A genetically modified Yersinia entomophila engineering bacterium and its application

Abstract

The present invention specifically relates to a genetically modified Yersinia entomophila engineered bacterium CSLH88-pCHSW and its application. The strain is classified and named as Yersinia entomophila engineered bacterium Yersinia entomophagous , which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 6, 2021, with the deposit number CGMCC NO.23127. The Yersinia entomophila engineered bacteria CSLH88‑pCHSW obtained by the present invention can stably nptII-cry3AaT-hasA The exogenous gene can express Cry3AaT protein exogenously, and can achieve the synergistic effect of Yersinia insectivora CSLH88 and Cry3AaT insecticidal toxin protein, can be used in the prevention and control of coleopteran pests, and has significant insecticidal activity.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically modified Yersinia entomophila engineering bacterium CSLH88-pCHSW and an application thereof. Background Art

[0002] Yersinia entomophaga ( Yersinia entomophaga ) is a Gram-negative bacillus belonging to the family Enterobacteriaceae. This bacterium is a strain with insecticidal activity against a variety of insects and is often used in biological pest control. Yersinia entomophila was first isolated from insects of the family Scarabaeidae in the order Coleoptera. Relevant literature reports that it has insecticidal activity against Coleoptera, Lepidoptera, and Diptera, mainly because Yersinia entomophila can secrete a multi-subunit toxin complex (Yen-Tc), and the toxin complex (Tc) protein is a toxic protein with toxic effects. The structure of the Tc toxin complex is mainly a complex formed by two smaller protein subunits (A1 and A2) and B and C subunits. The toxin complex also carries two chitinases, Chi1 and Chi2. Its insecticidal mechanism is mainly that after the strain is eaten by insects, the chitinase contained in the toxin complex will enzymatically hydrolyze the insect midgut peritrophic membrane. If the bacteria enter the insect hemocoel after being eaten by insects, it can cause the death of the insect.

[0003] In addition, studies have shown that the Cry toxins secreted by Bt strains have certain insecticidal activity against coleopteran pests such as larvae of Cerambycidae, red palm weevils, and red flour beetles. As an insect pathogen, Yersinia entomophila also has certain insecticidal activity against coleopteran pests. If the two can be combined and molecular modification methods are used to construct Yersinia entomophila engineered bacteria with higher insecticidal toxicity, it can provide a new and effective option for the prevention and control of coleopteran pests. The present invention uses Yersinia entomophila as a carrier strain of Cry3AaT toxin protein, and uses exocrine expression systems, conjugation transfer, transposons and other technologies to construct highly toxic engineered strains, achieve stable inheritance of toxin genes, and secrete toxins from the strain cells to the extracellular space, which is conducive to increasing the contact between insecticidal toxins and coleopteran pests, and obtaining a more efficient pest control method. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a genetically modified Yersinia entomophila engineered bacterium CSLH88-pCHSW and its application. The engineered bacterium can achieve synergistic enhancement of Yersinia entomophila CSLH88 and Cry3AaT insecticidal toxin protein, greatly improving the insecticidal activity of Yersinia entomophila against coleopteran pests.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A genetically modified Yersinia entomophila engineered bacterium CSLH88-pCHSW, the strain is classified and named as Yersinia entomophila engineered bacterium Yersinia entomophaga It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.23127.

[0007] The above-mentioned Yersinia entomophila engineered bacteria CSLH88-pCHSW can stably inherit nptII - cry3AaT-has A exogenous gene, and can secrete the insecticidal toxin Cry3AaT.

[0008] The above-mentioned Yersinia entomophila engineered bacteria CSLH88-pCHSW is obtained by molecularly modifying the genome of Yersinia entomophila CSLH88.

[0009] The method of the above molecular modification is: using pCHSW plasmid to transform the nptII - cry3AaT-has A foreign gene was inserted into the genome of Yersinia entomophila CSLH88.

