CN109874752B - Diapause regulation and storage method for waistband long body hornets - Google Patents
Diapause regulation and storage method for waistband long body hornets Download PDFInfo
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Abstract
The invention discloses a diapause regulation and storage method of a waistband long cocoon bee, which improves the diapause rate and the emergence rate by accurately controlling the diapause induction period, the diapause environmental condition, the storage condition, the diapause release time, the environmental condition and the like of the waistband long cocoon bee, has the advantages of concentrated and regular bee emergence, no degeneration of bee species, solves the storage and transportation problem of the bee species, prolongs the shelf life of natural enemy products, prepares sufficient bee sources for biological control and improves the biocontrol benefit of the natural enemy products.
Description
Technical Field
The application relates to the technical field of agricultural pest control, in particular to a diapause regulation and storage method of waistband long cocoon bee.
Background
Asiatic corn borer is a worldwide pest and is a main pest on corn in China. At present, China mainly relies on chemical prevention and control measures for preventing and controlling the corn borers, and a series of problems of rising of drug resistance of pests, increasing of pest outbreak probability, increasing of damage to agricultural ecological environment, increasing of pesticide residue of agricultural products, reduction of quality of agricultural products and the like are caused while harm loss of the pests is controlled. Severely restricting the agricultural and economic development of China.
Biological control is an important part in green control, and pest control by using natural enemy insects is one of important technologies in biological control. The biological control technology is an environment-friendly control strategy, can not cause secondary pollution to the environment while controlling the harm of pests, has strong specificity and long duration of killing the pests, and can simultaneously ensure grain harvest and environmental protection.
The anaglyphus waistband is one of important parasitic natural enemies of the larvae of the ostrinia nubilalis, the natural parasitic rate can reach 30% -40%, the control effect on the ostrinia nubilalis is good, and the anaglyphus waistband has high application value. However, the problems restricting the large-scale application of the waistband long body hornet are as follows: the breeding of the long body wasps in the waistband for one generation requires 1 month, and the bee species need to be preserved by means of subculture propagation, and even if the cocoons are preserved at low temperature, the number of the cocoons cannot exceed 15 d. Not only can cause a great deal of waste of manpower, material resources and financial resources, but also can cause the degeneration of bee species; meanwhile, in practical application, long-distance transportation is difficult to finish, and the task of providing sufficient natural enemy products is difficult in the pest emergence period.
Diapause is a bad environmental adaptation formed by the long-term evolution of insects, and the insects can survive for a long time without eating after entering the diapause state. Therefore, the problem of expanding propagation, storing and transporting and the like of the waistband long-body hornet is solved by exploring and manually regulating diapause, and the method has important significance for realizing large-scale production and application of natural enemy products.
Disclosure of Invention
In order to realize the artificial regulation and control of the parasitic natural enemy wasp solitaria, solve the long-term storage and transportation problem of the bee species and provide sufficient biocontrol products, the invention provides a diapause regulation and control and storage method of the wasp solitaria, which comprises the following steps:
(1) parasitizing third-instar Asiatic corn borer larvae with the waistband long body braconid bees after mating for 24 h; the inventor of the application finds that the larvae of the ostrinia nubilalis selected for the third instar are the best in parasitic state, the death rate of the selected larvae of the second instar is high, and the parasitic rate of the larvae of the fourth and fifth instar is low;
(2) after 48h of parasitization, the parasitized Asiatic corn borer larvae are taken out and placed in a lighting condition of 10L: 14D, inducing for 30-40D in an incubator with the humidity of 70-80%, the temperature of 20.5-21.5 ℃ or the light period of 20.5-21.5 ℃ and the dark period of 14.5-15.5 ℃, so that the anabolic brevicia girdle can enter a diapause state along with the host Asian corn borer; the breeding retardation induction is carried out after 48h of parasitism is selected, so that the parasitism rate is higher on the premise of ensuring the improvement of economic benefit, because the Asiatic corn borer 3d age is not greatly different at 24 ℃ under the feeding condition of 28 ℃, the larvae which are possibly selected when the hosts are selected are the early stage and the terminal stage of the 3 rd age, therefore, the parasitism is carried out for 48h, the parasitism rate of the Asiatic corn borers can be kept at a high level in the early stage of the 4 th age at most, the corncob slices serving as carriers are sufficient, the feeding and the growth of the Asiatic corn borers are not influenced, if the corn borer is parasitized for 24 hours, the parasitization rate is slightly low, the parasitization of the corn borer at the time of parasitization cannot be seen, the parasitization of the diapause corn borer after the development of the mature larva is difficult to be seen, therefore, if a lot of Asiatic corn borers are not parasitized, a great deal of economic loss is caused, and the number of the waistband long-body hornets cannot meet the requirement; if the corn ear slices are added, economic and labor costs are increased;
(3) placing the Asiatic corn borers parasitizing diapause wasps with waistband long body into a light cycle of 0L: 24D, storing for 60D-90D at the temperature of 4 ℃;
(4) taking the Asiatic corn borers stored in the step (3), and placing the Asiatic corn borers at the temperature of 27.5-28.5 ℃ under the illumination of 16L: and 8D, feeding sufficient water in an incubator with the humidity of 70-80%, adding water once every 3D, starting to collect the waistband long body braconid bees which drill cocoons from the host body at the 9 th D, collecting the waistband long body braconid bees once every 1-2D, and finishing the collection after 5-8D.
