Isolation and identification of cultivable symbiotic bacteria from the intestinal tract of Musca domestica during development

Abstract

Abstract:

Objective To identify the intestinal cultivable bacteria in Musca domestica at different development stages. Methods Five eggs or 5 larvae at day 1 after egg hatch were collected under aseptic conditions, and were homogenized in 1 ml sterile water. At day 2 or above, 5 larvae. 5 pupae, and 5 adult worms were also collected, and their intestinal tract was isolated and homogenized in 1 ml sterile water. The intestinal symbiotic bacteria were cultured from the homogenate. Single colonies with different morphology were picked up and cultured in LB medium for small-scale production. DNA was extracted to amplify 16S rDNA by PCR. The amplification products were sequenced and blasted in GenBank to determine the bacteria genus. The numbers of genera at different developmental stages were recorded. Results A total of 26 bacterial genera were obtained, comprising 11 in larvae stage, 19 in pupae stage, and 14 in adult worms. None cultivable symbiotic bacterium was isolated from eggs. There were genus overlaps between stages. The larvae and pupae stages shared 6 genera, consisting of Proteus, Kurthia, Bordetella, Microbacterium, Alcaligenes and Leuconostoc. The pupae and adult stages shared 3 genera, consisting of Sphingobacterium, Enterococcus and Ochrobactrum. The larvae and adult stages shared 1 genus, the Klebsiella. The three stages shared 4 genera, consisting of Providencia, Staphylococcus, Myroides and Pseudomonas. The proportions of gram-positive bacteria were 4/11, 6/19 and 3/14 in the larvae, pupae and adult stages, respectively. Conclusion The genus number of cultivable bacteria from Musca domestica shows a trend of increase followed by a decrease during the development from larvae to pupae stage, accompanied with a gradual decrease of proportions of gram-positive bacteria.

Key words: Musca domestica, Metamorphotic development, Intestine, Cultivable symbiotic bacteria

CLC Number:

R384.21

Jing LIU, Dan CHEN, Gui-fen ZHUANG, Zhen-dong HUANG, Zhi-jing XUE, Rui-ling ZHANG, Zhong ZHANG. Isolation and identification of cultivable symbiotic bacteria from the intestinal tract of Musca domestica during development[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(2): 120-124.

