Differential Microbial Diversity in Drosophila...
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Research Article Differential Microbial Diversity in Drosophila melanogaster : Are Fruit Flies Potential Vectors of Opportunistic Pathogens? Luis A. Ramírez-Camejo, 1,2 Génesis Maldonado-Morales, 2 and Paul Bayman 2 1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investigaciones Cient´ ıficas y Servicios de Alta Tecnolog´ ıa (INDICASAT AIP), Edificio 219, Ciudad del Saber, Apartado 0843-01103, Ciudad de Panam´ a, Panama 2 Department of Biology, University of Puerto Rico, R´ ıo Piedras, PR, USA Correspondence should be addressed to Luis A. Ram´ ırez-Camejo; [email protected] Received 11 April 2017; Revised 30 July 2017; Accepted 24 September 2017; Published 6 November 2017 Academic Editor: Michael McClelland Copyright © 2017 Luis A. Ram´ ırez-Camejo et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Drosophila melanogaster has become a model system to study interactions between innate immunity and microbial pathogens, yet many aspects regarding its microbial community and interactions with pathogens remain unclear. In this study wild D. melanogaster were collected from tropical fruits in Puerto Rico to test how the microbiota is distributed and to compare the culturable diversity of fungi and bacteria. Additionally, we investigated whether flies are potential vectors of human and plant pathogens. Eighteen species of fungi and twelve species of bacteria were isolated from wild flies. e most abundant microorganisms identified were the yeast Candida inconspicua and the bacterium Klebsiella sp. e yeast Issatchenkia hanoiensis was significantly more common internally than externally in flies. Species richness was higher in fungi than in bacteria, but diversity was lower in fungi than in bacteria. e microbial composition of flies was similar internally and externally. We identified a variety of opportunistic human and plant pathogens in flies such as Alcaligenes faecalis, Aspergillus flavus, A. fumigatus, A. niger, Fusarium equiseti/oxysporum, Geotrichum candidum, Klebsiella oxytoca, Microbacterium oxydans, and Stenotrophomonas maltophilia. Despite its utility as a model system, D. melanogaster can be a vector of microorganisms that represent a potential risk to plant and public health. 1. Introduction e microbiota of wild Drosophila melanogaster is distinct from that of flies from laboratory stocks [1–4]. A wide range of bacteria from Proteobacteria, Firmicutes, and Bacteroidetes phyla, among others, have been reported from Drosophila [2, 4]. In contrast, fungi are poorly characterized in Drosophila, with most studies focusing on taxonomy, ecology of yeast in the gut, and importance in the diet [5–7]. In the early 20th century, some Drosophila species were considered a potential vector of disease because its fre- quency near excrement and public toilets [8]. In a recent study, the Mediterranean fruit flies Ceratitis capitata and D. melanogaster were shown to transmit Escherichia coli to intact apple fruits, suggesting they are potential vectors of pathogens [9, 10]. is is a disturbing conclusion because D. melanogaster has a worldwide distribution and visits a wide variety of human foods [11]. Hence, fruit flies have been considered a common pest in the food industry [12]. In one instance, discovery of a population of fruit flies in an operating room at a New Jersey hospital resulted in the disruption of a dozen surgeries [13]. By the start of the 21st century, Drosophila had been established as a model system for immune studies aſter analysis of its genome revealed unsuspected sophistication and similarity to the mammalian innate immune system [14–16]. e use of Drosophila for studies of virulence and pathogen interactions requires a deeper knowledge of its microbial symbionts and their internal and external distribu- tions. In this study, we isolated microorganisms from wild D. melanogaster to answer the following questions: (1) how is the microbiota of Drosophila distributed spatially? We hypothesized that some microorganisms are found in both external and internal tissues of flies [17]; however, other bacteria and fungi will be limited to either internal or external surfaces. (2) Are culturable fungi and bacteria equally diverse in flies? We hypothesize that bacteria will be more diverse Hindawi International Journal of Microbiology Volume 2017, Article ID 8526385, 6 pages https://doi.org/10.1155/2017/8526385
Transcript of Differential Microbial Diversity in Drosophila...
Research ArticleDifferential Microbial Diversity in Drosophila melanogasterAre Fruit Flies Potential Vectors of Opportunistic Pathogens
Luis A Ramiacuterez-Camejo12 Geacutenesis Maldonado-Morales2 and Paul Bayman2
1Centro de Biodiversidad y Descubrimiento de Drogas Instituto de Investigaciones Cientıficas y Servicios deAlta Tecnologıa (INDICASAT AIP) Edificio 219 Ciudad del Saber Apartado 0843-01103 Ciudad de Panama Panama2Department of Biology University of Puerto Rico Rıo Piedras PR USA
Correspondence should be addressed to Luis A Ramırez-Camejo ramirezcamejogmailcom
Received 11 April 2017 Revised 30 July 2017 Accepted 24 September 2017 Published 6 November 2017
Academic Editor Michael McClelland
Copyright copy 2017 Luis A Ramırez-Camejo et alThis is an open access article distributed under theCreativeCommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited
Drosophila melanogaster has become a model system to study interactions between innate immunity and microbial pathogens yetmany aspects regarding itsmicrobial community and interactionswith pathogens remain unclear In this studywildDmelanogasterwere collected from tropical fruits in Puerto Rico to test how themicrobiota is distributed and to compare the culturable diversity offungi and bacteria Additionally we investigated whether flies are potential vectors of human and plant pathogens Eighteen speciesof fungi and twelve species of bacteria were isolated from wild flies The most abundant microorganisms identified were the yeastCandida inconspicua and the bacterium Klebsiella sp The yeast Issatchenkia hanoiensis was significantly more common internallythan externally in flies Species richness was higher in fungi than in bacteria but diversity was lower in fungi than in bacteriaThe microbial composition of flies was similar internally and externally We identified a variety of opportunistic human and plantpathogens in flies such as Alcaligenes faecalis Aspergillus flavus A fumigatus A niger Fusarium equisetioxysporum Geotrichumcandidum Klebsiella oxytocaMicrobacterium oxydans and Stenotrophomonas maltophilia Despite its utility as a model system Dmelanogaster can be a vector of microorganisms that represent a potential risk to plant and public health