Further, the method also comprises the step of placing the cocoons collected in the step (4) into a place with the temperature of 10 ℃ and the illumination of 0L: storing in 24D incubator or refrigerator, collecting at 24+ -0.5 deg.C, illuminating 16L: and (4) feathering in an incubator with humidity of 70-80% for 10 days at 8D.
Further, in the step (1), the number ratio of the wasps with long bodies in the waistband to the larvae of the three-instar Asiatic corn borers is 1: 7;
in the step (1), the illumination condition is 10L: 14D;
in the step (4), the soaked absorbent cotton is used for providing water.
The third-instar Asiatic corn borer larvae used in the step (1) are cultivated at the temperature of (24 +/-0.5) DEG C and the illumination of 10L: 14D, and feeding the mixture by using artificial feed in an artificial climate box with the humidity of 70-80 percent.
After the long cocoon bee of the waistband is eclosized, according to the female-male ratio of 1: 1 (female parent: 1), the ratio of the male parent to the female parent is controlled at the temperature (24 +/-0.5) DEG C, the illumination is 16L: and 8D, inoculating bees in a bee cage with the humidity of 70-80% for mating, and after mating for 24h, using the bee cage for parasitizing the ostrinia nubilalis in the third instar.
The beneficial effects of the invention include:
1. compared with the conventional method for selecting the larvae of the ostrinia nubilalis which are 4-5 th instar as hosts in the prior art, the method selects the larvae of the ostrinia nubilalis which are three th instar as hosts, and has lower parasitic mortality rate compared with 1-2 th instar larvae and higher parasitic rate compared with 4-5 th instar larvae.
2. The technical prejudice that diapause of the long hornet is possible to be realized under the condition that the diapause of the host Asiatic corn borer is possible is overcome, and the proper diapause condition of the long hornet is innovatively provided.
3. By adopting the diapause regulation and storage method, the diapause rate of the long body hornet on the waistband is high and can reach more than 98.95 percent; the bees grow regularly after diapause is removed, the bees emerge intensively and are mainly concentrated within 5-8 days, and the invention finds that the time from the diapause removal to the bee emergence of the first batch of bees is 8 days, so that only water needs to be added in the period without checking the state of the worms, thereby reducing the labor cost for manually checking whether cocoons appear every day; the emergence rate is as high as more than 90.6%, and bee species are not degenerated.
4. By adopting the method, the diapause release time of the waistband long cocoon bee can be accurately controlled, the shelf life of natural enemy products is prolonged, the storage and transportation problems of bee species are solved, sufficient bee sources are prepared for biological control, and the biocontrol benefit of the natural enemy products is improved.
Drawings
FIG. 1 is a graph of diapause rates of Braconidia elata and Ostrinia furnacalis under different treatment conditions;
FIG. 2 is a comparison of mortality at the same temperature for different storage times;
FIG. 3 is a comparison of mortality at different storage temperatures for the same storage time;
FIG. 4 is a comparison of initial bee emergence times at the same temperature for different storage times;
FIG. 5 is a comparison of initial bee emergence times at different storage temperatures for the same storage time;
FIG. 6 is a comparison of the concentrated bee emergence times at the same temperature for different storage times;
FIG. 7 is a comparison of concentrated bee emergence times at different temperatures for the same storage time;
FIG. 8 is a graph showing the comparison of parasitic amount of the long body of the wasps in the waistband after being stored at 4 ℃ for various periods of time;
fig. 9 is a comparison of female bee life of waistband long cocoon bees after storage at 4 ℃ for various times;
FIG. 10 is a comparison of emergence rates of the long body hornets of the waistband after storage at 4 ℃ for various periods of time.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 diapause induction experiment of Long body Braconidae in waistband
1. The material and the method are as follows:
1.1 sources of test insects
The Asiatic corn borer larva is bred by adopting standard insect breeding technology such as Zhoudangrong and the like and agar-free semi-artificial feed, and is cultured at the temperature of 28 +/-0.5 ℃ and the relative humidity of 70-80 percent, wherein the photoperiod is 16L: continuously feeding for multiple generations in 8D artificial climate chamber.
