Methods for introducing in vitro cultured gut commensal microbes into the body of a fall armyworm

Abstract

The application discloses a method for introducing in-vitro cultured intestinal symbiotic microorganisms into Spodoptera exigua, and comprises the following steps: isolating, purifying and shake-culturing the intestinal symbiotic microorganisms from the Spodoptera exigua, starving the larvae of the Spodoptera exigua, cutting corn leaves into small pieces, soaking the small pieces in the prepared bacterial liquid, and feeding the larvae of the Spodoptera exigua with the dried small pieces, so that the in-vitro cultured intestinal symbiotic microorganisms are introduced into the Spodoptera exigua. The method does not need a microinjector, is simple to operate, does not need continuous feeding of multiple generations, and has a short experimental period. The method can change the bacterial structure in the insects, so as to achieve the purpose of studying the influence of the symbiotic bacteria on the growth and development of the insects and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural pest and insect control, and particularly relates to a method for introducing in-vitro cultured intestinal symbiotic microorganisms into Spodoptera frugiperda. BACKGROUND

[0002] Spodoptera frugiperda is a transnational migratory major pest, which has extremely strong reproductive capacity, migratory capacity, environmental adaptability and destructive power. Since it first invaded Yunnan Province of China in 2018, it has gradually spread and broken out in China, causing great disturbance to China's agricultural production. As an invasive species, Spodoptera frugiperda also has the characteristics of fast evolution of drug resistance, and because of this characteristic, the effect of chemical pesticides on Spodoptera frugiperda is not very good.

[0003] The intestinal microorganisms of insects are a complex structure, which includes fungi, bacteria, archaea, viruses and other microorganisms. Since bacteria and fungi have the highest abundance in the intestinal microorganism structure of insects, these two are often used to study the influence of intestinal microorganisms of insects on insects. It is found through research that the symbiotic bacteria in insects can have the following influences on insects: 1. The main action mode of affecting the nutritional metabolism of insects is direct absorption, assisting the host in degrading high-molecular-structure compounds, synthesizing nutrients and regulating the host's nutrient cycle; 2. The main action mode of enhancing the detoxification capacity of insects is ① the detoxification capacity of bacteria itself to pesticide compounds; ② indirectly improving the detoxification capacity of the host itself by stimulating the host's immune system. 3. Other functions, including causing gender ratio imbalance and causing cytoplasmic incompatibility in insects. At present, researchers are committed to studying the symbiotic microorganisms in Spodoptera frugiperda, which not only affect the adaptability, toxin-carrying possibility, population formation, invasiveness and evolution of Spodoptera frugiperda, but also believe that in-depth research on Spodoptera frugiperda and its functions will help the comprehensive control of Spodoptera frugiperda.

[0004] In the prior art, the methods for studying the influence of symbiotic bacteria on insects usually include microinjection and antibiotic removal. The operation process of microinjection is as follows: by means of a syringe and a microinjection instrument, the in-vitro cultured symbiotic bacteria are introduced into the insect by injecting into the mouth of the insect. The process of antibiotic removal is as follows: a suitable antibiotic is selected, the selected antibiotic is added to the culture medium of the insect for continuous feeding for several generations, and finally PCR is performed on the offspring to verify whether the removal is successful. However, the existing microinjection method requires the use of a syringe and a microinjection instrument, which is expensive and requires skilled technology. The antibiotic removal method requires continuous feeding for several generations, which takes a long time.

[0005] Therefore, based on the defects of the existing method for studying the influence of insect symbiotic bacteria on insects, it is necessary to improve it. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art, and provides a method for introducing in vitro cultured intestinal symbiotic microorganisms into Spodoptera frugiperda, comprising the following steps:

[0007] Step one, isolate, purify and shake culture of bacteria liquid from Spodoptera frugiperda;

[0008] Step two, starvation treatment of Spodoptera frugiperda larvae;

[0009] Step three, cut corn leaves into small pieces, soak them in the bacteria liquid prepared in step one, dry them, and then use them to feed Spodoptera frugiperda larvae, so as to introduce in vitro cultured intestinal symbiotic microorganisms into Spodoptera frugiperda.

[0010] Preferably, the method for isolating, purifying and shake culture of bacteria liquid from Spodoptera frugiperda in step one is as follows: treat Spodoptera frugiperda with high-efficiency cypermethrin, 24 hours later, dissect the intestines of the surviving Spodoptera frugiperda, remove the intestinal contents, cut the clean insect body intestines in sterile water, stand for a while, take the supernatant and spread it on 1 / 10 LB solid medium, culture overnight, pick single colony products, use 1 / 10 LB liquid medium for single colony expansion culture, extract bacterial DNA for sequencing, determine the strain classification, select the bacteria liquid of the strain to be introduced into Spodoptera frugiperda, centrifuge to collect bacteria, wash with sterile distilled water, add sterile distilled water, shake and mix, and obtain the bacteria liquid for subsequent treatment.