[0010] The above molecular modification method comprises the following specific steps:

[0011] Constructing a vector capable of expressing insecticidal toxin protein exocrine nptII - cry3AaT-has The pCHSW plasmid carrying the A gene, the nucleotide sequence of which is shown in SEQ ID NO.1; the pCHSW plasmid carrying the A gene was transformed by heat shock. nptII - cry3AaT-has pCHSW plasmid containing A gene E.coli S17-1λpir strain was used to obtain the pCHSW plasmid. E.coli S17-1λpir strain; E.coli S17-1λpir strain and Yersinia entomophila CSLH88 were co-cultured to obtain the genetically modified strain that can stably inherit nptII - cry3AaT-has A is an engineered bacterium of Yersinia insectivora CSLH88-pCHSW that contains an exogenous gene and can secrete and express Cry3AaT protein.

[0012] The application of the above-mentioned Yersinia entomophila engineered bacterium CSLH88-pCHSW in killing coleopteran pests.

[0013] Furthermore, the combination of the multi-subunit toxin complex Yen-Tc and the Cry3AaT toxin in the Yersinia entomophila engineered bacterium CSLH88-pCHSW provides a synergistic effect in killing coleopteran pests.

[0014] Furthermore, the coleopteran pests include one or more of Monochamus alternatus, Anoplophora anterior, Anoplophora glabripennis, Red palm weevil, and Tribolium castaneum.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] Yersinia entomophila CSLH88 itself has certain insecticidal activity against coleopteran pests. The present invention will secrete the gene of insecticidal toxin protein nptII - cry3AaT-has A was inserted into the genome of Yersinia entomophila CSLH88, making the foreign gene nptII - cry3AaT-has A can be stably inherited; and the HasA heme transport system is used to achieve exocrine expression of the insecticidal toxin protein Cry3AaT, increasing the possibility of toxin contact with the insect body, so that the insecticidal toxin acts directly on the intestine after being eaten by insects. In addition, the CSLH88-pCHSW engineered bacteria can simultaneously exert the effects of the Yersinia entomophila multi-subunit toxin complex (Yen-Tc) and the Cry3AaT toxin, and the synergistic effect between the two greatly improves the insecticidal activity of Yersinia entomophila against coleopteran pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the pCHSW plasmid map.

[0018] Figure 2 It is the electrophoresis diagram of the genomic DNA of the engineered bacteria CSLH88-pCHSW of Yersinia entomophila and the PCR product; wherein, lane M is the DNA marker, lane 1 is the genomic DNA of the engineered bacteria CSLH88-pCHSW of Yersinia entomophila, and lane 2 is the PCR product.

[0019] Figure 3 SDS-PAGE and Western blotting were used to analyze the expression of Cry3AaT; lane M is a protein marker, lane 1 is Cry3AaT extracellular protein (SDS-PAGE), lane 2 is Cry3AaT intracellular protein (SDS-PAGE), lane 1 is the extracellular protein of BRC-CXG2-pCHSW strain (Western blotting), and lane 2 is the intracellular protein of BRC-CXG2-pCHSW strain (Western blotting). DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] The materials, reagents and culture medium used in the following examples are as follows:

[0022] The pine beetle, star beetle, clouded beetle, star beetle, red palm weevil, red flour beetle larvae, and Yersinia entomophila CSLH88 involved in the present invention are all provided by the Key Laboratory of Major Pest Control in Ecological Public Welfare Forests of Fujian Province. E.coli S17-1λpir was purchased from Biovector NTCC Plasmid Vector Bacteria Cell Gene Collection Center. The pCHSW plasmid involved in the present invention was synthesized by GenScript Biotech Co., Ltd. Other conventional reagents can be obtained from commercial sources.

