 1.5%, and the ratio of the stump and residual wing rate of the lucent-abdomen wasps is up to 0.4%. According to production practice, the body color shading rate of female lucent-abdomen glazed wasps in the lucent-abdomen glazed wasp product is not higher than 2.0%, and the incomplete wing rate of the female lucent-abdomen glazed wasps is not higher than 1.0%.
EXAMPLE 4 quantitative determination
Randomly selecting 5 bottles of small bright belly glaze bees for manual counting, recording the actual number of the small bright belly glaze bees in each bottle, calculating the ratio of the number of the small bright belly glaze bees in each bottle to the number of the packaging bottle identifiers (200 heads/bottle), detecting 5 samples in each bottle, and obtaining the detection results shown in table 4.
Randomly draw 5 bottles of glazed wasp products, the number of which accounts for the minimum 99.0% of the mark (table 4). According to the detection result and the production practice, the number ratio of the small glazed bees in the product package is not less than 95.0% of the identification number.
Example 5 female bee Rate detection
Randomly selecting 5 bottles of small glazed bees, recording the number of the female bees, counting the female bee rate, and detecting for 5 times, wherein the detection results are shown in table 5.
The female bee rate was the lowest 71.0% of 5 tests (table 5). The female bee rate in the specified product quality standard is not less than 70.0%.
Example 6 detection of mortality in storage of Ceratodes lucidus at different temperatures
Randomly selecting 15 bottles of belly glaze wasps and dividing the wasps into 3 groups, and taking 100 heads of each bottle; each group of small glazed bee was treated in an artificial climate box at 15 ℃ (low temperature), 25 ℃ (normal temperature) and 30 ℃ (high temperature) for 10 h, the number of deaths of the small glazed bee was recorded, the mortality was counted, and the detection results are shown in table 6.
As can be seen from Table 6, the death rate in the low-temperature storage at 15 ℃ is at most 3.0%, the death rate in the normal-temperature storage at 25 ℃ is at most 2.0%, and the death rate in the high-temperature storage at 30 ℃ is at most 4.0%. According to the detection result, the death rate of the small glazed bees at different temperatures in the specified product quality standard is not higher than 5.0%.
Example 7 parasitic Rate detection
Preparing a young tangerine with tender tips, selecting a 60-mesh insect-proof bag sleeved with 15 cm X15 cm at a proper position, and transferring 100-year-4-old terminal citrus psyllid nymphs into the bag. Randomly selecting 10 female worms from 1 bottle of the small glazed bee product, transferring the female worms into the insect-proof bag, and removing the small glazed bee after 24 h. And 7-9 d later, checking and counting the number of the parasitized diaphorina citri, and counting the parasitizing rate. The above experiment was carried out at 26 ℃ with a relative humidity of 60% and a photoperiod L: D of 14: 10, and the results are shown in Table 7, after repeating 5 times.
The parasitic rate of randomly extracted small glazed bees is 92.0 percent at the lowest and can reach 100.0 percent at the highest (Table 7). Thus, the quality standards specify that the parasitic rate of the lucent-abdomen glaze wasp product is not less than 90% under laboratory conditions. The detection result is used for quality evaluation indexes and cannot be used for field standard evaluation.
Example 8 establishing quality standards for Liangbelly glaze wasp products
According to the detection result, the quality detection items and detection indexes of the adult leucorrhea bees are determined, wherein the detection items comprise 7 quality indexes including the form and molecular characteristics of detected species, the appearance (body color shading rate, stump and residual wing rate), the product quantity, the female bee rate, the parasitic rate and the death rate of the leucorrhea bees stored at different temperatures (15 ℃,25 ℃ and 30 ℃). By detecting various indexes of the amelogia lucida produced by the animal research institute of the academy of sciences of Guangdong province, index parameters are determined for the first time, and the quality standard of the amelogia lucida imagoes is formulated and is shown in Table 8.
Claims (3)
1. Bee with glaze for brightening abdomenTamarixia radiata) The adult quality detection and evaluation method is characterized in that 7 quality indexes including species morphological characteristics, species molecular characteristics, appearance, product quantity, female bee rate, parasitic rate and death rate of amelogia nuda stored at different temperatures are detected and evaluated according to quality standards, wherein the quality standards are as follows:
the quality detection of the species molecular characteristics is to identify the species molecules by using cytochrome oxidase I, namely a conserved region of COI and an internal transcribed spacer 1,2, namely ITS-1 and ITS-2;
the quality detection of the appearance is to record the number of female bee bright abdomen glaze wasps with dark body color and incomplete limb and residual wing, and count the ratio of the number of the female bee bright abdomen glaze wasps to the total detection number;
the quality of the female bee rate is detected by manually counting, and the ratio of female bright belly glaze wasps in each bottle to the total number in each bottle is recorded;
the quality detection of the parasitic rate is carried out by adopting a 60-mesh insect-proof bag of 15 cm X15 cm, transferring 4-year-old terminal diaphorina citri nymphs and the female bees of the small glazed bee with the head ratio of 10: 1 and removing the small glazed bee after 24 h; checking and counting the number of the parasitized diaphorina citri after 7 to 9 days, and counting the parasitizing rate;
the quality detection of the death rate of the small glazed bee at different temperatures is to store 10 h in an artificial climate box at 15 ℃,25 ℃ and 30 ℃, record the death number of the small glazed bee and count the death rate.
2. The method of claim 1, wherein the quality test of the species morphological characteristics is the identification of male and female lucida ames, including body length, body color, tentacles, pedicles, clavicles, abdomen.
3. The method of claim 1, wherein the quality inspection of the product quantity is performed by manually counting the ratio of the actual quantity of the belly glaze wasps in each bottle to the quantity of the packaging bottle labels, wherein the quantity of the packaging bottle labels is 200/bottle.
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