[0011] Preferably, the method for treating Spodoptera frugiperda with high-efficiency cypermethrin in step one is as follows: soak corn leaves in high-efficiency cypermethrin, dry the corn leaves, and then feed them to Spodoptera frugiperda third instar larvae.

[0012] Preferably, in step one, the temperature of overnight culture is 37℃, the standing time is 3 min, and the strain to be introduced into Spodoptera frugiperda is Stenotrophomonas maltophilia.

[0013] Preferably, in step one, the bacteria liquid with OD600=1.0 is obtained for subsequent treatment.

[0014] Preferably, in step two, the Spodoptera frugiperda is corn type Spodoptera frugiperda, the larvae are third instar larvae, the body weight is 0.0100-0.0150g, and the starvation treatment time is 4h.

[0015] Preferably, in step three, the corn leaves are mature corn leaves, the corn leaves are cut into small pieces with an area of 1cm 2 , soaked in the bacteria liquid prepared in step one for 5s, and then taken out and dried for feeding Spodoptera frugiperda larvae on the same day.

[0016] Preferably, in step three, when feeding the Spodoptera exigua larvae, the single feeding method is adopted, and one piece of corn leaf is fed to each larva per day for two days before treatment, and 3-4 pieces of corn leaf are fed to each larva per day from the third day of treatment to the prepupal stage.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a method for introducing in-vitro cultured intestinal symbiotic microorganisms into Spodoptera exigua, which does not require a micropipette compared with traditional microinjection technology, and is simple to operate; compared with the antibiotic removal method for studying the influence of symbiotic bacteria on insects, the present application does not require continuous feeding of multiple generations, and the experimental period is short; the method of the present application can change the bacterial structure in insects, thereby achieving the purpose of studying the influence of symbiotic bacteria on insect growth and development and the like.

[0019] Drawings accompanying the specification

[0020] Figure 1 PCoA analysis diagram of intestinal microorganisms of the treatment group (S) and intestinal microorganisms of the control group (CK) at the OUT level.

[0021] Figure 2 SOD value statistical diagram of intestinal microbial symbiotic bacterial community sample sequencing of the treatment group (S-1, S-2, S-3) and the control group (C-1, C-2, C-3).

[0022] Figure 3 Rank diagram of intestinal microbial symbiotic bacterial community sample sequencing of the treatment group (S-1, S-2, S-3) and the control group (C-1, C-2, C-3).

[0023] Figure 4 Door level species distribution stack diagram of intestinal microorganisms of the treatment group (S-1, S-2, S-3) and intestinal microorganisms of the control group (C-1, C-2, C-3).

[0024] Figure 5 Class level species distribution stack diagram of intestinal microorganisms of the treatment group (S-1, S-2, S-3) and intestinal microorganisms of the control group (C-1, C-2, C-3).

[0025] Figure 6 Objective level species distribution stack diagram of intestinal microorganisms of the treatment group (S-1, S-2, S-3) and intestinal microorganisms of the control group (C-1, C-2, C-3).

[0026] Figure 7Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0027] Figure 8 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0028] Figure 9 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0029] Figure 10 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0030] Figure 11 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0031] Figure 12 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3).

[0032] Figure 13 Figure 6 is a stacked bar chart of the distribution of species at the genus level of the intestinal microorganisms of the treatment groups (S-1, S-2, S-3) and the control groups (C-1, C-2, C-3). DETAILED DESCRIPTION

[0033] The present application will be described in detail below with specific embodiments, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. The raw materials used in the examples are commercially available unless otherwise specified.

[0034] Example 1: Introducing in vitro cultured intestinal symbiotic microorganism Stenotrophomonas maltophilia into Spodoptera frugiperda

[0035] Step one, isolate, purify, shake culture and obtain bacterial solution from Spodoptera frugiperda; the specific steps are as follows:

[0036] S11, soak corn leaves in 150 mg / L (half lethal concentration LC50 obtained by experiment) 10% high-efficiency cypermethrin, dry and feed 30 Spodoptera frugiperda third instar larvae with a body weight of 0.0100-0.0150 g, 8 died after 24 h, 22 survived (3 times repeated);