1 Introduction
The microbiota of wild Drosophila melanogaster is distinctfrom that of flies from laboratory stocks [1ndash4] Awide range ofbacteria from Proteobacteria Firmicutes and Bacteroidetesphyla among others have been reported from Drosophila [24] In contrast fungi are poorly characterized in Drosophilawith most studies focusing on taxonomy ecology of yeast inthe gut and importance in the diet [5ndash7]
In the early 20th century some Drosophila species wereconsidered a potential vector of disease because its fre-quency near excrement and public toilets [8] In a recentstudy the Mediterranean fruit flies Ceratitis capitata andD melanogaster were shown to transmit Escherichia coli tointact apple fruits suggesting they are potential vectors ofpathogens [9 10] This is a disturbing conclusion becauseD melanogaster has a worldwide distribution and visits awide variety of human foods [11] Hence fruit flies havebeen considered a common pest in the food industry [12]
In one instance discovery of a population of fruit flies inan operating room at a New Jersey hospital resulted in thedisruption of a dozen surgeries [13]
By the start of the 21st century Drosophila had beenestablished as a model system for immune studies afteranalysis of its genome revealed unsuspected sophisticationand similarity to the mammalian innate immune system[14ndash16] The use of Drosophila for studies of virulence andpathogen interactions requires a deeper knowledge of itsmicrobial symbionts and their internal and external distribu-tions
In this study we isolated microorganisms from wild Dmelanogaster to answer the following questions (1) howis the microbiota of Drosophila distributed spatially Wehypothesized that some microorganisms are found in bothexternal and internal tissues of flies [17] however otherbacteria and fungi will be limited to either internal or externalsurfaces (2) Are culturable fungi and bacteria equally diversein flies We hypothesize that bacteria will be more diverse
HindawiInternational Journal of MicrobiologyVolume 2017 Article ID 8526385 6 pageshttpsdoiorg10115520178526385
2 International Journal of Microbiology
than fungi in D melanogaster because they form stablerelationships with flies in nature and are important foodsources for larvae [4] (3) Are fruit flies potential vectors ofopportunistic pathogens Because fruit flies can transportmicroorganisms of human concern [9 10 18] we hypothesizethat some fungi and bacteria isolated from wild flies will bepotential vectors of plant and animal pathogens
2 Methodology
21 Sampling CultureMedia and Isolation ofMicroorganismsWild females of Drosophila melanogaster were collectedin Puerto Rico from tropical fruits (mango orange star-fruit and jackfruit) Only culturable microorganisms wereincluded in order to obtain data and isolates for a relatedproject on probiotics [19] Flies were attracted using glass jarswith fruit and jars were covered with gauze after enough fliesentered the jar This was repeated four times at one-monthintervals for a total of 160 flies
Fungi were isolated on Potato Dextrose Agar (PDA) andbacteria on Tryptic Soy Agar (TSA) they are common andnonselective media that provide enough nutrients to encour-age growth of a range of fungi and bacteria respectivelyFungi and bacteria were isolated externally and internallyfrom 40 flies per sample as follows 5 flies each were placedon plates of PDA and TSA and allowed to walk on thesurface for five minutes Another 10 flies per sample werethen anesthetized with CO
2 placed in a microcentrifuge
tube with sterile water and Tween 80 (001) and mixed ina vortex for 1 minute to release microbial cells from bodysurfaces [20] The wash solution was then streaked on theculture media described above Another 10 flies were surface-sterilized in 70 ethanol for 1min rinsed three times withsterile water and placed on PDA and TSA five per plate Theguts of another 10 flies surface-sterilized were extracted usinga sterile forceps and needle Guts were rinsed in sterile waterand streaked with a sterile loop on PDA and TSA (5 guts perplate) Plates were incubated at 28∘C for seven days to allowmicrobial growth
Microorganisms were isolated every day and plated ina 2mL glass vial with PDA or TSA Microorganisms weregrouped by morphospecies based on morphological charac-teristics for example colony size color texture and type ofmargin
22 DNAExtraction One fungal isolate from eachmorphos-pecies was cultured in Potato Dextrose Broth (PDB) filteredand macerated in liquid nitrogen DNA was extracted usinga phenol-chloroform method [21] The same procedure wasused for Drosophila One bacterial isolate from each mor-phospecies was cultured in liquid Nutrient broth for 24ndash48hours cells were then lysed by heat-shock and suspendedin 1mL of sterile distilled water and DNA was diluted to4ndash30 ng120583L for PCR
23 Polymerase Chain Reaction and Sequencing For fungithe nuclear ribosomal Internal Transcribed Spacer (ITS) wasamplified using primers ITS 1F and ITS 4 [21 22] Forbacteria part of the 16S ribosomal gene was amplified using
Fungi
Bacteria
02468
101214161820
Num
ber o
f mor
phos
peci
es
21 41 61 81 101 121 141 1611Number of isolates
Figure 1 Species accumulation curves for fungal and bacterialmorphospecies isolated from female Drosophila melanogaster inPuerto Rico Species order was randomized 100 times Fungaland bacterial species are represented with dashed and solid linesrespectively
