Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly

doi:10.12140/j.issn.1000-7423.2022.03.012

Abstract

Abstract:

Objective To isolate cultivable bacteria from the intestine of housefly larvae, investigate the activity of digestive enzymes produced by these bacteria, and explore the effects of intestinal bacteria on the digestion of food and the growth and development of housefly larvae. Methods The bacteria in the intestine of housefly larvae were isolated and purified by traditional bacterial isolation and culture method, and 16S rRNA gene sequence was analyzed for molecular identification of identify species. Using the bacteria culture medium of different property to screen the bacteria producing amylase, cellulase, protease, or lipase, and the diameter ratio of hydrolytic circle (D) to bacterial colony (d) was measured to compare the activities of digestive enzymes produced by different bacteria. SPSS 20.0 software was used for statistical analysis. A one-way analysis of variance was used for comparison between groups. Results A total of 9 species of facultative anaerobic bacteria, which belongs to 6 genera, were isolated from the intestines of housefly larvae cultured in an anaerobic environment, among which there were 3 species in Providence (Providencia sneebia DSM 19967, P. rettgeri and P. stuartii), 2 species in Enterococcus (E. casseliflavus and E. faecalis), and 1 specie each in Pseudocitrobacter (P. faecalis), Morganella (M. morganii), Enterobacter (E. hormaechei) and Klebsiella (K. pneumoniae). A total of 10 species of bacteria belonging to 8 genera were isolated from the intestines of housefly larvae cultured in an aerobic environment, among which there were 2 species in Enterobacter (E. hormaechei and E. cloacae), 2 species in Providence (P. stuartii and P. vermicola), and 1 specie each in Klebsiella (K. pneumoniae), Pseudomonas (P. aeruginosa), Acinetobacter (A. bereziniae), Lactococcus (L. lactis), Lysinibacillus (L. fusiformis) and Bacillus (B. safensis). Among these facultative anaerobes were isolated from the intestines of housefly larvae cultured in an anaerobic environment, 9 produced amylase, and 8 produced cellulase and 1 produced lipase; no bacteria produced protease under anaerobic conditions. Among these facultative anaerobes, 9 produced amylase and 8 produced cellulase; no bacteria produced protease and lipase detected in aerobic condition. Under anaerobic conditions, there was no difference in amylase activity among 9 facultative anaerobic culture bacteria (F = 1.953, P > 0.05); among the cellulase activities of 8 facultative anaerobes, E. casseliflavus lead had the strongest cellulase activity, with a D/d value of 1.36 ± 0.06 (F = 3.367, P < 0.05); and only P. rettgeri produced lipase, with a D/d value of 2.28 ± 0.16. In the aerobic state, among the amylase activities of 9 facultative anaerobic bacteria, E. faecalis had the strongest amylase-producing activity, and the D/d value of 1.42 ± 0.06 (F = 3.881, P < 0.05). Among the cellulase activities of 8 facultative anaerobes, E. casseliflavus had the strongest cellulase activity, with a D/d value of 1.29 ± 0.01（F = 6.633, P < 0.05）. Among aerobic bacteria, P. aeruginosa and B. safensis produced protease, with D/d values of 3.67 ± 0.25 and 3.58 ± 0.31 (F = 0.087, P > 0.05); and only K. pneumoniae produced amylase, with a D/d value of 4.83 ± 0.12; no bacteria produced cellulase and lipase. Conclusion The composition of cultivable bacteria in the intestine of housefly larvae is relatively unitary, but most anaerobic bacteria and aerobic bacteria have the function of secreting digestive enzymes.

Key words: Housefly larvae, Intestinal anaerobic bacteria, Intestinal aerobic bacteria, Digestive enzyme-producing bacteria

CLC Number:

R384.21

ZHANG Ke-xin, LIU Wen-juan, ZHANG Xin-yu, ZHANG Qian, ZHANG Rui-ling, ZHANG Zhong. Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(3): 355-361.