[0037] S12, in the super-clean sterile environment, the surviving armyworms are subjected to intestinal dissection, the intestinal contents are removed, the clean insect intestinal tracts are cut into pieces in 100 μL sterile water, the supernatant is taken after standing for 3 min and is coated on 1 / 10 LB solid medium, and the culture is incubated at 37°C overnight; the single colony product is picked and expanded in 1 / 10 LB liquid medium; the expansion culture is carried out at 37°C and 200 r / min on a shaker;

[0038] S13, the bacterial DNA is extracted by CTAB-lysozyme method: 1-5 mL of bacterial culture (106-108 cells, not more than 2 x 109 cells at most) is placed in a centrifuge tube, centrifuged at 12000 r / min (13400 x g) for 1 min, and the supernatant is carefully aspirated; 500 pL TE buffer is added for washing twice, then centrifuged at 12000 rpm for 3 min, and the precipitated bacterial bodies are collected; the precipitated bacterial bodies are resuspended with 300 μL TE buffer, 50 μL of 50 mg / mL lysozyme is added, and the mixture is gently mixed every 15 min for 30 min at 37°C; 10 μL of 20 mg / mL proteinase K and 60 μL of 10% SDS solution are added, and the mixture is gently mixed and incubated at 37°C for 1 h; 100 μL of 5 mol / L NaCl solution is added, and the centrifuge tube is inverted several times to mix thoroughly; 80 μL of CTAB / NaCl solution is added, and the mixture is gently mixed and incubated at 65°C for 10 min; 600 μL of chloroform / isoamyl alcohol (24:1) is added, and the mixture is gently mixed and centrifuged at 12000 r / min for 2 min; the supernatant is transferred to another clean centrifuge tube, an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) is added, and the mixture is inverted and mixed, then centrifuged at 12000 r / min for 2 min; the supernatant is transferred to another clean centrifuge tube, and 2 times the volume of ice-cold anhydrous ethanol is added to precipitate the DNA, and the mixture is gently mixed; centrifuged (12000 r / min, 30 min, 4°C) to completely remove the supernatant; centrifuged (12000 r / min, 30 min, 4°C) to completely remove the supernatant; the precipitate is washed with 300 μL of 70% pre-cooled ethanol, centrifuged (12000 r / min, 15 min, 4°C), the supernatant is discarded, and the precipitate is again centrifuged for a few seconds, then the alcohol is completely removed with a pipette, and the precipitate is air-dried; the precipitate is dissolved in 100 ul TE solution and stored at -20°C; the DNA concentration and quality are detected by ultraviolet spectrophotometry and 0.8% agarose gel electrophoresis;

[0039] S14, the extracted DNA is sequenced to determine that the screened strain is Xanthomonas maltophilia; the bacterial liquid is centrifuged to collect the bacterial bodies, washed with sterile distilled water 3 times, then anhydrous distilled water is added and mixed to obtain a bacterial liquid with OD600 = 1.0 for subsequent processing.

[0040] Step two, three larvae of Spodoptera exigua with a weight of 0.0100-0.0150 g were starved for 4 hours, and a total of 30 larvae of Spodoptera exigua were treated, and the treatment was repeated three times.

[0041] Step three, the mature leaves of the silver butterfly corn No. 9 were cut into small pieces with an area of 1 cm 2 , soaked in the bacterial solution prepared in step one for 5 seconds, and dried, and then used to feed the larvae of Spodoptera exigua on the same day. When feeding the larvae of Spodoptera exigua, the larvae were fed individually, and each larva was fed with one piece of corn leaf piece per day two days before treatment, and 3-4 pieces of corn leaf piece per day from the third day of treatment to the pre-pupa stage. The intestines of five larvae of Spodoptera exigua were removed and used as samples for intestinal 16sRNA microbial sequencing.

[0042] In this experiment, a control group was set up, and the control group used sterile water instead of bacterial solution to soak the corn leaves, and the rest of the operations were the same as steps two and three.

[0043] Analysis of the results of experiment example 2

[0044] I. Beta diversity analysis of the intestinal 16sRNA microbial sequencing results of Spodoptera exigua into which the Stenotrophomonas maltophilia was introduced (treatment group) and the intestinal 16sRNA microbial sequencing results of untreated blank Spodoptera exigua (control group) Figure 1 ). Figure 1 It can be seen that the OUT level of the intestinal microbial symbiotic flora of Spodoptera exigua changed significantly, indicating that the intestinal microbial community structure of Spodoptera exigua into which the Stenotrophomonas maltophilia was introduced by the method of the present application changed.