[0023] Example 1

[0024] A method for constructing a genetically modified Yersinia entomophila engineered bacterium, the specific steps are as follows:

[0025] (1) Construction of a donor strain carrying the nptII-cry3AaT-hasA expression element

[0026] The present invention uses the pBAM1 plasmid (GenBank: HQ908071) as a reference. The pBAM1 plasmid is a suicide plasmid that can only grow in specific strains. The plasmid components include: Kanna resistance gene KanR ; Transposable gene TNP ; Ampicillin resistance gene promoter AmpR promoter ; Ampicillin resistance gene AmpR ; Conjugative transfer gene oriT ; Replication origin R6KOriV ; Mosaic ME-O and ME-I Since the test strain Yersinia entomophila CSLH88 of the present invention is resistant to ampicillin (Amp), it is not possible to select AmpR gene as a resistance gene. However, the test strain Yersinia entomophila CSLH88 has no resistance to spectinomycin (Spec). Therefore, the present invention replaces the pBAM1 plasmid with AmpR Gene replacement with spectinomycin resistance gene ( SmR ) for subsequent donor strain screening and KanR Gene upstream insertion by nptII Promoter, cry3AaT Genes and hasAExpression elements composed of sequential gene splicing—— nptII-cry3AaT-hasA Gene, thereby constructing the pCHSW plasmid (see the plasmid map for details) Figure 1 ), and commissioned GenScript Biotech Co., Ltd. to synthesize the pCHSW plasmid. The nucleotide sequence of the pCHSW plasmid is shown in SEQ ID NO.1. Among them, the nptII promoter and hasA gene are from the pGHKW4 plasmid (Genbank: MK562404). cry3AaT The gene has the same nucleotide sequence as the gene encoding the BRC1912 toxin protein in patent CN111647058A [Bt toxin highly toxic to Monochamus alternatus and other Monochamus alternatus insects].

[0027] Heat shock transformation was used to transform the pCHSW plasmid into E.coli S17-1λpir (transformation method refers to the literature: Guo Peiyi, Chen Xiangdong, Xie Zhixiong, et al. A simple and rapid Escherichia coli plasmid transformation method [J]. Heredity, 1999, 21(4):4.), and the transformation product was obtained. The transformation product was spread on LB solid medium containing Spec antibiotics (final concentration of 100 μg / mL) and cultured in a 37 ℃ constant temperature incubator for 12 h. The positive transformants on the plate were picked and inoculated into LB liquid medium containing Spec antibiotics (final concentration of 100 μg / mL), and cultured at 150 r / min and 37 ℃ for 12 h. Subsequently, the plasmid was extracted according to the instructions of the plasmid extraction kit Plasmid MiniProtocol I of OMEGA, and the selection BamH I and Bag I restriction endonuclease double digestion, and finally sequencing verification, to obtain the pCHSW plasmid E.coli S17-1λpir strain, carrying the pCHSW plasmid E.coli The S17-1λpir strain was used as the donor strain in the next conjugative transfer experiment.

[0028] (2) Co-cultivation to obtain the engineered strain of Yersinia entomophila CSLH88-pCHSW

[0029] The donor bacteria carrying the pCHSW plasmid E.coli S17-1λpir and the recipient bacterium Yersinia entomophila CSLH88 were cultured to the logarithmic growth phase, and the cells were washed with 10 mM MgSO4 solution and resuspended to OD 600The volume ratio of the donor bacteria was 0.1, and then the bacterial suspension of the donor bacteria and the bacterial suspension of the recipient bacteria were mixed at a volume ratio of 1:1, and spread on an LB solid culture medium plate without any antibiotics and cultured at 37 °C for 5 hours. The bacterial lawn on the plate was scraped and cultured in an LB liquid culture medium containing Amp antibiotics (concentration of 200ug / ml) at 30 °C for 12 hours, and then streaked on an LB solid culture medium plate containing Kan antibiotics (concentration of 200ug / ml) at 30 °C for 12 hours, and a single colony was picked to obtain the Yersinia entomophila engineered bacteria.

