Genetic improvement of some microorganisms to increase the effect of bio-control on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)
Egyptian Journal of Biological Pest Control volume 33, Article number: 12 (2023)
During the last five decades, chemical synthetic pesticides have been used extensively to control pests and protect crops. Use of synthetic pesticide has caused some unfortunate consequences like environmental pollution, pest resistance and toxicity to other non-target organisms. Due to the hazardous effects of their chemical residues to human and animal health, several studies have been carried out to determine effective alternative control methods. One of methods is the usage of entomopathogens such as bacteria, virus and fungi. Entomopathogenic bacteria have unique insecticidal properties mainly due to the production of larvicidal proteins that accumulate as parasporal crystalline inclusions within the cell. The bio-insecticidal bacterium Bacillus thuringiensis (Bt) has been widely used in agriculture for the control of pest insects which attack crops.
Thirteen genetically stable fusants strains were obtained as a result of protoplast fusion technique between a local Bacillus thuringiensis (Bt) and each of B. subtilis subsp. subtilis strain (Bs1), Bacillus licheniformis strain (Bl) or B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were obtained, including three fusants (group B) from Bt::Bs1 fusion, six fusants (group C) from Bt::Bl fusion and four fusants (group D) from Bt::Bs2 fusion. All fusants were chosen for bioassay treatments against the potato tuber moth (PTM) Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) which recorded a high mortality percentage of PTM ranged from 75 to 80% in F1 and F2 of first attempt B (Bt::Bs1). The accumulative larval mortality was notation the highest percentage reached to 90% in case of treatment by fusants F4 followed by F7, F8 and F9 which gave 75, 70 and 80% larval mortality, respectively, for second attempt C (Bt::Bl). The third attempt D (Bt::Bs2) fusants F10, F11, F13 achieved the highest mortality percentage up to 60, 60 and 70%, respectively. Expression of apoptosis-related encoding genes in PTM was determined in three fusants B (Bt::Bs1), C (Bt::Bl) and D (Bt::Bs2) and compared with A (Bt) and Control. The results showed a high expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes in tissues of the (PTM) treated with different biological pesticides.
The study used protoplast fusion technique between a local Bacillus thuringiensis (Bt) and B. subtilis subsp. subtilis strain (Bs1), B. licheniformis strain (Bl) and B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were chosen for bioassay treatments against PTM. Bacterial fusant, F1, F2, F4, F7, F9 and F13, achieved the highest mortality rates against PTM ranged 75–90% under laboratory conditions. The highest expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes was recorded in insect tissues treated with different bio-insecticides. As a result of the effected on the genes responsible for expression the vital processes (genes of apoptosis) in the insect as a result of treatment with bacteria, this led to deformities and death of PTM.
Potato (Solanum tuberosum L.) (Solanaceae) is one of the major food crops all over the globe. Potato crop comes at the fourth rank after wheat, maize and rice. Egypt is producing around 4,800,000 tons of potatoes per year, and about 180–200 thousand feddan are cultivated in three different plantations seasons throughout the year. Egypt is also playing an important role in potato production, and it is considered one of the largest exporters to Europe and Arab countries (Abou Elmaaty 2012). Different numbers of insect pests are infesting the potato tubers either in the field or in the storage. The potato tuber moth Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) is considered as the most destructive pest (Sabbour et al. 2012). It causes severe damage to potato foliage and tubers. The production loss may reach 30–70% in some areas like India, North Africa and the Middle East. Several control methods including insecticides have been used in order to reduce the damage caused by PTM. Due to their negative side effects on non-targeted organisms, other control means as the biopesticide.
Bacillus thuringiensis as a spore forming Bacilli have received the most attention as biological control agent. Many isolates produce proteinaceous insect protoxins during sporulation. Members of Bt have been used as a successful microbial pesticide against several insect pests, particularly Lepidopterans, Coleopterans and Dipterans (Tsedaley 2015). The isolation of new Bt strains from soil or other environments received the attention of scientists worldwide according to the recommendation of UNEP (United Nations of Environmental Program 1992) to isolate indigenous isolates for pest control. Sabbour and Moharam (2015) isolated seven bacterial strains of Bt from the Egyptian soil and tested them against the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae).