Figures/Tables 5

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References 35

[1]	Engel P,, Moran NA. The gut microbiota of insects-diversity in structure and function[J]. FEMS Microbiol Rev, 2013, 37(5): 699-735. doi: 10.1111/1574-6976.12025
[2]	Cai Z,, Yu Q,, Cheng G. Progress towards mosquito microbiome on regulating the transmission of mosquito-borne diseases[J]. Chin J Parasitol Parasit Dis, 2019, 37(5): 603-608. (in Chinese)
	( 蔡珍,, 余茜,, 程功. 蚊肠道微生物调节蚊媒传染病传播的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(5): 603-608.)
[3]	Wang XM,, Wu K,, Chen XG, et al. Research advances on diversity and function of mosquito-bacteria symbiosis[J]. Chin J Parasitol Parasit Dis, 2017, 35(3): 305-312. (in Chinese)
	( 王晓明,, 吴焜,, 陈晓光, 等. 蚊虫共生微生物群多样性及功能的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 305-312.)
[4]	Ren SE,, Nan XN,, Xu M, et al. Comparison of intestinal bacterial diversity of Gansu zokor under wild and artificial feeding conditions[J]. Acta Microbiol Sin, 2020, 60(4): 826-838. (in Chinese)
	( 任世恩,, 南小宁,, 许淼, 等. 野生和人工饲喂条件下甘肃鼢鼠肠道细菌多样性比较[J]. 微生物学报, 2020, 60(4): 826-838.)
[5]	Gao HH,, Qin DY,, Dai XY, et al. Effects of intestinal bacteria Citrobacter freundi and Klebsiella oxytoca on the development and substance metabolism of Drosophila suzukii (Diptera ∶ Drosophilidae)[J]. Acta Entomol Sin, 2020, 63(4): 462-469. (in Chinese)
	( 高欢欢,, 覃冬云,, 代晓彦, 等. 肠道细菌弗氏柠檬酸杆菌和产酸克雷伯氏菌对斑翅果蝇生长发育和物质代谢的影响[J]. 昆虫学报, 2020, 63(4): 462-469.)
[6]	Guo XX,, Wang HW. Research progress on the molecular mechanisms of mosquito innate immunity[J]. Chin J Parasitol Parasit Dis, 2015, 33(1): 52-57. (in Chinese)
	( 郭秀霞,, 王怀位. 蚊虫先天免疫分子机制的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(1): 52-57.)
[7]	Huang SW. Molecular diversity of bacterial community in the gut of Holotrichia parallela (Coleoptera∶ Scarabaeidae)[D]. Wuhan: Huazhong Agricultural University, 2009: 1-77. (in Chinese)
	( 黄胜威. 暗黑鳃金龟肠道共生菌分子多态性研究[D]. 武汉: 华中农业大学, 2009: 1-77.)
[8]	Yang YT,, Guo JY,, Long CY, et al. Advances in endosymbionts and their functions in insects[J]. Acta Entomol Sin, 2014, 57(1): 111-122. (in Chinese)
	( 杨义婷,, 郭建洋,, 龙楚云, 等. 昆虫内共生菌及其功能研究进展[J]. 昆虫学报, 2014, 57(1): 111-122.)
[9]	Zhou F,, Pang ZC,, Yu XQ, et al. Insect gut microbiota research: progress and applications[J]. Chin J Appl Entomol, 2020, 57(3): 600-607. (in Chinese)
	( 周帆,, 庞志倡,, 余小强, 等. 昆虫肠道微生物的研究进展和应用前景[J]. 应用昆虫学报, 2020, 57(3): 600-607.)
[10]	Feng S,, Xie XC,, Huang ZD, et al. The activities of digestive enzymes in culturable bacteria isolated from intestinal tract of Alphitobius laevigatus[J]. Chin J Microecol, 2019, 31(11):1255-1259. (in Chinese)
	( 丰硕,, 谢晓晨,, 黄振东, 等. 小菌虫肠道可培养细菌的分离鉴定及产消化酶活性分析[J]. 中国微生态学杂志, 2019, 31(11): 1255-1259.)
[11]	He ZW,, Dong QY,, Liu R, et al. Isolation and identification of culturable intestinal bacteria from Clogmia albipunctata larvae and analysis of their digestive enzyme-producing activities[J]. Chin J Vector Biol Control, 2021, 32(2): 224-229. (in Chinese)