primers fD1 and rP2 [23] For Drosophila the mitochondrialcytochrome oxidase subunit I was amplified with primersLCO1490 and HCO2198 [24] Amplicons were 600ndash1300nucleotides for bacteria 200ndash500 nucleotides for fungi andsim600 nucleotides forDrosophila PCR products were cleanedusing Exo-Sap (Fermentas) and sequenced in the Sequenc-ing and Genotyping Facility (SGF) at UPR-RP Sequencesfrom flies bacteria and fungi (GenBank accession numbersKU238836ndashKU238862) were correctedwith Sequencher soft-ware and identified by BLASTn searches in GenBank Namesassigned were based on gt98 identity (Table 1)
24 Statistical Analysis EstimateS (version 910 forMac) wasused to compare the richness (Chao 1) diversity (Shannonindex) and composition (Bray-Curtis index) of communi-ties in flies (httpviceroyeebuconneduestimates) Speciesaccumulation curves were obtained using the variable 119878(est) Chi square (1205942) tests were used to compare differencesbetween external versus internal microbial communities
3 Results
31 Distribution andDiversity ofMicroorganisms Isolated fromDrosophila melanogaster We isolated 314 microorganismsfrom wild Drosophila melanogaster including 171 fungi and143 bacteria which were grouped into 18 and 12 morphos-pecies respectively (Table 1) The most abundant fungusidentified was the yeast Candida inconspicua which repre-sented 49 of fungi isolated The most common bacterialgenus was Klebsiella (22)
Species richness estimated as Chao 1 was 20 and 12in fungal and bacterial communities respectively Speciesaccumulation curves showed that for bacteria sampling wassufficient assuming that our morphospecies did not containcryptic species For fungi the sampling was insufficient(Figure 1) The microbial diversity estimated with Shannonrsquosindex (1198671015840) was 187 for fungi and 223 for bacteria
Only the yeast Issatchenkia hanoiensis (1205942 = 62 119875 lt0013) was significantly more common in fly guts thanexternal surfaces (Table 1) The remaining microorganisms
International Journal of Microbiology 3Ta
ble1Identifi
catio
nanddistrib
utionof
fung
iand
bacteriaon
andin
fruitfl
iesGenBa
nkaccessionnu
mbersarefor
ITSsequ
enceso
ffun
giand16SrD
NAsequ
encesfor
bacteriaTh
elast
sixcolumns
show
numbersof
each
microorganism
isolatedwith
thefollowingprotocolsflies
thatwalkedacrosspetriplatesflies
washedin
001Tw
een80surface-sterilized
fliesand
guts
removed
from
flies
Strain
Species
GenBa
nkaccessionID
Highesthit
GenBa
nkaccessionID
identity
Externaliso
latio
nInternaliso
latio
nTo
tal119875value
Walked
Washed
Insect
Gut
Fung
usH137
Cand
idainconspicua
KU238836
Cand
idainconspicua
KT207004
100
2720
2314
840275
H35
Penicilliu
mcrustosumcom
mun
eKU
238837
Penicilliu
mcommun
eKR
012904
100
105
67
280705
H127
Issatchenkiahanoien
sisKU
238838
Issatchenkiahanoien
sisFJ153178
991
19
213
0013lowast
H66
Aspergillus
versico
lorsydowiinidu
lans
KU238839
Aspergillus
sydowii
KT989398
100
31
04
810
00
H11
Aspergillus
fumiga
tus
KU238840
Aspergillus
fumiga
tus
AB2
98709
100
21
20
50655
H37
Fusariu
msp
KU238841
Fusariu
mequiseti
HQ332532
100
11
21
50655
H44
Galactomyces
cand
idum
geotrichium
KU238842
Galactomycescand
idum
KJ579946
991
11
14
1000
H102
Pichiamem
branifa
ciens
KU238843
Pichiamem
branifa
ciens
FJ231462
993
01
04
0317
H7
Rhizopus
sp
mdashmdash
mdashmdash
03
00
30083
H24
Fusariu
mequisetioxysporum
KU238844
Fusariu
mequiseti
KJ174399
100
20
01
30564
H77
Penicilliu
mcitrin
umgriseofulvum
KU238845
Penicilliu
mcitrin
umKU
681430
980
21
03
0564
H31
Aspergillus
niger
KU238846
Aspergillus
niger
KP748369
991
11
03
0564
H46
Geotrichum
cand
idum
KU238847
Geotrichum
cand
idum
KF713518
100
11
00
20157
H69
Aspergillus
flavuso
ryzae
KU238848
Aspergillus
flavus
KU360621
100
01
00
10317
H70
Aspergillus
nigertubingensis
KU238849
Aspergillus
niger
EU64
5723
980
01
01
0317
Bacteria
B82
Klebsiella
sp
KU238850
Klebsiella
sp
FN178363
995
68
1332
0077
B5Ba
cillussp
KU238851
Bacillusp
umilu
sKU
517819
997
54
622
0670
B30
Klebsiella
oxytoca
KU238852
Klebsiella
mich
iganensis
KP717391
996
63
520
0371
B44
Klebsiella
pneumoniaevariicola
KU238853
Klebsiella
variicola
KT895843
993
14
614
0109
B22
Erwiniasp
KU238854
Uncultu
redErwiniasp
HE5
75588
993
43
111
0366
B105
Bacillusp
umilu
ssafensis
KU238855
Bacillusp
umilu
sKU
239978
992
03
49
0096
B39
Stenotrophom
onas
maltophilia
KU238856
Stenotrophom
onas
maltophilia
LT222226
992
23
29
0739
B84
Micrococcus
luteusyun
nanensis
KU238857
Micrococcusluteus
KT901825
100
40
40
810
00
B43
Alcaligenesfaecalis
KU238858
Alcaligenesfaecalis
KU179370
992
11
26
1000
B8Microbacteriu
moxydan
sKU
238859
Microbacteriu
moxydan
sKT
580637
990
10
01
0317
Drosoph
ilaD1
Drosophila
mela
nogaste
rKU
238860
Drosophila
mela
nogaste
rKP
161877
99D2
Drosophila
mela
nogaste
rKU
238861
Drosophila
mela
nogaste
rKP
161877
99D3
Drosophila
mela
nogaste
rKU
238862
Drosophila
mela
nogaste
rKP
161877
99Aste
riskrepresentssig
nificantd
ifferencesb
yCh
isqu
aretest
4 International Journal of Microbiology
did not differ significantly in frequency between external andinternal origin (119875 gt 005)
The species composition did not differ significantlybetween internal and external microbiotas either for fungi(Bray-Curtis = 068) or for bacteria (Bray-Curtis = 071)
32 Potential Opportunistic Pathogens Isolated from Dro-sophila melanogaster Bacteria and fungi isolated from Dro-sophila melanogaster included opportunistic pathogens ofhumans and animals includingKlebsiella oxytocaAlcaligenesfaecalis Microbacterium oxydans Stenotrophomonas malto-philia Aspergillus fumigatus A flavus and A niger (Table 1)Also A flavus A niger Fusarium equisetioxysporum andGeotrichum candidum are considered opportunistic plantpathogens (Table 1)
4 Discussion
41 Differences between Bacteria and Fungi Isolated fromDrosophila melanogaster This study was limited to cultur-able microorganisms which were used for experiments onprobiotics [19] However our protocol excluded the majorityof bacteria and many fungi which are nonculturable orrequire specialized media or culture conditions [25]