[0030] (3) DNA extraction and detection of engineered strains

[0031] The single colony of Yersinia entomophila obtained above was inoculated into 5 mL of LB liquid medium containing Kan antibiotic and Amp antibiotic (final concentration of 100 μg / mL) and cultured at 150 r / min and 37 ℃ for 12 h. Subsequently, the DNA of the engineered bacteria was extracted by referring to the Bacterial DNA Kit D3350 of OMEGA Company, and finally PCR verification was performed using the extracted DNA as a template. PCR verification nptII-cry3AaT The primers required for the gene sequence are: forward primer (SEQ ID NO.2): 5′-CGAGCTCgtcaggctgttacagctcagagaaa-3′, reverse primer (SEQ ID NO.3): 3′-GCggatccGGATACGGCACCGGCGCAC-5′. The total PCR reaction system is 25 μL: template DNA 2 μL, forward and reverse primers (10 mM) 1.25 μL each, Q5® hot start super fidelity enzyme (NEB) 2× Master Mix 12.5 μL, ddH2O 8 μL. The PCR amplification system is: 98℃ 5 min; 98℃ 1 min, 66.1℃ 30 s, 72℃ 2 min, 30 cycles; 72℃ 10min. The electrophoresis results are as follows Figure 2 As shown, a 2000 bp target band can be observed, indicating that the exogenous gene nptII-cry3AaT-has A was successfully inserted into the genome of Yersinia entomophila, and the engineered strain of Yersinia entomophila was successfully constructed. The engineered strain was named CSLH88-pCHSW.

[0032] Example 2

[0033] The genetic stability test of the engineered strain of Yersinia entomophila CSLH88-pCHSW is as follows:

[0034] Take the CSLH88-pCHSW strain (bacterial solution OD 600The value was 0.6) and the inoculation volume was transferred to LB liquid medium without antibiotics at 1% (v / v), and the culture was continuously shaken at 37 ℃ and 150 r / min, and the tube was transferred every 12 hours. The bacterial liquid after the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, 19th, and 21st transfers was diluted and spread on LB solid medium without antibiotics, and cultured at 30 ℃ for 24 hours. 100 single colonies were picked and inoculated into LB solid medium containing Kan antibiotics (final concentration of 100 μg / ml) and LB solid medium without antibiotics, and the number of colonies was counted after culturing for 12 hours. Then the gene carrying rate was calculated by dividing the number of colonies in LB solid medium containing Kan antibiotics (concentration of 100 μg / ml) by the number of colonies in LB solid medium without antibiotics, so as to detect the genetic stability of the recombinant plasmid under non-selective pressure, and the CSLH88 strain treated under the same conditions was used as a negative control.

[0035] The results are shown in Table 1. Under the condition of no antibiotic selection pressure, the gene carrying rate of CSLH88-pCHSW strains in the 148th generation was 100%, indicating that the exogenous gene nptII-cry3AaT-has A can be stably inherited in the host strain. The present invention solves the problem of genetic instability existing in traditional transgenic engineering bacteria.

[0036] Table 1 Genetic stability test of strains

[0037]

[0038] The above-mentioned Yersinia entomophila engineered bacteria CSLH88-pCHSW was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021, with the deposit number CGMCC NO.23127.

[0039] Example 3

[0040] The steps for extraction and verification of the exocrine insecticidal toxin protein of the engineered strain CSLH88-pCHSW of Yersinia entomophila are as follows:

[0041] (1) Pipette 30 μL of engineered Yersinia entomophila CSLH88-pCHSW (bacterial solution OD 600 The bacterial suspension was inoculated into 3 mL of LB liquid medium containing Kan (final concentration was 100 μg / mL) and cultured at 30°C and 150 r / min for 12 h. Then, the bacterial suspension was transferred into 3 mL of new LB liquid medium containing Kan antibiotic (final concentration was 100 μg / mL) at a 1 vol% inoculum and cultured at 30°C and 150 r / min for 24 h.

[0042] (2) Centrifuge the cultured bacterial liquid in step (1) at 8000 r / min for 10 min at 4°C to collect the supernatant, slowly add the supernatant to an ice-pretreated ultrafiltration tube (Amicon Ultra-4 centrifugal filter devices), and centrifuge at 5000 r / min for 20 min at 4°C. Repeat this operation until all the supernatant is concentrated.

[0043] (3) After the centrifugation in step (2) is completed, the protein solution in the ultrafiltration tube is gently mixed with a pipette and collected into a centrifuge tube. This is the extracellular protein and is stored at -20°C for later use.

[0044] (4) Resuspend the cells centrifuged in step (2) above in PBS buffer (1.0 M, pH = 7.3), centrifuge at 4 °C and 8000 r / min for 8 min, and discard the supernatant. Repeat this step 3 times.