[0045] II. Alpha diversity analysis of the intestinal 16sRNA microbial sequencing results of Spodoptera exigua into which the Stenotrophomonas maltophilia was introduced (treatment group) and the intestinal 16sRNA microbial sequencing results of untreated blank Spodoptera exigua (control group) Figure 2 and Figure 3 ), from Figure 2 and Figure 3It can be seen that the bacterial flora abundance of the grass caterpillar fed with the corn leaves soaked with the bacterial liquid is obviously changed compared with the untreated blank group, mainly reflected in the SOD and rank abundance curves of sample sequencing, the intestinal flora abundance of the treated group is higher than that of the untreated blank group; compared with the untreated group, the species abundance in the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia by the method of the application is obviously changed, and the corresponding horizontal axis value of the Rank curve in the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia by the method of the application is obviously increased compared with the untreated blank CK group, which further indicates that the intestinal microbial species abundance of the grass caterpillar introduced with the Stenotrophomonas maltophilia by the method of the application is increased.

[0046] III. Compared the 16sRNA microbial sequencing results of the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia (treated group) with the 16sRNA microbial sequencing results of the intestine of the untreated blank grass caterpillar (control group), the intestinal microbial symbiotic flora community of the grass caterpillar at the door level changes obviously, mainly manifested as the changes of Proteobacteria, Patescibacteria, Fusobacteriota, etc. Figure 4 Compared with the control group, the abundance of Proteobacteria and Patescibacteria in the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia by the method of the application is increased, and the abundance of Fusobacteriota is reduced.

[0047] IV. Compared the 16sRNA microbial sequencing results of the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia (treated group) with the 16sRNA microbial sequencing results of the intestine of the untreated blank grass caterpillar (control group), the intestinal microbial symbiotic flora community of the grass caterpillar at the class level changes obviously, mainly manifested as the changes of Gammaproteobacteria, Actinobacteria, Saccharimonadia, Fusobacteriia, Negativicutes, etc. Figure 5 Compared with the control group, the abundance of Proteobacteria and Patescibacteria in the intestine of the grass caterpillar introduced with the Stenotrophomonas maltophilia by the method of the application is increased, and the abundance of Fusobacteriota is reduced.

[0048] V. Compared with the 16sRNA microbial sequencing results of the intestines of the untreated blank Spodoptera exigua (control group), the 16sRNA microbial sequencing results of the intestines of the Spodoptera exigua into which the Streptomyces avermitilis was introduced (treatment group) showed that the abundance of the intestinal microbial symbiotic flora community at the order level of the Spodoptera exigua changed obviously, mainly manifested as the orders of Lactobacillales, Pseudomonadales, Saccharimonadales, etc. Figure 6 Compared with the control group, the abundance of the orders of Pseudomonadales and Saccharimonadales in the intestines of the Spodoptera exigua into which the Streptomyces avermitilis was introduced by the method of the present application increased obviously, and the abundance of the order of Lactobacillales decreased slightly.

[0049] VI. Compared with the 16sRNA microbial sequencing results of the intestines of the untreated blank Spodoptera exigua (control group), the 16sRNA microbial sequencing results of the intestines of the Spodoptera exigua into which the Streptomyces avermitilis was introduced (treatment group) showed that the abundance of the intestinal microbial symbiotic flora community at the family level of the Spodoptera exigua changed obviously, mainly manifested as the families of Streptococcaceae, Neisseriaceae, Burkholderiaceae, Saccharimonadaceae, etc. Figure 7 Compared with the control group, the abundance of the families of Streptococcaceae and Burkholderiaceae in the intestines of the Spodoptera exigua into which the Streptomyces avermitilis was introduced by the method of the present application increased obviously, and the abundance of the families of Streptococcaceae and Neisseriaceae decreased obviously.

[0050] VII. Compared with the 16sRNA microbial sequencing results of the intestines of the untreated blank Spodoptera exigua (control group), the 16sRNA microbial sequencing results of the intestines of the Spodoptera exigua into which the Streptomyces avermitilis was introduced (treatment group) showed that the abundance of the intestinal microbial symbiotic flora community at the genus level of the Spodoptera exigua changed obviously, mainly manifested as the genera of TM7a and Enterobacter increased obviously, and the abundance of the genera of Streptococcus and Neisseria decreased obviously. Figure 8

[0051] ​Eight, compared the 16sRNA microbial sequencing results of the gut of the Spodoptera exigua into which the Methylobacterium was introduced (treatment group) with the 16sRNA microbial sequencing results of the gut of the untreated blank Spodoptera exigua (control group), the abundance of the intestinal microbial symbiotic flora of the Spodoptera exigua at the genus level changed obviously, and the comparison between the treatment group and the control group; there were 322 genera in common between the two, of which 78 genera were unique to the CK group, and the number of genera unique to the treatment group was 137; this indicated that after the method was used for treatment, the types of the intestinal microbial community of the Spodoptera exigua at the genus level increased Figure 9 ).