The richness of fungal morphospecies was higher thanthat of bacteria (Figure 1) The accumulation curves for flieslevelled off suggesting that nearly all the culturable bacterialspecies present in flies were detected but not for fungiThese results contradict a previous study where the fungalcommunities associated with different Drosophila speciessampled around theworldwere less rich that those of bacteria[26]However that study only focused on yeasts isolated fromguts of flies which constitute the vast majority of knownDrosophila-associated fungi
In contrast even though the fungal community is richerin species the bacteria community is more diverse in Dmelanogaster (1198671015840 fungi = 187 versus bacteria = 223) Thissuggests that the population sizes of different bacterial speciesin the flies are more equitable This is supported by twostudies in which bacterial diversity exceeds fungal diversityin Drosophila populations [2 26]
The yeast Issatchenkia hanoiensis was more abundant ininternal parts of flies than externally (119875 lt 0013) Yeasts arecommon Drosophila symbionts and some are food sourcesfor Drosophila [26] Yeast like Saccharomyces cerevisiae cansurvive passage through the digestive tract of flies because theconstituents of spore walls are more resistant than vegetativecells [27] It would be interesting to examine if I hanoiensisprovides any benefit to flies for example food source forlarvae roles in attraction ovoposition development orprotection against pathogens [5 7 28ndash31] I hanoiensis wasfirst described in 2003 from insect frass it has not previouslybeen reported from Drosophila [32]
Apart from Issatchenkia species composition internallyversus externally in flies was similar for fungi (Bray-Curtis =068) and for bacteria (Bray-Curtis = 071) This result con-trasts with a previous microbiome study where the internalbacterial communities were a reduced subset of the external
bacterial communities suggesting that flies can control themicroorganisms in the digestive tract and internal tissues [3]
42 Drosophila melanogaster as a Potential Vector of Path-ogens Drosophila melanogaster can carry opportunisticpathogens of humans [18] We isolated the Gram-negativebacterium Klebsiella oxytoca which has been reported asa causal agent of hemorrhagic colitis and Alcaligenes fae-calis was previously associated with infections in newborns[33 34] Other microorganisms isolated in this study werealso reported as emerging clinical pathogens for exampleMicrobacterium oxydans and Stenotrophomonas maltophilia[35ndash38]We also isolated three opportunistic pathogens capa-ble of causing animal and human aspergillosis Aspergillusfumigatus A flavus and A niger [39ndash42] Their presenceis not surprising because they are ubiquitous in nature withabundant airborne conidia [43]
Fruit flies as sources of contamination could represent apublic health risk especially to patients with compromisedimmune systems For example Mediterranean fruit flies(Ceratitis capitata) exposed to fecal material enriched withGFP-tagged Escherichia coli are capable of transmitting E colito intact apples in a cage model system [10] The same wasseen in D melanogaster [9]
In addition plant pathogens of agricultural concern weredocumented in the sampled flies for exampleA niger Fusar-ium equisetioxysporum and Geotrichum candidum [44ndash47]A flavus causes substantial problems in agriculture as asource of aflatoxins and frequently enters plants throughinsect-induced wounds [40 48]
Almost one hundred years ago D melanogaster com-monly found in exposed fruit in grocery stores and houseswas reported as ldquonot an efficient disease carrierrdquo [8] Thiswas based on the fact that D melanogaster is rarely attractedto excrement However the studies mentioned support ourhypothesis that flies might serve as vectors for opportunisticpathogens to humans and plants More experiments arenecessary to clarify the identity and virulence of the oppor-tunistic pathogens found in this study
5 Conclusions
The isolation of culturable microorganisms from wild Dmelanogaster suggests that its microbiota is rich diverseand distributed throughout internal and external surfacesIssatchenkia hanoiensiswas identified as common componentof the fly microbiota Other microorganisms are relatedto opportunistic human pathogens which may represent apublic health risk indicating D melanogaster is a potentialvector of disease
Conflicts of Interest
The authors report no conflicts of interest
Acknowledgments
This study was part of the doctoral thesis of Luis ARamırez-Camejo [49] and was supported by the following
International Journal of Microbiology 5
entities National Science Foundation-Center for AppliedTropical Ecology and Conservation (NSF-CRESTHRD0734826) National Institutes of Health-Supportof Continuous Research Excellence (NIH SCORE2S06GM08102) Secretarıa Nacional de Ciencia Tecnologıa eInnovacion of Panama (SENACYT ITE15-030) and SistemaNacional de Investigacion of Panama (SNI-NM2017-062)Special thanks are due to undergraduates Ana P Torres-Ocampo Ivana Serrano-Lachapel Michael Garcıa-Aliceaand Luisa Bernacet for help in the lab
References
[1] V Corby-Harris A C Pontaroli L J Shimkets J L BennetzenK E Habel and D E L Promislow ldquoGeographical distributionand diversity of bacteria associated with natural populations ofDrosophila melanogasterrdquo Applied and Environmental Microbi-ology vol 73 no 11 pp 3470ndash3479 2007
[2] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophila melanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007
[3] J A Chandler J Lang S Bhatnagar J A Eisen and A KoppldquoBacterial communities of diverse Drosophila species ecologi-cal context of a host-microbemodel systemrdquo PLoS Genetics vol7 no 9 p e1002272 2011
[4] N A Broderick and B Lemaitre ldquoGut-associated microbes ofDrosophila melanogasterrdquo Gut Microbes vol 3 no 4 pp 307ndash321 2012
[5] M A Ebbert J L Marlowe and J J Burkholder ldquoProtozoanand intracellular fungal gut endosymbionts in Drosophila Pre-valence and fitness effects of single and dual infectionsrdquo Journalof Invertebrate Pathology vol 83 no 1 pp 37ndash45 2003
[6] P B Morais M-A Lachance and C A Rosa ldquoSaturnisporahagleri sp nov a yeast species isolated from Drosophila flies inAtlantic rainforest in Brazilrdquo International Journal of Systematicand Evolutionary Microbiology vol 55 no 4 pp 1725ndash17272005