Figures/Tables 2

References 36

[1]	Dillon RJ, Dillon VM.The gut bacteria of insects: nonpathogenic interactions[J]. Annu Rev Entomol, 2004, 49(1): 71-92.
[2]	Schloss PD, Delalibera I, Handelsman J, et al. Bacteria associated with the guts of two wood-boring beetles: Anoplophora glabripennis and Saperda vestita(Cerambycidae)[J]. Environ Entomol, 2006, 35(3): 625-629.
[3]	Behar A, Yuval B, Jurkevitch E.Enterobacteria-mediated nitrogen fixation in natural populations of the fruit fly Ceratitis capitata[J]. Mol Ecol, 2005, 14(9): 2637-2643.
[4]	Behar A, Yuval B, Jurkevitch E.Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity[J]. J Insect Physiol, 2008, 54(9): 1377-1383.
[5]	薛宝燕, 程新胜, 陈树仁, 等. 昆虫共生菌研究进展[J]. 中国微生态学杂志, 2004, 16(3): 189-191.
[6]	Zheng W, Zhao Y, Zhang H.Morphology and ultrastructure of the hindgut fermentation chamber of a melolonthine beetle Holotrichia parallela (Coleoptera∶ Scarabaeidae) during larval development[J]. Micron, 2012, 43(5): 638-642.
[7]	Brinza L, Viñuelas J, Cottret L, et al. Systemic analysis of the symbiotic function of Buchnera aphidicola, the primary endosymbiont of the pea aphid Acyrthosiphon pisum[J]. C R Biol, 2009, 332(11): 1034-1049.
[8]	Haines LR, Haddow JD, Aksoy S, et al. The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia[J]. Insect Biochem Molec Biol, 2002, 32(11): 1429-1438.
[9]	Lu ZX, Yu XP, Chen JM, et al. Dynamics of yeast-like symbiote and its relationship with the virulence of brown planthopper, Nilaparvata lugens St<inline-graphic xlink:href="1000-7423-35-2-120/img_2.tif"/>l, to resistant rice varieties[J]. J Asia-Pac Entomol, 2004, 7(3): 317-323.
[10]	Thomas P, Kenny N, Eyles D, et al. Infection with the wMel and wMelPop strains of Wolbachia leads to higher levels of melanization in the hemolymph of Drosophila melanogaster, Drosophila simulans and Aedes aegypti[J]. Dev Comp Immunol, 2011, 35(3): 360-365.
[11]	Russell JA, Latorre A, Sabater-Muñoz B, et al. Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea[J]. Mol Ecol, 2003, 12(4): 1061-1075.
[12]	Knapp BA, Podmirseg SM, Seeber J, et al. Diet-related composition of the gut microbiota of Lumbricus rubellus as revealed by a molecular fingerprinting technique and cloning[J]. Soil Biol Biochem, 2009, 41(41): 2299-2307.
[13]	Cho MJ, Kim YH, Shin K, et al. Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: low endo-β-1,4-glucanase activity[J]. Biochem Bioph Res Commun, 2010, 395(3): 432-435.
[14]	Farikou O, Njiokou F, Cuny G, et al. Microsatellite genotyping reveals diversity within populations of Sodalis glossinidius, the secondary symbiont of tsetse flies[J]. Vet Microbiol, 2011, 150(1-2): 207-210.
[15]	Morrison M, Pope PB, Denman SE, et al. Plant biomass degradation by gut microbiomes: more of the same or something new[J]. Curr Opin Biotechnol, 2009, 20(3): 358-363.
[16]	Zhang D, Allen AB, Lax AR.Functional analyses of the digestive β-glucosidase of Formosan subterranean termites (Coptotermes formosanus)[J]. J Insect Physiol, 2012, 58(1): 205-210.
[17]	Dillon RJ, Vennard CT, Charnley AK.A note: gut bacteria produce components of a locust cohesion pheromone[J]. J Appl Microbiol, 2002, 92(4): 759-763.
[18]	相辉, 黄勇平. 肠道微生物与昆虫的共生关系[J]. 昆虫知识, 2008, 45(5): 687-693.
[19]	Fiołka MJ, Zagaja MP, Piersiak TD, et al. Gut bacterium of Dendrobaena veneta (Annelida ∶ Oligochaeta) possesses antimycobacterial activity[J]. J Invertebr Pathol, 2010, 105(1): 63-73.
[20]	Zhang YL, Kong LC, Jiang DH, et al. Phytotoxic and antifungal metabolites from Curvularia FH01 isolated from the gut of Atractomorpha sinensis[J]. Bioresour Technol, 2011, 102(3): 3575-3577.
[21]	Khajuria C, Buschman LL, Chen MS, et al. Characterization of six antibacterial response genes from the European corn borer (Ostrinia nubilalis) larval gut and their expression in response to bacterial challenge[J]. J Insect Physiol, 2011, 57(3): 345-355.
[22]	谭周进, 肖启明, 谢丙炎, 等. 昆虫内共生菌研究概况[J]. 微生物学通报, 2005, 32(4): 140-143.
[23]	Weiss B, Aksoy S.Microbiome influences on insect host vector competence[J]. Trends Parasitol, 2011, 27(11): 514-522.
[24]	Beard CB, Cordon-Rosales C, Durvasula RV.Bacterial symbionts of the triatominae and their potential use in control of Chagas disease transmission[J]. Annu Rev Entomol, 2002, 47: 123-141.
[25]	Bosshard PP, Abels S, Zbinden R, et al. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory(an 18-month evaluation)[J]. J Clin Microbiol, 2003, 41(9): 4134-4140.
[26]	Raj A, Kumar S, Singh SK, et al. Characterization of a new Providencia strain X1 producing multiple xylanases on wheat bran[J]. Sci World J, 2013, 2013(4): 386769.
[27]	Su Z, Zhang M, Liu X, et al. Comparison of bacterial diversity in wheat bran and in the gut of larvae and newly emerged adult of Musca domestica (Diptera : Muscidae) by use of ethidium monoazide reveals bacterial colonization[J]. J Econ Entomol, 2010, 103(5): 1832-1841.
[28]	Spiteller D, Dettner K, Bolan W.Gut bacteria may be involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles[J]. Biol Chem, 2000, 381(8): 755-762.
[29]	Dharne MS, Gupta AK, Rangrez AY, et al. Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies(Diptera ∶ sarcophagidae) are independent of metallo beta-lactamase gene[J]. Braz J Microbiol, 2008, 39(2): 397-404.
[30]	刘杨, 钟玮, 费忠安, 等. 家蝇中肠细菌群落组成分析[J]. 应用与环境生物学报, 2013, 19(5) : 800-804.
[31]	Scott JG, Liu N, Kristensen M, et al. A case for sequencing the genome of Musca domestica (Diptera ∶ Muscidae)[J]. J Med Entomol, 2009, 46(2): 175-182.
[32]	Petridis M, Bagdasarian M, Waldor MK, et al. Horizontal transfer of Shiga toxin and antibiotic resistance genes among Escherichia coli strains in house fly (Diptera ∶ Muscidae) gut[J]. J Med Entomol, 2006, 43(2): 288-295.
[33]	Ben Ami E, Yuval B, Jurkevitch E.Manipulation of the microbiota of mass-reared Mediterranean fruit flies Ceratitis capitata (Diptera ∶ Tephritidae) improves sterile male sexual performance[J]. ISME J, 2010, 4(1): 28-37.
[34]	Shi Z, Wang L, Zhang H.Low diversity bacterial community and the trapping activity of metabolites from cultivable bacteria species in the female reproductive system of the Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera ∶ Tephritidae)[J]. Int J Mol Sci, 2012, 13(5): 6266-6278.
[35]	Wang H, Jin L, Peng T, et al. Identification of cultivable bacteria in the intestinal tract of Bactrocera dorsalis from three different populations and determination of their attractive potential[J]. Pest Manag Sci, 2014, 70(1): 80-87.
[36]	Robacker DC, Bartelt RJ.Chemicals attractive to Mexican fruit fly from Klebsiella pneumoniae and Citrobacter freundii cultures sampled by solid-phase microextraction[J]. J Chem Ecol, 1997, 23(12): 2897-2915.