The waistband long cocoon bee is picked from parasitized Asiatic corn borer aged larvae in a field test base in the city of the public main ridge of Jilin province, and the Asiatic corn borer 3 rd larvae are used as hosts for subculture propagation indoors. After parasitic, culturing at the temperature (28 +/-0.5) DEG C, the relative humidity of 70-80 percent and the photoperiod of 16L: 8D artificial climate box; after cocooning, culturing at the temperature of (24+0.5) DEG C, the relative humidity of 60% -70%, the photoperiod of 16L: 8D climatic chamber. The temperature of the adult wasps with long cocoons on the waistband is (24 +/-0.5) DEG C, the relative humidity is 60-70%, and the light cycle is 16L: and breeding in 8D acrylic bee cages, and feeding 20% of honey water.
1.2 Experimental methods
As shown in table 1, a total of 4 treatments were set. Each treated 1 24-well bioassay plate, the parasitized asian corn borers were replicated 5 times, and the non-parasitized asian corn borers were replicated 3 times. Firstly, putting part of the ostrinia furnacalis guenee in a culture dish with a corn cob slice, putting the ostrinia furnacalis in a beekeeping cage with a waistband long body of braconid bees in advance for parasitism, wherein the raising conditions still maintain the experimental conditions before parasitism in the table 1. After parasitizing for 48h, the parasitized Asiatic corn borers and the Asiatic corn borers of which the rest parts are not used for parasitizing are simultaneously inoculated into a 24-hole plate with artificial feed, and the parasitized Asiatic corn borers and the Asiatic corn borers are placed into an incubator for breeding according to 4 different postparasitized conditions. And in the midway, if the feed in the 24-well plate is broken or dried, the Asiatic corn borers in the feed are put into a new 24-well plate. At present, the international universal method for judging diapause of the small parasitic wasps is mainly a development calendar method, namely, if the insects cannot complete development in the known calendar of the state of the insects, the diapause is generated. The method is characterized in that the wasps with long horns can drill out of the bodies of the Asiatic corn borers at the temperature of 28 ℃ for 11 days, the temperature is low under the experimental conditions, the development duration is prolonged, and therefore, in order to guarantee the accuracy of the experimental results, 25d, 30d and 40d are selected to respectively observe the change of the number of diapause individuals once, and the result shows that the ratio of the number of the diapause individuals to 25d still changes at 30d, and the result is unchanged as that of 30d at 40d, so that the wasps with long horns can completely enter the diapause state at 30 d. Under the condition of induced diapause, the energy consumption of the ostrinia furnacalis and the wasps with long body still exists, so if the ostrinia furnacalis and the wasps with long body are placed under the condition for too long time, the consumption of the bodies of the insects is too large, the diapause storage and the development after the diapause is removed are not facilitated, therefore, in practical application, the storage induction time cannot be too long, 30-40 days are taken as the optimal diapause induction time, and the specific diapause induction time can be selected in the range according to the actual production requirements.
TABLE 1 design of the experiment
2. Results and analysis
As can be seen from fig. 1, the four treatment conditions can make more than 90% of the individuals of the asian corn borer which is not parasitized enter a diapause state, but under the treatment conditions of a1 and a4, only about 10% of the individuals of the parasitized asian corn borer, namely the wasps loidenticulata enter diapause, so that the conditions capable of making the diapause of the host asian corn borer do not necessarily make the wasps loidenticulate, which is not in accordance with the conventional cognitive viewpoint of the skilled person, and it is also explained that the wasps loidenticulate is diapause along with the host in the host asian corn borer, but the desire to make the wasps loidenticulate diapause is not simply realized according to the diapause conditions of the host. Under the treatment conditions of A2 and A3, more than 98% of individuals of the wasps with long horny bodies enter diapause, so experiments prove that the low temperature is the main factor for diapause of the wasps with long horny bodies relative to the photoperiod. 10L: 14D, light phase 21 ℃ dark phase 15 ℃ and 10L: 14D and 21 ℃ are good conditions for diapause of the waistband long cocoon bee, and the diapause rate can reach 100 percent and 98.95 percent respectively.