	( 何志伟,, 董起钰,, 刘锐, 等. 白斑蛾蚋幼虫肠道可培养细菌的分离鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 2021, 32(2): 224-229.)
[12]	Wang Y,, Wan LH,, Li XB. Effect of feeding on different tissues on larva development of Lucilia sericata[J]. Chin J Parasitol Parasit Dis, 2013, 31(2): 124-126, 130. (in Chinese)
	( 王尧,, 万立华,, 李学博. 不同组织源食物对丝光绿蝇生长发育的影响[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(2): 124-126, 130.)
[13]	Bing XL,, Gerlach J,, Loeb G, et al. Nutrient-dependent impact of microbes on Drosophila suzukii development[J]. mBio, 2018, 9(2): e02199-17.
[14]	Khamesipour F,, Lankarani KB,, Honarvar B, et al. A systematic review of human pathogens carried by the housefly (Musca domestica L.)[J]. BMC Public Health, 2018, 18(1): 1049. doi: 10.1186/s12889-018-5934-3 pmid: 30134910
[15]	Davies MP,, Anderson M,, Hilton AC. The housefly Musca domestica as a mechanical vector of Clostridium difficile[J]. J Hosp Infect, 2016, 94(3): 263-267. doi: S0195-6701(16)30381-4 pmid: 27671221
[16]	Förster M,, Klimpel S,, Sievert K. The house fly (Musca domestica) as a potential vector of metazoan parasites caught in a pig-pen in Germany[J]. Vet Parasitol, 2009, 160(1/2): 163-167. doi: 10.1016/j.vetpar.2008.10.087
[17]	Barin A,, Arabkhazaeli F,, Rahbari S, et al. The housefly, Musca domestica, as a possible mechanical vector of Newcastle disease virus in the laboratory and field[J]. Med Vet Entomol, 2010, 24(1): 88-90. doi: 10.1111/j.1365-2915.2009.00859.x pmid: 20377736
[18]	Cafarchia C,, Lia RP,, Romito D, et al. Competence of the housefly, Musca domestica, as a vector of Microsporum canis under experimental conditions[J]. Med Vet Entomol, 2009, 23(1): 21-25. doi: 10.1111/j.1365-2915.2008.00785.x pmid: 19239611
[19]	Xue ZJ,, Zhang JL,, Zhang RL, et al. Comparative analysis of gut bacterial communities in housefly larvae fed different diets using a high-throughput sequencing approach[J]. FEMS Microbiol Lett, 2019, 366(11): fnz126. doi: 10.1093/femsle/fnz126
[20]	Zhao Y,, Wang WQ,, Zhu F, et al. The gut microbiota in larvae of the housefly Musca domestica and their horizontal transfer through feeding[J]. AMB Express, 2017, 7(1): 147. doi: 10.1186/s13568-017-0445-7 pmid: 28697583
[21]	de Jonge N,, Michaelsen TY,, Ejbye-Ernst R, et al. Housefly (Musca domestica L.) associated microbiota across different life stages[J]. Sci Rep, 2020, 10(1): 7842. doi: 10.1038/s41598-020-64704-y
[22]	Zhang Q,, Wang SM,, Zhang XY, et al. Negative impact of Pseudomonas aeruginosa Y12 on its host Musca domestica[J]. Front Microbiol, 2021, 12: 691158. doi: 10.3389/fmicb.2021.691158
[23]	Wan Q,, Huang ZD,, Xue ZJ, et al. Screening for intestinal bacterial strains against Beauveria bassiana from Musca domestica larvae[J]. Chin J Vector Biol Control, 2020, 31(1): 36-40. (in Chinese)
	( 万晴,, 黄振东,, 薛志静, 等. 家蝇幼虫肠道细菌拮抗球孢白僵菌菌株的筛选[J]. 中国媒介生物学及控制杂志, 2020, 31(1): 36-40.)
[24]	Zhang XY,, Wang SM,, Li T, et al. Bacteriophage: a useful tool for studying gut bacteria function of housefly larvae, Musca domestica[J]. Microbiol Spectr, 2021, 9(1): e0059921.