[0052] Nine, compared the 16sRNA microbial sequencing results of the gut of the Spodoptera exigua into which the Methylobacterium was introduced (treatment group) with the 16sRNA microbial sequencing results of the gut of the untreated blank Spodoptera exigua (control group), there were differences between the two in the LEfSe analysis diagram Figure 10 ), which indicated that in the intestinal microbial community of the Spodoptera exigua, the indicator species of the intestinal microbial community of the Spodoptera exigua of the treatment group changed from the indicator species of the intestinal microbial community of the Spodoptera exigua of the control group.

[0053] Ten, compared the 16sRNA microbial sequencing results of the gut of the Spodoptera exigua into which the Methylobacterium was introduced (treatment group) with the 16sRNA microbial sequencing results of the gut of the untreated blank Spodoptera exigua (control group), the proportion of the Xanthomonas in which the Methylobacterium was located in the individual microbial community increased from 0.2% to 0.3%, and the proportion of the protective bacteria in the insect increased from 0.9% to 1% Figure 11 and Figure 12 ).

[0054] Eleven, compared the 16sRNA microbial sequencing results of the gut of the Spodoptera exigua into which the Methylobacterium was introduced (treatment group) with the 16sRNA microbial sequencing results of the gut of the untreated blank Spodoptera exigua (control group), the flora function of the Spodoptera exigua of the treatment group changed from the control group, and some metabolic pathways and physiological functions in the insect were enhanced, including signal transduction, cell growth and death, and replication and repair Figure 13 ).

[0055] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. It is intended that the scope of the application be limited not with this detailed description, but rather by the claims appended hereto.

Claims

1. A method of introducing an in vitro cultured gut commensal microorganism into Spodoptera frugiperda, characterized in that, It comprises the following steps: Step one, isolate, purify, shake bacteria culture from the Spodoptera frugiperda to obtain a bacterial solution; Step two, starve the larvae of the Spodoptera frugiperda; Step three, cut the corn leaves into small pieces, soak them in the bacterial solution prepared in step one, dry them, and then feed the Spodoptera frugiperda larvae with them, so as to introduce the in vitro cultured intestinal symbiotic microorganism into the Spodoptera frugiperda; The method for isolating, purifying, and shaking bacteria culture from the Spodoptera frugiperda in step one is as follows: treat the Spodoptera frugiperda with high-efficiency cypermethrin, dissect the intestinal tract of the surviving Spodoptera frugiperda after 24 hours, remove the intestinal contents, cut the clean insect body into small pieces in sterile water, stand for a while, take the supernatant, and spread it on 1 / 10 LB solid medium for overnight culture, pick single colony products, and use 1 / 10 LB liquid medium for single colony expansion culture, extract bacterial DNA for sequencing, determine the strain classification, select the bacterial solution of the strain to be introduced into the Spodoptera frugiperda, centrifuge to collect the bacterial cells, wash them with sterile distilled water, add sterile distilled water, shake and mix, and obtain the bacterial solution for subsequent treatment; The method for treating the Spodoptera frugiperda with high-efficiency cypermethrin is as follows: soak corn leaves in high-efficiency cypermethrin, dry the corn leaves, and then feed the third instar larvae of the Spodoptera frugiperda with them; The temperature for overnight culture is 37℃, the standing time is 3 minutes, and the strain to be introduced into the Spodoptera frugiperda is Stenotrophomonas maltophilia; The bacterial solution with OD600=1.0 is obtained for subsequent treatment; In step three, the corn leaves are mature corn leaves, the corn leaves are cut into small pieces with an area of 1 cm 2 , soaked in the bacterial solution prepared in step one for 5 seconds, and then taken out and air-dried. The corn leaves are used to feed the corn earworm larvae on the same day. In step three, when feeding the Spodoptera frugiperda larvae, the single feeding method is adopted, one piece of corn leaf piece is fed to each larva per day for two days before treatment, and 3-4 pieces of corn leaf piece are fed to each larva per day from the third day of treatment to the pre-pupa stage; It does not need to continuously feed multiple generations, the experimental period is short, and it can change the bacterial structure in insects, so as to achieve the purpose of studying the influence of symbiotic bacteria on the growth and development of insects.

2. The method of claim 1, wherein: In step two, the Spodoptera frugiperda is corn type Spodoptera frugiperda, the larvae are third instar larvae, the body weight is 0.0100-0.0150g, and the starvation treatment time is 4 hours.

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