Bacillus thuringiensis (Bt), B. subtilis subsp. subtilis (Bs1), B. licheniformis (Bl) and B. subtilis subsp. spizizeniie (Bs2) strains were grown on LB medium at 37C0 overnight with shaking 250 rpm/24 h and tested for their antimicrobial susceptibility. Eleven antibiotics were used with final concentrations as follows: Rifampicin (Rif), 100 μg/ml; ampicillin (Amp), 100 μg/ml; amikacin (Amk), 30 μg/ml; streptomycin (Sm), 200 μg/ml; kanamycin (Km), 40 μg/ml; tetracycline (Tc), 15 μg/ml; chloramphenicol (Cm), 35 μg/ml; gentamicin (Gm), 15 μg/ml; polymyxin (Pmx), 50 μg/ml; neomycin (Nm), 40 μg/ml; and erythromycin (Erm), 20 μg/ml. The Kirby–Bauer disk diffusion method for antimicrobial susceptibility test was used National Committee for Clinical Laboratory Standards (NCCLS 1999).
Protoplast fusion of Bacillus thuringiensis (Bt), B. subtilis subsp. subtilis (Bs1), B. licheniformis (Bl) and B. subtilis subsp. spizizeniie (Bs2) was carried out according to Mohamed et al. (2022).
Different stages of the PTM were collected from the laboratory colony kept at the National Research Centre (NRC), Pests and Plant Protection Department, Giza, Egypt. Infected tubers were placed in plastic containers (25 × 12 × 10 cm) with a thin layer of sterile sand on top to act as a pupation substrate. The pupae were collected out of the sand each day and placed in (750 ml) mating cups covered with black muslin. The eggs were removed from the muslin, which serves as the oviposition site for adult females (Moawad and Ebadah 2007).
Effect of bacterial strains and their fusions against the potato tuber moth
The experiments tested four bacterial strains and their fusions in vitro conditions. Potato tuber was washed well and left to dry. Each of the 20 tubers was sprayed by the tested bacteria strains; one concentration was used (1.5 × 107 cells/ml) (original concentration) and left for 5 min to dry. Each tuber was then placed in a plastic cup (300 ml) containing thin layer of sterile sand. Each treated tuber was infested by adding 5 eggs (at last stage before hatching) on it. Each treatment was examined daily to count the larval mortality percentage. Also, penetration% of 1st instar larvae, pupal and adult malformation were studied. The two controls were the untreated one and the other was a Bt parent. Each test was replicated four times. The mortality percent in treatments was corrected according to Abbott’s formula (Abbott 1925). Mortality% = (T − C)/(100 − C) × 100.
Expression of apoptosis related encoding genes in insects
Analyzing the expression of apoptotic genes
Total RNA was isolated from entire tissues of the PTM by the standard TRIzol® Reagent extraction method (Invitrogen, Germany). Briefly, tissue samples were homogenized in 1 ml of TRIzol® Reagent per 50–100 mg of the tissue. Afterward, the homogenized sample was incubated for 15 min at room temperature. A volume of 0.2 ml of chloroform per 1 ml of TRIzol® Reagent was added. Then, the samples were vortexed vigorously for 15 s and incubated at room temperature for 3 min. The samples were centrifuged for no more than 12,000 ×g for 15 min at 4 °C. Following centrifugation, the mixture was separated into low red, phenol–chloroform phase, an interphase and a colorless upper aqueous phase. RNA was remained exclusively in the aqueous phase. Therefore, the upper aqueous phase was carefully transferred without disturbing the interphase into a fresh tube. The RNA was precipitated from the aqueous phase by mixing with isopropyl alcohol. A volume of 0.5 ml of isopropyl alcohol was added per 1 ml of TRIzol® Reagent used for the initial homogenization. Afterward, the samples were incubated at 15–30 °C for 10 min and centrifuged at not more than 12,000 ×g for 10 min at 4 °C. The precipitated RNA as often invisible before centrifugation formed a gel-like pellet on the side and bottom of the tube. The supernatant was removed completely. The RNA pellet was washed once with 1 ml of 75% ethanol. The samples were mixed by vortexing and centrifuged at no more than 7500 ×g for 5 min at 4 °C. The supernatant was removed, and RNA pellet was air-dried for 10 min. RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water by passing the solution a few times through a pipette tip.