The above results demonstrate that the illumination is 10L: 14D, the humidity is 70-80%, and the conditions of the temperature of 21 ℃ or the light period of 21 ℃ and the dark period of 15 ℃ are all the dominant diapause induction conditions of the waistband long body cocoons.
Example 2 diapause storage and release experiment of Braconidae longated in waistband
1. Materials and methods
1.1 sources of test insects
The raising method of Asiatic corn borer and wasp with long body is the same as experiment 1
1.2 Experimental methods
According to the result of the above example 1, the experiment selects the illumination of 10L: 14D, using diapause Asiatic corn borers parasitizing diapause girdle and hornet parasitizing in vivo obtained by inducing under the conditions that the humidity is 70-80% and the temperature is 21 ℃ in the light period and the temperature is 15 ℃ in the dark period as a test object. The diapause waistband long body hornet is respectively treated as follows:
1) firstly, treating diapause waistband long body cocoons at 10 ℃, 4 ℃ and-4 ℃ for 15, 30, 60 and 90 days:
2) and (3) taking out the long body wasps in the waistband after the treatment time is reached, and placing the wasps in a place with the temperature of 28 +/-0.5 ℃ and the illumination of 16L: 8D, in an incubator with the humidity of 70-80%, and sufficient moisture is fed to the host through absorbent cotton;
3) and after the long body cocoons on the waistband are drilled out of the host cocoons, counting the host mortality, the initial bee emergence time and the centralized bee emergence time. Each 30 asian corn borers were treated and the experiment was repeated 4 times.
2. Results and analysis
As can be seen from fig. 2, the mortality rate gradually increased with the storage time at a constant temperature, but the mortality rate did not significantly differ between the storage times at 4 ℃ and-4 ℃, and the mortality rate was significantly higher in 90 days at a storage temperature of 10 ℃ than at other times.
As can be seen from FIG. 3, the mortality rate at 10 ℃ was significantly higher than that at 4 ℃ and that at-4 ℃ and that at 4 ℃ was slightly lower than that at-4 ℃ for a given storage time, but there was no significant difference between the two. Therefore, the mortality rate can show that the 10 ℃ is not beneficial to the storage of diapause waistband long body braconid bees; there were no significant differences between days 15, 30, 60, 90 for 4 ℃ and-4 ℃.
As can be seen from FIG. 4, the initial bee emergence time was shortened with the increase of the storage time at a constant temperature, and the initial bee emergence times at 60d and 90d were not significantly different at 4 ℃.
As can be seen from fig. 5, the initial bee emergence time at-4 ℃ was significantly longer than 4 ℃ and 10 ℃ for a given storage time, while there was no significant difference between 4 ℃ and 10 ℃.
The shorter the initial bee-exiting time is, the shorter the time required for diapause of the diapause waistband long cocoon-lifting bees after being taken out from the low temperature is, so the less the consumed resources are, and the higher the economic benefit is. Thus, -4 ℃ is not the optimum temperature for storage relative to 4 ℃ and 10 ℃. For 4 ℃ and 10 ℃, 60 and 90 days are better than 15 days and 30 days.
The concentrated bee emergence time refers to the whole bee emergence degree, so the shorter the concentrated bee emergence time is, the better the diapause resolving effect is.
As can be seen from FIG. 6, the time for collecting bees is shortened with the increase of the storage time at a constant temperature, the time for collecting bees is not significantly different at-4 ℃ under different storage times, and the time for collecting bees is significantly better than that for 60 and 90 days at 4 ℃ and 10 ℃ for 15 and 30 days.
As can be seen from fig. 7, the peak emergence times at the three temperatures were not significantly different at 15 and 30 days for a given storage time, while peak emergence times at 4 ℃ and 10 ℃ were significantly less than-4 ℃ at 60 and 90 days.
Therefore, in conclusion, the diapause of the waistband of the hornet is relieved most favorably after the hornet is stored for 60-90 days at the temperature of 4 ℃. Meanwhile, as can be seen from FIGS. 2 to 7, the initial bee emergence time is 8 days and the concentrated bee emergence time is 5 to 8 days when the bee is stored at 4 ℃ for 60 to 90 days.