[25]	Zhang Q,, Wang SM,, Zhang XY, et al. Enterobacter hormaechei in the intestines of housefly larvae promotes host growth by inhibiting harmful intestinal bacteria[J]. Parasit Vectors, 2021, 14(1): 598. doi: 10.1186/s13071-021-05053-1 pmid: 34876203
[26]	Huang ZD,, Wan Q,, Xue ZJ, et al. Isolation and identification of culturable aerobic bacteria from the intestines of Blattella germanica and the activity of digestive enzymes produced by these bacteria[J]. Chin J Vector Biol Control, 2019, 30(4): 409-413. (in Chinese)
	( 黄振东,, 万晴,, 薛志静, 等. 德国小蠊肠道可培养非厌氧细菌的分离、鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 2019, 30(4): 409-413.)
[27]	Xue ZJ,, Zhuang GF,, Huang ZD, et al. Screening and identification of digestive enzyme-producing bacteria from the intestine of Anomala corpulenta larvae[J]. Chin J Microecol, 2017, 29(8): 878-883. (in Chinese)
	( 薛志静,, 庄桂芬,, 黄振东, 等. 铜绿丽金龟蛴螬肠道产消化酶细菌的分离与鉴定[J]. 中国微生态学杂志, 2017, 29(8): 878-883.)
[28]	Hani YMI,, Marchand A,, Turies C, et al. Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): influence of body size and temperature[J]. PLoS One, 2018, 13(4): e0194932.
[29]	Wei DF. The digestive enzyme activities and bacterial diversity in the gut of Hyphantria cunea(Lepidoptera∶ Arctiidae) larvae[D]. Beijing: Beijing Forestry University, 2017: 1-47. (in Chinese)
	( 魏丹峰. 美国白蛾幼虫肠道消化酶活性和细菌多样性研究[D]. 北京: 北京林业大学, 2017: 1-47.)
[30]	Zhang Q,, Ma RM,, Xie XM, et al. Culture optimization of Bacillus sp. B59 for the production of carboxymethyl-cellulase[J]. J Ludong Univ Nat Sci Ed, 2021, 37(2): 146-150, 156. (in Chinese)
	仉倩,, 马瑞梅,, 解孝满, 等. 产羧甲基纤维素酶的Bacillus sp.B59培养优化研究[J]. 鲁东大学学报(自然科学版), 2021, 37(2):146-150, 156.)
[31]	Zhang LJ,, Zhang HT,, Li YL, et al. Isolation and identification of amylase producing Bacillus from kitchen waste and its enzymatic properties[J]. Feed Res, 2021, 44(10): 69-73. (in Chinese)
	( 张林吉,, 张海涛,, 李云龙, 等. 厨余中产淀粉酶芽孢杆菌的筛选、鉴定及酶学性质研究[J]. 饲料研究, 2021, 44(10): 69-73.)
[32]	Xie QX,, Hou NN,, Lu XH, et al. Enzyme-producing characteristic and antibiotic tolerance of Bacillus[J]. Chin Brew, 2021, 40(5): 91-96. (in Chinese)
	( 谢全喜,, 侯楠楠,, 鹿晓慧, 等. 芽孢杆菌的产酶特性及其对抗生素的耐受性[J]. 中国酿造, 2021, 40(5): 91-96.)
[33]	Xiang Y. Improving the thermal stability of Bacillus subtilis lipase A via computational protein designing[D]. Wuxi: Jiangnan University, 2020: 1-48. (in Chinese)
	( 向玉. 计算设计提高枯草芽孢杆菌脂肪酶A的热稳定性[D]. 无锡: 江南大学, 2020: 1-48.)
[34]	Sun XX,, Li JJ,, Ning N, et al. Isolation and identification of chitin-degrading bacteria from the hindgut of Macrotermes barneyi[J]. Microbiol China, 2017, 44(7): 1649-1654. (in Chinese)
	孙新新,, 李净净,, 宁娜, 等. 黄翅大白蚁后肠几丁质降解微生物的分离与鉴定[J]. 微生物学通报, 2017, 44(7): 1649-1654.)
[35]	Zhang GZ. Characterization of dietary fiber and XOS extraction from wheat bran[D]. Wuhan: Hubei University of Technology, 2015: 1-65. (in Chinese)
	( 张国真. 麦麸膳食纤维、低聚木糖制备及物化特性研究[D]. 武汉: 湖北工业大学, 2015: 1-65.)