菌属 Genus	幼虫期 Larvae	蛹期 Pupae	成蝇 Adult
变形杆菌属Proteus	+	+
普罗威登斯菌属Providencia	+	+	+
库特氏菌属Kurthia	+	+
葡萄球菌属Staphylococcus	+	+	+
香味菌属Myroides	+	+	+
博德特氏菌属Bordetella	+	+
微杆菌属Microbacterium	+	+
产碱杆菌属 Alcaligenes	+	+
克雷伯菌属 Klebsiella	+		+
明串球菌属 Leuconostoc	+	+
假单胞菌属 Pseudomonas	+	+	+
柠檬酸杆菌属 Citrobacter		+
血杆菌属 Sanguibacter		+
鞘氨醇杆菌属 Sphingobacterium		+	+
肠球菌属 Enterococcus		+	+
乳球菌属 Lactococcus		+
拉乌尔菌属 Raoultella		+
短波毛单胞菌属 Brevundimonas		+
俊片菌属 Lampropedia		+
苍白杆菌属 Ochrobactrum		+	+
肠杆菌属 Enterobacter			+
不动杆菌属 Acinetobacter			+
寡养单胞菌属 Stenotrophomonas			+
土壤杆菌 Agrobacterium			+
代尔夫特菌属 Delftia			+
漫游球菌属 Vagococcus			+
合计 Total	11	19	14