Total RNA was treated by 1 U of RQ1 RNase-free DNase (Invitrogen, Germany) to digest DNA residues, re-suspended in DEPC-treated water. Purity of total RNA was assessed by the 260/280 nm ratio (between 1.8 and 2.1). Additionally, integrity was assured with ethidium bromide stain analysis of 28S and 18S bands by formaldehyde-containing agarose gel electrophoresis. Aliquots were used immediately for reverse transcription (RT), otherwise stored at − 80 °C.
Reverse transcription (RT) reaction
The complete Poly(A)+ RNA isolated from insect tissues was reverse transcribed into cDNA in a total volume of 20 μl using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, Germany). An amount of total RNA (5 μg) was used with a reaction mixture, termed as master mix (MM). The MM was consisted of 50 mM MgCl2, 5 × reverse transcription (RT) buffer (50 mM KCl; 10 mM Tris–HCl; pH 8.3); 10 mM of each dNTP, 50 μM oligo-dT primer, 20 U ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 U M-MuLV reverse transcriptase. The mixture of each sample was centrifuged for 30 s at 1000 g and transferred to the thermocycler (Biometra GmbH, Göttingen, Germany). The RT reaction was carried out at 25 °C for 10 min, followed by 1 h at 42 °C, and the reaction was stopped by heating for 5 min at 99 °C. Afterward, the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for DNA amplification through real-time polymerase chain reaction (RT-PCR).
Real-time quantitative PCR (RT-qPCR)
A StepOne Real-Time PCR System (Applied Biosystems, USA) was used to assess the copy of the cDNA of moth insect tissues to detect the expression values of the tested genes. The PCR reaction was prepared containing 12.5 μl of SYBR® green (TaKaRa, Biotech. Co. Ltd.), 0.5 μl of 0.2lM forward and reverse primers, 6.5 μl DNA-RNA free water and 2.5 μl of the synthesized cDNA. The cDNA was propagated using reaction program consisted of 3 steps. In the first step, the PCR tubes were incubated at 95 °C for 3 min. The second step consisted of 50 cycles. Each cycle consisted of 3 sub-steps: (a) 15 s at 95 °C; (b) 30 s at 60 °C; and (c) 30 s at 72 °C. The third step consisted of 71 cycles. The first cycle of them started at 60 °C for 10 s, and then, the followed cycles increased about 0.5 °C every 10 s up to 95.0 °C. A melting curve of the reaction was performed for each RT-qPCR termination at 95.0 °C to assess the quality of the primers. To verify that the reaction of the RT-qPCR had not any contamination tubes containing non-template were used as control.
Real-time polymerase chain reaction (RT-PCR)
Thermo Fisher Scientific, Waltham, MA, USA StepOne™ Real-Time PCR System was utilized to ascertain the insect’s cDNA copy number. PCR reactions were set up in 25 μl containing 12.5 μl 1 × SYBR® Premix ExTaqTM (TaKaRa, Biotech. Co. Ltd.), 0.5 μl 0.2 μM sense primer, 0.5 μl 0.2 μM antisense primer, 6.5 μl distilled water and 5 μl of cDNA template. The reaction program was allocated to 3 steps. First step was at 95.0 °C for 3 min. Second step consisted of 40 cycles each cycle divided into 3 steps: (a) at 95.0 °C for 15 s; (b) at 55.0 °C for 30 s; and (c) at 72.0 °C for 30 s. The third step consisted of 71 cycles which started at 60.0 °C and then increased about 0.5 °C every 10 s up to 95.0 °C. At the end of each sqRT-PCR, a melting curve analysis was performed at 95.0 °C to check the quality of the used primers. Each experiment included a distilled water control. According to Ocampo et al. (2013), the sequences of specific primers of the genes used are listed in Table 1. The relative quantification of the target to the reference was determined by using the 2−ΔΔCT method as follows:
All data obtained from biochemical and molecular genetics studies were expressed as means ± SEM. The data were investigated with the Statistical Package for Social Sciences (SPSS 0.26 for windows). The outcomes were dissected utilizing one route investigation of difference (ANOVA) trailed by Duncan’s test for examination between various treatment gatherings, and statistical significance was set at P < 0.05.