细菌种类 Bacterial species	蛋白酶Protease		淀粉酶Amylase		纤维素酶Cellulase		脂肪酶Lipase
细菌种类 Bacterial species	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture
普罗威登斯菌属Providencia
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	-	-	+	+	+	+	-	-
雷氏普罗维登斯菌P. rettgeri	-	-	+	+	+	+	+	-
斯氏普罗威登斯菌P. stuartii	-	-	+	+	+	+	-	-
肠球菌属Enterococcus
铅黄肠球菌E. casseliflavus	-	-	+	+	+	+	-	-
粪肠球菌E. faecalis	-	-	+	+	+	+	-	-
假柠檬酸杆菌属Pseudocitrobacter
粪假柠檬酸杆菌 P. faecalis	-	-	+	+	+	+	-	-
摩根菌属Morganella
摩根摩根菌M. morganii	-	-	+	+	+	+	-	-
肠杆菌属Enterobacter
霍氏肠杆菌E. hormaechei	-	-	+	+	-	+	-	-
克雷伯氏菌属Klebsiella
肺炎克雷伯菌K. pneumoniae	-	-	+	+	+	-	-	-

细菌种类Bacterial species	蛋白酶Protease	淀粉酶Amylase	纤维素酶Cellulase	脂肪酶Lipase
肠杆菌属Enterobacter
霍氏肠杆菌E. hormaechei	-	-	-	-
阴沟肠杆菌E. cloacae	-	-	-	-
普罗威登斯菌属Providencia
斯氏普罗威登斯菌P. stuartii	-	-	-	-
居幼虫普罗威登斯菌P. vermicola	-	-	-	-
克雷伯氏菌属Klebsiella
肺炎克雷伯菌K. pneumoniae	-	+	-	-
不动杆菌属Acinetobacter
别雷斯不动杆菌A. bereziniae	-	-	-	-
假单胞菌属Pseudomonas
铜绿假单胞菌P. aeruginosa	+	-	-	-
乳球菌属Lactococcus
乳酸乳球菌L. lactis	-	-	-	-
赖氨酸芽胞杆菌属Lysinibacillus
纺锤形赖氨酸芽孢杆菌L. fusiformis	-	-	-	-
芽孢杆菌属Bacillus Cohn
沙福芽孢杆菌B. safensis	+	-	-	-

细菌种类 Bacterial species	厌氧培养的D/d值 D/d value of anaerobic culture	需氧培养的D/d值 D/d value of aerobic culture
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	1.31 ± 0.02^a	1.26 ± 0.05^b
粪假柠檬酸杆菌 P. faecalis	1.39 ± 0.08^a	1.26 ± 0.05^b
摩根摩根菌M. morganii	1.32 ± 0.05^a	1.32 ± 0.01^ab
霍氏肠杆菌E. hormaechei	1.31 ± 0.04^a	1.32 ± 0.08^ab
铅黄肠球菌E. casseliflavus	1.37 ± 0.03^a	1.35 ± 0.05^ab
雷氏普罗维登斯菌株 P. rettgeri	1.30 ± 0.08^a	1.27 ± 0.01^ab
肺炎克雷伯菌 K. pneumoniae	1.27 ± 0.00^a	1.26 ± 0.03^b
斯氏普罗威登斯菌 P. stuartii	1.43 ± 0.02^a	1.41 ± 0.02^a
粪肠球菌E. faecalis	1.33 ± 0.05^a	1.42 ± 0.06^a

细菌种类 Bacterial species	厌氧培养的D/d值 D/d value of anaerobic culture	需氧培养的D/d值 D/d value of aerobic culture
铅黄肠球菌E. casseliflavus	1.36 ± 0.06^a	1.29 ± 0.01^a
雷氏普罗维登斯菌株 P. rettgeri	1.23 ± 0.05^ab	1.13 ± 0.01^c
霍氏肠杆菌E. hormaechei	-	1.13 ± 0.02^c
肺炎克雷伯菌 K. pneumoniae	1.15 ± 0.01^b	-
粪肠球菌E. faecalis	1.20 ± 0.04^ab	1.24 ± 0.05^ab
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	1.23 ± 0.04^ab	1.17 ± 0.03^bc
粪假柠檬酸杆菌P. faecalis	1.31 ± 0.06^ab	1.18 ± 0.04^bc
摩根摩根菌M. morganii	1.35 ± 0.08^a	1.17 ± 0.03^bc
斯氏普罗威登斯菌 P. stuartii	1.24 ± 0.07^ab	1.13 ± 0.04^c