[0045] (5) The precipitate obtained in step (4) was placed on ice for ultrasonic disruption (50 Hz) at a frequency of 10 s and an interval of 5 s. The disruption time for each tube of bacteria was approximately 30 min. The disrupted cell solution was centrifuged at 4 °C and 8000 r / min for 8 min. The supernatant obtained was the intracellular protein and was stored at -20 °C for later use.

[0046] (6) Prepare SDS-PAGE gel according to the method of molecular cloning experiment guide, and prepare samples of extracellular protein and intracellular protein obtained in steps (3) and (5) for SDS-PAGE electrophoresis. Place the SDS-PAGE gel in R250 Coomassie brilliant blue staining solution and decolorizing solution for slow shaking for staining and decolorization, and finally analyze the SDS-PAGE situation using a two-dimensional electrophoresis scanning imager. At the same time, perform Western blotting analysis, first perform SDS-PAGE, then place the SDS-PAGE gel at 200 mA for 1 h to transfer the protein to the NC membrane, and then perform conventional 5wt% skim milk powder blocking, primary antibody incubation and secondary antibody incubation operations, wherein the primary antibody solution is Cry3Aa-Tag rabbit polyclonal antibody (Cry3Aa-TagRabbitePolycionalAntibody) diluted 1:4000, and the secondary antibody solution is goat anti-rabbit IgG (Goat Anti-Rabbit IgG) diluted 1:5000 by volume. Finally, a multifunctional imaging system (FluorChem Q, ProteinSimple) was used to detect the fluorescent bands on the NC membrane and analyze the expression of Cry3AaT protein.

[0047] The results are as follows Figure 3As shown: SDS-PAGE results show that the engineered Yersinia entomophila CSLH88-pCHSW can secrete proteins both intracellularly and extracellularly; Western blotting results show that the engineered Yersinia entomophila CSLH88-pCHSW has secretory expression of insecticidal toxin protein Cry3AaT both intracellularly and extracellularly. The above results show that the HasA heme transport system can function in the engineered Yersinia entomophila CSLH88-pCHSW and can successfully perform exocrine expression of the target protein.

[0048] Embodiment 4:

[0049] The toxicity of the engineered strain of Yersinia entomophila CSLH88-pCHSW to larvae of Cerambycidae (Monochromis alternatus, Anoplophora spheniscus, Anoplophora glabripennis, etc.) was determined as follows:

[0050] Place the second-instar Cerambycidae larvae in a 1.5 mL centrifuge tube containing sterilized, dried and single-distilled water-soaked paper towels, and feed them for 3 days at a constant temperature of 28°C to ensure the vitality of the larvae. After 3 days, transfer the Cerambycidae larvae with better vitality to a new 1.5 mL centrifuge tube that has been sterilized by high pressure, activate the culture with the isolated and purified bacterial liquid (Yersinia entomophila CSLH88 and Yersinia entomophila engineered bacteria CSLH88-pCHSW), and feed them with sterile paper towels. Prepare 15 replicates, and use single-distilled water feeding as a blank control. Observe and count the number of deaths of Cerambycidae larvae every day, and conduct LC after 10 days of observation. 50 calculate.

[0051] As shown in Table 2, the LC of CSLH88-pCHSW strain was 50 The LC of the unmodified Yersinia entomophila CSLH88 was 50 The decrease was exponential, which showed that Yersinia spp. CSLH88 itself had certain insecticidal activity. cry3AaT After the toxin protein gene was added, the virulence of the engineered bacteria was greatly improved, indicating that the Yersinia entomophila CSLH88 and the Cry3AaT toxin protein had a significant synergistic effect, and also indicating that the engineered bacteria CSLH88-pCHSW constructed by the present invention had extremely high insecticidal activity against the larvae of the family Cerambycidae.