Antibiotic resistance pattern of Bt, Bs1, Bl and Bs2 strains
The antibiotic resistance of the Bt, Bs1, Bl and Bs2 strains is presented in Table 2. The results showed that all strains were resistant to Streptomycin (Smr), while they are sensitive to three different antibiotics, i.e., Gentamicin (Gms), Kanamycin (Kms) and Neomycin (Nms). The two strains Bs1 and Bl revealed similar antibiotic resistance pattern characterized by their resistance to three antibiotics, i.e., Tetracycline (Tcr), Ampicillin (Ampr) and Chloramphenicol (Cmr). The Bl strain was sensitive to four antibiotics: amikacin (Amks), erythromycin (Erms), rifampicin (Rifs) and polymyxin (Pmxs). The strain (Bt) revealed antibiotic resistance pattern, where it was resistant to four antibiotics (Rifr, Amkr, Ampr and Pmxr), while it was sensitive to three antibiotics (Tcs, Cms and Erms). On the other hand, one strain Bs1 was resistance to three antibiotics (Amkr, Ermr and Rifr), while it was sensitive to one antibiotic (Pmxs) and the other Bs2 resistance to one antibiotic (Cmr), while it was sensitive to six antibiotics (Tcs, Amks, Erms, Rifs, Amps and Pmxs).
Protoplast fusion between Bt and each of Bs1, Bl and Bs2.
Three attempts of protoplast fusion technique were used between B. thuringiensis (Bt), B. subtilis subsp. subtilis strain (Bs1) and each of B. licheniformis strain (Bl) or B. subtilis subsp. spizizeniie (Bs2). Protoplast fusion technique is used to produce genetically stable modified microorganisms 3 fusants B (Bt::Bs1) after 40 min, 6 fusants C (Bt::Bl) after 50 min (F4,F5 and F6) and 70 min (F7, F8 and F9), 4 fusants D (Bt::Bs2) after 50 min (F10),60 min (F11 and F12) and 120 min (F13).
Effect of different fusants on PTM on some biological aspects of the potato tuber moth Phthorimaea operculella
Effect of (Bt::Bs1) fusants on PTM
Fusant strains of Bt and Bs1 (Bt::Bs1) had more toxic impact on larvae of the PTM compared to their parents. The highly impact was F2 more than F1 and F3 were recorded mortality percentage 80, 75 and 60% compare to their parents Bt and Bs1 which recorded 30 and 0.0% mortality, respectively. Also, data in (Table 3) indicated that the treatment by fusion B (Bt::Bs1) caused reduction in % penetration of larvae to potato tuber reached to 40% in F2 fusant than its parents which record 75 and 83% penetrations to PTM larvae, respectively. On the other hand, all treatments caused in appearance pupal and adult malformations.
Effect of (Bt::Bl) fusants on PTM larvae
The fusant F4 C(Bt::B1) had the fusants high impact on larvae that led to increase mortality percentage reaching 90% compared to Bt and Bl parents 30 and 35%, respectively (Table 4). On the other hand, all treatments recoded pupal and adult malformations, but the parents were recorded malformations more than their genetically stable fusants.
Effect of (Bt::Bs2) fusants on PTM larvae
Treatment of potato tubers by fusants no F10, F12 and F13 produced from experiment D (Bt::Bs2) caused effect on 1st instar larvae of PTM, but the high impact was fusion F13 followed by F10 and F12 which recorded 70 and 60% mortality, respectively, than their parents Bt and Bs2 which caused 30 and 40% mortality, respectively (Table 5). The pupal and adult malformation were appeared in all cases of treatment at different level degrees.