[7] C AnagnostouMDorsch andM Rohlfs ldquoInfluence of dietaryyeasts on Drosophila melanogaster life-history traitsrdquo Entomo-logia Experimentalis et Applicata vol 136 no 1 pp 1ndash11 2010
[8] A H Sturtevant ldquoFlies of the genus Drosophila as possibledisease carriersrdquo The Journal of Parasitology vol 5 no 2 pp84-85 1918
[9] W J Janisiewicz W S Conway M W Brown G M SapersP Fratamico and R L Buchanan ldquoFate of Escherichia coliO157H7 on fresh-cut apple tissue and its potential for trans-mission by fruit fliesrdquo Applied and Environmental Microbiologyvol 65 no 1 pp 1ndash5 1999
[10] S Sela D Nestel R Pinto E Nemny-Lavy and M Bar-Joseph ldquoMediterranean fruit fly as a potential vector of bacterialpathogensrdquo Applied and Environmental Microbiology vol 71no 7 pp 4052ndash4056 2005
[11] A Keller ldquoDrosophila melanogasterrsquos history as a human com-mensalrdquo Current Biology vol 17 no 3 pp R77ndashR81 2007
[12] Chartered Institute of Environmental Health ldquoPest control proce-dures in the food industryrdquo London pp52 2009
[13] J Landau Fruit flies disrupt surgeries at AtlantiCare hospital inGallowayrdquo Press of Atlantic City 2013 httpwwwpressof-atlanticcitycomnewspressatlanticfruit-flies-disrupt-surgeries-at-atlanticare-hospital-in-gallowayarticle 2f2a7fe6-3779-11e3-9754-001a4bcf887ahtml
[14] P Irving L Troxler T S Heuer et al ldquoA genome-wide analysisof immune responses inDrosophilardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 98 no26 pp 15119ndash15124 2001
[15] A-M Alarco A Marcil J Chen B Suter D Thomas andM Whiteway ldquoImmune-deficient Drosophila melanogaster amodel for the innate immune response to human fungalpathogensrdquoThe Journal of Immunology vol 172 no 9 pp 5622ndash5628 2004
[16] M S Lionakis andD P Kontoyiannis ldquoThe growing promise oftoll-deficient Drosophila melanogaster as a model for studyingAspergillus pathogenesis and treatmentrdquo Virulence vol 1 no 6pp 488ndash499 2010
[17] Y Tanada and H K Kaya ldquoAssociations between insects andnonpathogenicmicroorganismsrdquo in Insect Pathology AcademicPress San Diego Calif USA 1993
[18] W D Munyikombo ldquoPotential for transmission of Pseu-domonas aeruginosa and other bacterial and parasitic infectiousagents by Drosophila sp (fruit flies) as mechanical vectorsrdquoJournal of Biology Agriculture and Healthcare vol 4 no 20 pp154ndash170 2014
[19] L A Ramırez-Camejo M Garcıa-Alicea and G Maldonado-Morales ldquoProbiotics may protect Drosophila from infectionby Aspergillus flavusrdquo International Journal of PharmaceuticalSciences and Research vol 8 no 4 pp 1624ndash1632 2017
[20] A D Banjo O A Lawal andO O Adeduji ldquoBacteria and fungiisolated from housefly (Musca domestica L) larvaerdquo AfricanJournal of Biotechnology vol 4 no 8 pp 780ndash784 2005
[21] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA Genes forphylogeneticsrdquo in PCR Protocols a Guide to Methods andApplicationsW T J InnisM D GelfandH and J Sninsky Edspp 315ndash322 Academic Press Inc New York NY USA 1990
[22] M Gardes and T D Bruns ldquoITS primers with enhancedspecificity for basidiomycetesmdashapplication to the identificationof mycorrhizae and rustsrdquo Molecular Ecology vol 2 no 2 pp113ndash118 1993
[23] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991
[24] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMole-cular Marine Biology and Biotechnology vol 3 no 5 pp 294ndash299 1994
[25] P Bayman ldquoDiversity scale and variation of endophytic fungiin leaves of tropical plantsrdquo inMicrobiol Ecology of Aerial PlantSurfaces B MJ L AK T-W TM and P T N Spencer-PhillipsEds pp 37ndash50 CABI Publishing UK 2006
[26] J A Chandler J A Eisen and A Kopp ldquoYeast communities ofdiverseDrosophila species Comparison of two symbiont groupsin the samehostsrdquoApplied andEnvironmentalMicrobiology vol78 no 20 pp 7327ndash7336 2012
[27] A E Coluccio R K Rodriguez M J Kernan and A MNeiman ldquoThe yeast spore wall enables spores to survive passagethrough the digestive tract ofDrosophilardquo PLoS ONE vol 3 no8 p e2873 2008
[28] J H Northrop ldquoThe role of yeast in the nutrition of an insectDrosophilardquoThe Journal of Biological Chemistry vol 30 pp 30ndash181 1917
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 201
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Signal TransductionJournal of
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Evolutionary BiologyInternational Journal of
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Genetics Research International
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Advances in
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Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
2 International Journal of Microbiology
than fungi in D melanogaster because they form stablerelationships with flies in nature and are important foodsources for larvae [4] (3) Are fruit flies potential vectors ofopportunistic pathogens Because fruit flies can transportmicroorganisms of human concern [9 10 18] we hypothesizethat some fungi and bacteria isolated from wild flies will bepotential vectors of plant and animal pathogens
2 Methodology
21 Sampling CultureMedia and Isolation ofMicroorganismsWild females of Drosophila melanogaster were collectedin Puerto Rico from tropical fruits (mango orange star-fruit and jackfruit) Only culturable microorganisms wereincluded in order to obtain data and isolates for a relatedproject on probiotics [19] Flies were attracted using glass jarswith fruit and jars were covered with gauze after enough fliesentered the jar This was repeated four times at one-monthintervals for a total of 160 flies
Fungi were isolated on Potato Dextrose Agar (PDA) andbacteria on Tryptic Soy Agar (TSA) they are common andnonselective media that provide enough nutrients to encour-age growth of a range of fungi and bacteria respectivelyFungi and bacteria were isolated externally and internallyfrom 40 flies per sample as follows 5 flies each were placedon plates of PDA and TSA and allowed to walk on thesurface for five minutes Another 10 flies per sample werethen anesthetized with CO