[0052] Table 2 Insecticidal activity of CSLH88-pCHSW strain against Monochamus alternatus larvae

[0053]

[0054] Embodiment 5:

[0055] The toxicity of the engineered strain of Yersinia entomophila CSLH88-pCHSW to red palm weevils is determined as follows:

[0056] The artificial diet of red palm weevils was added to a 24-well plate, and a second-instar red palm weevil larva was placed in each well, with 3 replicates and 10 larvae in each replicate. Single distilled water was used as a blank control. The plates were placed in an artificial climate chamber for culture. Culture conditions: temperature was 28 °C, relative humidity was above 70%, and the photoperiod was 8 h / 16 h (day / night). They were initially fed for 3 days to ensure the vitality of the larvae. After 3 days, the red palm weevil larvae with better vitality were transferred to a new 24-well plate containing artificial diet for red palm weevils, and then the isolated and purified bacterial liquid (Yersinia entomophila CSLH88 and Yersinia entomophila engineered bacteria CSLH88-pCHSW) were activated and added to the 24-well plate. 15 replicates were prepared, and the blank control was used for feeding with single distilled water. The number of deaths of red palm weevil larvae was observed and counted every day. LC was performed after 10 days of observation. 50 calculate.

[0057] As shown in Table 3, the LC of the CSLH88-pCHSW strain 50 is 2.365×10 4 CFU / μL, LC of unmodified Yersinia entomophila CSLH88 50 is 3.274×10 6 CFU / μL. Compared with the two, the virulence of the CSLH88-pCHSW strain was greatly improved, indicating that the CSLH88-pCHSW strain had extremely high insecticidal activity against the larvae of red palm weevils, and the Yersinia insectophila CSLH88 and Cry3AaT toxin protein had a significant synergistic effect in preventing and controlling red palm weevils.

[0058] Table 3 Insecticidal activity of CSLH88-pCHSW strain against red palm weevil larvae

[0059]

[0060] Embodiment 6:

[0061] The toxicity of the engineered strain of Yersinia entomophila CSLH88-pCHSW to Tribolium castaneum was determined as follows:

[0062] The artificial diet of red flour beetle was added to a 24-well plate, and one I-instar red flour beetle larva was placed in each well, with 3 replicates and 10 larvae in each replicate. Single distilled water was used as a blank control. The culture was placed in an artificial climate chamber. Culture conditions: temperature was 28°C, relative humidity was above 70%, and the photoperiod was 8 h / 16 h (day / night). The larvae were initially fed for 3 days to ensure the vitality of the larvae. After 3 days, the red flour beetle larvae with better vitality were transferred to a new 24-well plate containing artificial diet of red flour beetle, and then the isolated and purified bacterial liquid (Yersinia entomophila CSLH88 and Yersinia entomophila engineered bacteria CSLH88-pCHSW) were activated and added to the 24-well plate. 15 replicates were prepared, and the blank control was used for feeding with single distilled water. The number of deaths of red flour beetle larvae was observed and counted every day. LC was performed after 10 days of observation. 50 calculate.

[0063] The results are shown in Table 4. The LC of CSLH88-pCHSW strain 50 is 4.564×10 4 CFU / μL, LC of unmodified Yersinia entomophila CSLH88 50 is 3.672×10 5 CFU / μL. Compared with the two, the virulence of the CSLH88-pCHSW strain was greatly improved, indicating that the CSLH88-pCHSW strain had extremely high insecticidal activity against the larvae of red flour beetle, and the Yersinia insectophila CSLH88 and Cry3AaT toxin protein had a significant synergistic effect in preventing and controlling red flour beetle.

[0064] Table 4 Insecticidal activity of CSLH88-pCHSW strain against Tribolium castaneum larvae

[0065]

Claims

1. A genetically modified Yersinia entomophila engineered bacterium CSLH88-pCHSW, characterized in that: The engineering bacteria CSLH88-pCHSW of Yersinia entomophila is named as Yersinia entomophila ( Yersinia entomophaga ), was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.23127.

2. The use of the engineered Yersinia entomophila CSLH88-pCHSW according to claim 1 in killing coleopteran pests, characterized in that: The coleopteran pests are one or more of Monochamus alternatus, Anoplophora anterior, Anoplophora glabripennis, Red palm weevil and Red flour beetle.

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