Analyzing gene expression with quantitative real-time (RT-qPCR)
The results of the gene expression analysis using RT-qPCR are summarized in Figs. 1, 2, 3. The genes encoding apoptotic proteins were determined in four fusants treatments in which the results showed the highest levels of expression of Caspase-16 gene (Fig. 1) to the treatment of fusants D (group) (Bt:Bs2). However, the levels of Dronc (Fig. 2) and Dredd (Fig. 3) genes expression in fusant-treated insects’ tissues D (Bt:Bs2) were lower than those in Caspase-16. The expression levels of all tested genes Caspase-16, Dronc and Dredd genes in group A (Bt parent) were overexpressed but in lower levels than with group D. Group C exhibited expression levels of Caspase-16, Dronc and Dredd genes lower than those shown in Groups D and A. Additionally, Group B revealed the lowest expression levels of Caspase-16, Dronc and Dredd genes compared to Group D, Group A and Group C. Contrarily, compared to other treated groups, the expression levels of the Caspase-16, Dronc and Dredd genes in the control group were at baseline levels.
Recombinant fusant strains obtained from protoplast fusion experiment between B. thuringiensis (Bt) and three Bacillus strains revealed higher toxicity than the parental strains with more than threefold. Compared to insecticidal Bt strains, recombinant fusants are more effective and offer greater potential for use in agriculture against the PTM larvae. The strain (Bt) revealed antibiotic resistance pattern, where it was resistant to four antibiotics (Rifr, Amkr, Ampr and Pmxr), which showed 30% mortality against the PTM larvae. The two parental strains’ different patterns of antibiotic resistance were employed as selectable markers to screen for the fusant strains. A total of 3 Bt::Bs1 fusants were obtained after 2 h of fusion time on selective medium containing the three antibiotics, Pmx, Erm and Tc. A total of 6 Bt::Bl fusants were obtained after 2 h of fusion time on selective medium containing the three antibiotics, Pmx, Amk and Tc. A total of four Bt::Bs2 fusants were obtained after 2 h of fusion time on selective medium containing the three antibiotics, Pmx, Amk and Cm. The three Bacillus strains revealed Bs1 0% and Bl 35% Bs2 40%, respectively, mortality against the PTM larvae. This showed that each of the two parental strains of the fusant strains combined their respective antibiotic properties. In order to identify the recombinants of the combined protoplasts of B. cereus and B. thuringiensis subsp. galleriae that carry the plasmid pBC16 responsible for Tc resistance, Belykh et al. 1983 utilized a medium containing Rif and Tc. antibiotic resistance pattern as a selectable marker. Similar to this, fusing of B. cereus protoplasts led to the isolation of hybrid cells with a high frequency of the Tcr phenotype (harboring pBC16) (Kovtunenko et al. 1985). Mohamed et al. (2022) constructed protoplast fusions between B. amyloliquefaciens and L. sphaericus based on the antibiotic markers to improve the nematicidal activity for fusants compared to their parental strains.
The 13 recombinant fusants showed that mortality percentages against the PTM larvae ranged from 45 to 90%. Seven of the 13 generated fusants were chosen because they had high mortality rates to P. operculella larvae. This is demonstrated by the newly developed fusant strains’ expression of a few insecticidal crystal protein genes (cry) derived from Bt genes agree with Mohamed et al. (2016) studied that produce cry proteins and able to naturally colonize the phylloplane of host plants to increase either the effect of biocontrol on pests and or persistence for long time. The results are consistent with those of Mohamed et al. (2022), who investigated the impact of protoplast fusion between two antibiotic-resistant mutants of Bt subsp. israelensis (H14) on the production of δ-endotoxin. They discovered that the fusion type has higher capacity for protein synthesis (δ-endotoxin concentration) than the wild type, with a ratio of 1.48. In general, numerous researchers have employed the protoplast fusion approach to create recombinant strains using novel combinations from various bacteria. For instance, Agbessi et al. (2003) produced stable prototrophic recombinants of Streptomyces melanosporofaciens, a biocontrol agent of plant disease that produces geldanamycin, and characterized two recombinant strains (FP-54 and FP-60) that differed in their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390. Hussein et al. (2010) used novel Bacillus strains with a broader spectrum of action against various insects by fusing protoplasts from the Bt strain I977 and the Bs strain GHAI. Their findings showed that most fusants expressed cry genes from Bt genes that encode insecticidal crystal proteins. The obtained results agree with Bouthaina and Merdanand (2018) treating larval diet with Bt isolates affected larval and pupal mortality. Reduction in number of deposited eggs and adult emergence with mild elongation in larval duration and adult longevity were recorded. The effect on fecundity and fertility of resulted females was detected.