2 placed in a microcentrifuge
tube with sterile water and Tween 80 (001) and mixed ina vortex for 1 minute to release microbial cells from bodysurfaces [20] The wash solution was then streaked on theculture media described above Another 10 flies were surface-sterilized in 70 ethanol for 1min rinsed three times withsterile water and placed on PDA and TSA five per plate Theguts of another 10 flies surface-sterilized were extracted usinga sterile forceps and needle Guts were rinsed in sterile waterand streaked with a sterile loop on PDA and TSA (5 guts perplate) Plates were incubated at 28∘C for seven days to allowmicrobial growth
Microorganisms were isolated every day and plated ina 2mL glass vial with PDA or TSA Microorganisms weregrouped by morphospecies based on morphological charac-teristics for example colony size color texture and type ofmargin
22 DNAExtraction One fungal isolate from eachmorphos-pecies was cultured in Potato Dextrose Broth (PDB) filteredand macerated in liquid nitrogen DNA was extracted usinga phenol-chloroform method [21] The same procedure wasused for Drosophila One bacterial isolate from each mor-phospecies was cultured in liquid Nutrient broth for 24ndash48hours cells were then lysed by heat-shock and suspendedin 1mL of sterile distilled water and DNA was diluted to4ndash30 ng120583L for PCR
23 Polymerase Chain Reaction and Sequencing For fungithe nuclear ribosomal Internal Transcribed Spacer (ITS) wasamplified using primers ITS 1F and ITS 4 [21 22] Forbacteria part of the 16S ribosomal gene was amplified using
Fungi
Bacteria
02468
101214161820
Num
ber o
f mor
phos
peci
es
21 41 61 81 101 121 141 1611Number of isolates
Figure 1 Species accumulation curves for fungal and bacterialmorphospecies isolated from female Drosophila melanogaster inPuerto Rico Species order was randomized 100 times Fungaland bacterial species are represented with dashed and solid linesrespectively
primers fD1 and rP2 [23] For Drosophila the mitochondrialcytochrome oxidase subunit I was amplified with primersLCO1490 and HCO2198 [24] Amplicons were 600ndash1300nucleotides for bacteria 200ndash500 nucleotides for fungi andsim600 nucleotides forDrosophila PCR products were cleanedusing Exo-Sap (Fermentas) and sequenced in the Sequenc-ing and Genotyping Facility (SGF) at UPR-RP Sequencesfrom flies bacteria and fungi (GenBank accession numbersKU238836ndashKU238862) were correctedwith Sequencher soft-ware and identified by BLASTn searches in GenBank Namesassigned were based on gt98 identity (Table 1)
24 Statistical Analysis EstimateS (version 910 forMac) wasused to compare the richness (Chao 1) diversity (Shannonindex) and composition (Bray-Curtis index) of communi-ties in flies (httpviceroyeebuconneduestimates) Speciesaccumulation curves were obtained using the variable 119878(est) Chi square (1205942) tests were used to compare differencesbetween external versus internal microbial communities
3 Results
31 Distribution andDiversity ofMicroorganisms Isolated fromDrosophila melanogaster We isolated 314 microorganismsfrom wild Drosophila melanogaster including 171 fungi and143 bacteria which were grouped into 18 and 12 morphos-pecies respectively (Table 1) The most abundant fungusidentified was the yeast Candida inconspicua which repre-sented 49 of fungi isolated The most common bacterialgenus was Klebsiella (22)
Species richness estimated as Chao 1 was 20 and 12in fungal and bacterial communities respectively Speciesaccumulation curves showed that for bacteria sampling wassufficient assuming that our morphospecies did not containcryptic species For fungi the sampling was insufficient(Figure 1) The microbial diversity estimated with Shannonrsquosindex (1198671015840) was 187 for fungi and 223 for bacteria
Only the yeast Issatchenkia hanoiensis (1205942 = 62 119875 lt0013) was significantly more common in fly guts thanexternal surfaces (Table 1) The remaining microorganisms
International Journal of Microbiology 3Ta
ble1Identifi
catio
nanddistrib
utionof
fung
iand
bacteriaon
andin
fruitfl
iesGenBa
nkaccessionnu
mbersarefor
ITSsequ
enceso
ffun
giand16SrD
NAsequ
encesfor
bacteriaTh
elast
sixcolumns
show
numbersof
each
microorganism
isolatedwith
thefollowingprotocolsflies
thatwalkedacrosspetriplatesflies
washedin
001Tw
een80surface-sterilized
fliesand
guts
removed
from
flies
Strain
Species
GenBa
nkaccessionID
Highesthit
GenBa
nkaccessionID
identity
Externaliso
latio
nInternaliso
latio
nTo
tal119875value
Walked
Washed
Insect
Gut
Fung
usH137
Cand
idainconspicua
KU238836
Cand
idainconspicua
KT207004
100
2720
2314
840275
H35
Penicilliu
mcrustosumcom
mun
eKU
238837
Penicilliu
mcommun
eKR
012904
100
105
67
280705
H127
Issatchenkiahanoien
sisKU
238838
Issatchenkiahanoien
sisFJ153178
991
19
213
0013lowast
H66
Aspergillus
versico
lorsydowiinidu
lans
KU238839
Aspergillus
sydowii
KT989398
100
31
04
810
00
H11
Aspergillus
fumiga
tus
KU238840
Aspergillus
fumiga
tus
AB2
98709
100
21
20
50655
H37
Fusariu
msp
KU238841
Fusariu
mequiseti
HQ332532
100
11
21
50655
H44
Galactomyces
cand
idum
geotrichium
KU238842
Galactomycescand
idum
KJ579946
991
11
14
1000
H102
Pichiamem
branifa
ciens
KU238843
Pichiamem
branifa
ciens
FJ231462
993
01
04
0317
H7
Rhizopus
sp
mdashmdash
mdashmdash
03
00
30083
H24
Fusariu
mequisetioxysporum
KU238844
Fusariu
mequiseti
KJ174399
100
20
01
30564
H77
Penicilliu
mcitrin
umgriseofulvum
KU238845
Penicilliu
mcitrin
umKU
681430
980
21
03
0564
H31
Aspergillus
niger
KU238846
Aspergillus
niger
KP748369
991
11
03
0564
H46
Geotrichum
cand
idum
KU238847
Geotrichum
cand
idum
KF713518
100
11
00
20157
H69
Aspergillus
flavuso
ryzae
KU238848
Aspergillus
flavus
KU360621
100
01
00
10317
H70
Aspergillus
nigertubingensis
KU238849
Aspergillus
niger
EU64
5723
980
01
01
0317
Bacteria
B82
Klebsiella
sp
KU238850
Klebsiella
sp
FN178363
995
68
1332
0077
B5Ba
cillussp
KU238851
Bacillusp
umilu
sKU
517819
997
54
622
0670
B30
Klebsiella
oxytoca
KU238852
Klebsiella
mich
iganensis
KP717391
996
63
520
0371
B44
Klebsiella
pneumoniaevariicola
KU238853
Klebsiella
variicola
KT895843
993
14
614
0109
B22
Erwiniasp
KU238854
Uncultu
redErwiniasp
HE5
75588
993
43
111
0366
B105
Bacillusp