Example 3 Effect of Low temperature storage on biology after diapause of diapause girdle Long body Conomonas
3.1 Experimental methods
Placing the long body of the cocoon bee cocoons of the waistband cocoon bee which are released and bred out of the cocoons under the storage condition of 4 ℃ at the temperature of 24 ℃ and the photoperiod of 16L: 8D, in an artificial climate box with the humidity of 70-80%, after the emergence of the long body cocoons on the waistband, performing the following treatment: 1) inoculating a pair of male and female adult wasps (female: male ═ 1: 1) of a long-bodied cocoon bee within 24h in a bee-keeping cage of 15cm × 15cm × 15cm, simultaneously feeding 20% of honey water, after mating for 24h, taking fresh corn cob slices as a carrier, inoculating 3-instar larvae of Asiatic corn borers into each bee-keeping cage, after parasitizing for 24h, taking out the Asiatic corn borers, putting the Asiatic corn borers into artificial feed for feeding, after 10d, counting the parasitizing conditions, and repeating each treatment for 6 times; 2) 10 female bees of the waistband long-body cocoons within 24h are inoculated into the bee-keeping cage, 20% of honey water is fed, the survival condition of the female bees is observed and recorded every day until all the female bees die, and each treatment is repeated for 3 times; 3) after all the long body hornets of the waistband are emerged, the number of emerging hornets and the number of hornets are counted, and accordingly emergence rate data are obtained. And finally, comparing and analyzing the data with the non-diapause waistband long body bombesia.
3.2 results and analysis
As can be seen from fig. 8, after diapause and eclosion, the parasitism amount of diapause wasps with long body in the diapause period has no significant difference to the asian corn borer larvae, and has no significant difference to the control, that is, the parasitism amount of the conventional rearing wasps with long body in the diapause period, and the average parasitism amount at 15d is even slightly larger than that of the control group. The low-temperature storage has no adverse effect on the fertility of the long body wasps in the waistband.
As can be seen from fig. 9, the life of the female bees was slightly reduced with the increase of the storage time in the cold storage, but the difference from the control group was not significant, and the lives were all 9d or more.
As can be seen from fig. 10, although the emergence rate of the wasps with long hornets after the low-temperature storage at 4 ℃ was lower than that of the control group, the emergence rates were not significantly different at the respective storage times and were all 90.6% or more.
The above description is only a preferred embodiment of the present invention, and it should be understood that the present invention is not limited to the above examples. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (5)
1. A diapause regulation and control and storage method of a waistband long cocoon bee is characterized by comprising the following steps:
(1) parasitizing third-instar Asiatic corn borer larvae with the waistband long body braconid bees after mating for 24 h;
(2) after 48h of parasitization, the parasitized Asiatic corn borer larvae are taken out and placed in a lighting condition of 10L: 14D, inducing for 30-40D in an incubator with the humidity of 70-80%, the temperature of 20.5-21.5 ℃ or the light period of 20.5-21.5 ℃ and the dark period of 14.5-15.5 ℃, so that the anabolic brevicia girdle can enter a diapause state along with the host Asian corn borer;
(3) placing the Asiatic corn borers parasitizing diapause wasps with waistband long body into a light cycle of 0L: 24D, storing for 60D-90D at the temperature of 4 ℃;
(4) taking the Asiatic corn borers stored in the step (3), and placing the Asiatic corn borers at the temperature of 27.5-28.5 ℃ under the illumination of 16L: and 8D, feeding sufficient water in an incubator with the humidity of 70-80%, adding water once every 3D, starting to collect the waistband long body braconid bees which drill cocoons from the host body at the 9 th D, collecting the waistband long body braconid bees once every 1-2D, and finishing the collection after 5-8D.
2. The diapause regulation and control and storage method for the braconid wasps with long waist bodies as claimed in claim 1, wherein the method further comprises the step of placing the braconid wasps with long waist bodies collected in the step (4) at a temperature of 10 ℃, with a light intensity of 0L: storing in 24D incubator or refrigerator, collecting at 24+ -0.5 deg.C, illuminating 16L: and (4) feathering in an incubator with humidity of 70-80% for 10 days at 8D.
3. The method for controlling diapause and storing the braconid wasps according to claim 1 or 2, wherein in the step (1), the number ratio of the braconid wasps to the third-instar Asiatic corn borer larvae is 1: 7.
4. The diapause control and storage method for the waistband long cocoon bee as claimed in claim 1 or 2, wherein in the step (4), absorbent cotton soaked in water is used for providing water.
5. The diapause control and storage method of the waist belt long cocoon bee according to claim 1 or 2, characterized in that, in the step (1), the illumination condition is 10L: 14D.
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