The genes encoding apoptotic proteins were determined in three fusants treatments in which the results showed the high expression levels of Caspase-16 gene to the treatment of fusants D (Bt:Bs2). However, the expression levels of Dronc and Dredd genes in tissues of the insects treated with fusants D (Bt:Bs2) were lower than those in Caspase-16. The expression levels of all tested genes Caspase-16, Dronc and Dredd genes in group A (Bt parent) were overexpressed but in lower levels compared with group D. Group C exhibited expression levels of Caspase-16, Dronc and Dredd genes lower than those shown in Groups D and A. Additionally, Group B revealed the lowest expression levels of Caspase-16, Dronc and Dredd genes compared to Group D, Group A and Group C. On the other hand, the expression levels of Caspase-16, Dronc and Dredd genes in control group were in baseline levels compared to other treated groups. In the same line with our findings, (Hussein et al. 2014) telomerase reverse transcriptase and caspase-8m-RNA expression levels were measured using real-time PCR. The melting curves of both TERT and caspase-8 mRNA as well as the amplification plots of fluorescence intensity against PCR cycle were obtained from tissue samples. When compared to the control group, all groups’ expression levels of caspase-8 were considerably low (p1 0.001). Group III significantly increased caspase-8 expression levels compared to group II (p2 = 0.031); however, groups IV and V did not (p2 = 0.992, 1.000 and 1.000, respectively). Group IV did not substantially differ from group III in terms of caspase-8 expression level (p3 = 0.098); however, group V considerably decreased it (p3 = 0.038). When compared to group IV, group V did not exhibit any discernible difference (p4 = 0.996). Moreover, Alam et al. (2016) proved that the ethanolic extract of U. lactuca appears to be more efficient in down-regulations of apoptotic genes Bax and Caspase-3 than the aqueous one. They suggested that total phenolics, flavonoids and sulfated polysaccharides, as well known in U. lactuca, are thought to be the main drivers responsible for this potent antioxidant defense mechanism. The widely distributed green alga U. lactuca seems to be a promising antioxidant and anti-apoptotic tool to overcome the lethal effects of γ-irradiation. In Awad et al. (2018), analysis of gene expression (qRT-PCR), DNA fragmentation test and apoptosis assay were conducted to determine the Feline panleukopenia disease in cat tissues.
It was concluded that protoplast fusion technique was used between a local B. thuringiensis (Bt) and each of B. subtilis subsp. subtilis strain (Bs1), B. licheniformis strain (Bl) and B. subtilis subsp. spizizeniie (Bs2). All fusants were chosen for bioassay treatments against the P. operculella which recorded a high mortality percentage of (PTM) of first attempt B (Bt::Bs1). The highest larval mortality rate was notation in case of fusants treatments for second attempt C (Bt::Bl). The third attempt D (Bt::Bs2) fusants F10, F11, F13 achieved the highest mortality percentages. The genes encoding were determined in three treatments by fusants B (Bt::Bs1), C (Bt::Bl), D (Bt::Bs2), A (Bt) and Control. The highest expression of gene apoptosis was in fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes in tissues of the insects treated with different bio-insecticides.
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Mohamed, S.AH., Kh, AEA.S., Moawad, S.S. et al. Genetic improvement of some microorganisms to increase the effect of bio-control on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). Egypt J Biol Pest Control 33, 12 (2023). https://doi.org/10.1186/s41938-023-00648-5