umilu
ssafensis
KU238855
Bacillusp
umilu
sKU
239978
992
03
49
0096
B39
Stenotrophom
onas
maltophilia
KU238856
Stenotrophom
onas
maltophilia
LT222226
992
23
29
0739
B84
Micrococcus
luteusyun
nanensis
KU238857
Micrococcusluteus
KT901825
100
40
40
810
00
B43
Alcaligenesfaecalis
KU238858
Alcaligenesfaecalis
KU179370
992
11
26
1000
B8Microbacteriu
moxydan
sKU
238859
Microbacteriu
moxydan
sKT
580637
990
10
01
0317
Drosoph
ilaD1
Drosophila
mela
nogaste
rKU
238860
Drosophila
mela
nogaste
rKP
161877
99D2
Drosophila
mela
nogaste
rKU
238861
Drosophila
mela
nogaste
rKP
161877
99D3
Drosophila
mela
nogaste
rKU
238862
Drosophila
mela
nogaste
rKP
161877
99Aste
riskrepresentssig
nificantd
ifferencesb
yCh
isqu
aretest
4 International Journal of Microbiology
did not differ significantly in frequency between external andinternal origin (119875 gt 005)
The species composition did not differ significantlybetween internal and external microbiotas either for fungi(Bray-Curtis = 068) or for bacteria (Bray-Curtis = 071)
32 Potential Opportunistic Pathogens Isolated from Dro-sophila melanogaster Bacteria and fungi isolated from Dro-sophila melanogaster included opportunistic pathogens ofhumans and animals includingKlebsiella oxytocaAlcaligenesfaecalis Microbacterium oxydans Stenotrophomonas malto-philia Aspergillus fumigatus A flavus and A niger (Table 1)Also A flavus A niger Fusarium equisetioxysporum andGeotrichum candidum are considered opportunistic plantpathogens (Table 1)
4 Discussion
41 Differences between Bacteria and Fungi Isolated fromDrosophila melanogaster This study was limited to cultur-able microorganisms which were used for experiments onprobiotics [19] However our protocol excluded the majorityof bacteria and many fungi which are nonculturable orrequire specialized media or culture conditions [25]
The richness of fungal morphospecies was higher thanthat of bacteria (Figure 1) The accumulation curves for flieslevelled off suggesting that nearly all the culturable bacterialspecies present in flies were detected but not for fungiThese results contradict a previous study where the fungalcommunities associated with different Drosophila speciessampled around theworldwere less rich that those of bacteria[26]However that study only focused on yeasts isolated fromguts of flies which constitute the vast majority of knownDrosophila-associated fungi
In contrast even though the fungal community is richerin species the bacteria community is more diverse in Dmelanogaster (1198671015840 fungi = 187 versus bacteria = 223) Thissuggests that the population sizes of different bacterial speciesin the flies are more equitable This is supported by twostudies in which bacterial diversity exceeds fungal diversityin Drosophila populations [2 26]
The yeast Issatchenkia hanoiensis was more abundant ininternal parts of flies than externally (119875 lt 0013) Yeasts arecommon Drosophila symbionts and some are food sourcesfor Drosophila [26] Yeast like Saccharomyces cerevisiae cansurvive passage through the digestive tract of flies because theconstituents of spore walls are more resistant than vegetativecells [27] It would be interesting to examine if I hanoiensisprovides any benefit to flies for example food source forlarvae roles in attraction ovoposition development orprotection against pathogens [5 7 28ndash31] I hanoiensis wasfirst described in 2003 from insect frass it has not previouslybeen reported from Drosophila [32]
Apart from Issatchenkia species composition internallyversus externally in flies was similar for fungi (Bray-Curtis =068) and for bacteria (Bray-Curtis = 071) This result con-trasts with a previous microbiome study where the internalbacterial communities were a reduced subset of the external
bacterial communities suggesting that flies can control themicroorganisms in the digestive tract and internal tissues [3]
42 Drosophila melanogaster as a Potential Vector of Path-ogens Drosophila melanogaster can carry opportunisticpathogens of humans [18] We isolated the Gram-negativebacterium Klebsiella oxytoca which has been reported asa causal agent of hemorrhagic colitis and Alcaligenes fae-calis was previously associated with infections in newborns[33 34] Other microorganisms isolated in this study werealso reported as emerging clinical pathogens for exampleMicrobacterium oxydans and Stenotrophomonas maltophilia[35ndash38]We also isolated three opportunistic pathogens capa-ble of causing animal and human aspergillosis Aspergillusfumigatus A flavus and A niger [39ndash42] Their presenceis not surprising because they are ubiquitous in nature withabundant airborne conidia [43]
Fruit flies as sources of contamination could represent apublic health risk especially to patients with compromisedimmune systems For example Mediterranean fruit flies(Ceratitis capitata) exposed to fecal material enriched withGFP-tagged Escherichia coli are capable of transmitting E colito intact apples in a cage model system [10] The same wasseen in D melanogaster [9]
In addition plant pathogens of agricultural concern weredocumented in the sampled flies for exampleA niger Fusar-ium equisetioxysporum and Geotrichum candidum [44ndash47]A flavus causes substantial problems in agriculture as asource of aflatoxins and frequently enters plants throughinsect-induced wounds [40 48]
Almost one hundred years ago D melanogaster com-monly found in exposed fruit in grocery stores and houseswas reported as ldquonot an efficient disease carrierrdquo [8] Thiswas based on the fact that D melanogaster is rarely attractedto excrement However the studies mentioned support ourhypothesis that flies might serve as vectors for opportunisticpathogens to humans and plants More experiments arenecessary to clarify the identity and virulence of the oppor-tunistic pathogens found in this study
5 Conclusions
The isolation of culturable microorganisms from wild Dmelanogaster suggests that its microbiota is rich diverseand distributed throughout internal and external surfacesIssatchenkia hanoiensiswas identified as common componentof the fly microbiota Other microorganisms are relatedto opportunistic human pathogens which may represent apublic health risk indicating D melanogaster is a potentialvector of disease
Conflicts of Interest
The authors report no conflicts of interest
Acknowledgments
This study was part of the doctoral thesis of Luis ARamırez-Camejo [49] and was supported by the following
International Journal of Microbiology 5
entities National Science Foundation-Center for AppliedTropical Ecology and Conservation (NSF-CRESTHRD0734826) National Institutes of Health-Supportof Continuous Research Excellence (NIH SCORE2S06GM08102) Secretarıa Nacional de Ciencia Tecnologıa eInnovacion of Panama (SENACYT ITE15-030) and SistemaNacional de Investigacion of Panama (SNI-NM2017-062)Special thanks are due to undergraduates Ana P Torres-Ocampo Ivana Serrano-Lachapel Michael Garcıa-Aliceaand Luisa Bernacet for help in the lab
References
[1] V Corby-Harris A C Pontaroli L J Shimkets J L BennetzenK E Habel and D E L Promislow ldquoGeographical distributionand diversity of bacteria associated with natural populations ofDrosophila melanogasterrdquo Applied and Environmental Microbi-ology vol 73 no 11 pp 3470ndash3479 2007
[2] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophila melanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007
[3] J A Chandler J Lang S Bhatnagar J A Eisen and A KoppldquoBacterial communities of diverse Drosophila species ecologi-cal context of a host-microbemodel systemrdquo PLoS Genetics vol7 no 9 p e1002272 2011
[4] N A Broderick and B Lemaitre ldquoGut-associated microbes ofDrosophila melanogasterrdquo Gut Microbes vol 3 no 4 pp 307ndash321 2012
[5] M A Ebbert J L Marlowe and J J Burkholder ldquoProtozoanand intracellular fungal gut endosymbionts in Drosophila Pre-valence and fitness effects of single and dual infectionsrdquo Journalof Invertebrate Pathology vol 83 no 1 pp 37ndash45 2003
[6] P B Morais M-A Lachance and C A Rosa ldquoSaturnisporahagleri sp nov a yeast species isolated from Drosophila flies inAtlantic rainforest in Brazilrdquo International Journal of Systematicand Evolutionary Microbiology vol 55 no 4 pp 1725ndash17272005
[7] C AnagnostouMDorsch andM Rohlfs ldquoInfluence of dietaryyeasts on Drosophila melanogaster life-history traitsrdquo Entomo-logia Experimentalis et Applicata vol 136 no 1 pp 1ndash11 2010
[8] A H Sturtevant ldquoFlies of the genus Drosophila as possibledisease carriersrdquo The Journal of Parasitology vol 5 no 2 pp84-85 1918
[9] W J Janisiewicz W S Conway M W Brown G M SapersP Fratamico and R L Buchanan ldquoFate of Escherichia coliO157H7 on fresh-cut apple tissue and its potential for trans-mission by fruit fliesrdquo Applied and Environmental Microbiologyvol 65 no 1 pp 1ndash5 1999
[10] S Sela D Nestel R Pinto E Nemny-Lavy and M Bar-Joseph ldquoMediterranean fruit fly as a potential vector of bacterialpathogensrdquo Applied and Environmental Microbiology vol 71no 7 pp 4052ndash4056 2005
[11] A Keller ldquoDrosophila melanogasterrsquos history as a human com-mensalrdquo Current Biology vol 17 no 3 pp R77ndashR81 2007
[12] Chartered Institute of Environmental Health ldquoPest control proce-dures in the food industryrdquo London pp52 2009
[13] J Landau Fruit flies disrupt surgeries at AtlantiCare hospital inGallowayrdquo Press of Atlantic City 2013 httpwwwpressof-atlanticcitycomnewspressatlanticfruit-flies-disrupt-surgeries-at-atlanticare-hospital-in-gallowayarticle 2f2a7fe6-3779-11e3-9754-001a4bcf887ahtml
[14] P Irving L Troxler T S Heuer et al ldquoA genome-wide analysisof immune responses inDrosophilardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 98 no26 pp 15119ndash15124 2001
[15] A-M Alarco A Marcil J Chen B Suter D Thomas andM Whiteway ldquoImmune-deficient Drosophila melanogaster amodel for the innate immune response to human fungalpathogensrdquoThe Journal of Immunology vol 172 no 9 pp 5622ndash5628 2004
[16] M S Lionakis andD P Kontoyiannis ldquoThe growing promise oftoll-deficient Drosophila melanogaster as a model for studyingAspergillus pathogenesis and treatmentrdquo Virulence vol 1 no 6pp 488ndash499 2010
[17] Y Tanada and H K Kaya ldquoAssociations between insects andnonpathogenicmicroorganismsrdquo in Insect Pathology AcademicPress San Diego Calif USA 1993
[18] W D Munyikombo ldquoPotential for transmission of Pseu-domonas aeruginosa and other bacterial and parasitic infectiousagents by Drosophila sp (fruit flies) as mechanical vectorsrdquoJournal of Biology Agriculture and Healthcare vol 4 no 20 pp154ndash170 2014
[19] L A Ramırez-Camejo M Garcıa-Alicea and G Maldonado-Morales ldquoProbiotics may protect Drosophila from infectionby Aspergillus flavusrdquo International Journal of PharmaceuticalSciences and Research vol 8 no 4 pp 1624ndash1632 2017
[20] A D Banjo O A Lawal andO O Adeduji ldquoBacteria and fungiisolated from housefly (Musca domestica L) larvaerdquo AfricanJournal of Biotechnology vol 4 no 8 pp 780ndash784 2005
[21] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA Genes forphylogeneticsrdquo in PCR Protocols a Guide to Methods andApplicationsW T J InnisM D GelfandH and J Sninsky Edspp 315ndash322 Academic Press Inc New York NY USA 1990
[22] M Gardes and T D Bruns ldquoITS primers with enhancedspecificity for basidiomycetesmdashapplication to the identificationof mycorrhizae and rustsrdquo Molecular Ecology vol 2 no 2 pp113ndash118 1993
[23] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991
[24] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMole-cular Marine Biology and Biotechnology vol 3 no 5 pp 294ndash299 1994
[25] P Bayman ldquoDiversity scale and variation of endophytic fungiin leaves of tropical plantsrdquo inMicrobiol Ecology of Aerial PlantSurfaces B MJ L AK T-W TM and P T N Spencer-PhillipsEds pp 37ndash50 CABI Publishing UK 2006
[26] J A Chandler J A Eisen and A Kopp ldquoYeast communities ofdiverseDrosophila species Comparison of two symbiont groupsin the samehostsrdquoApplied andEnvironmentalMicrobiology vol78 no 20 pp 7327ndash7336 2012
[27] A E Coluccio R K Rodriguez M J Kernan and A MNeiman ldquoThe yeast spore wall enables spores to survive passagethrough the digestive tract ofDrosophilardquo PLoS ONE vol 3 no8 p e2873 2008
[28] J H Northrop ldquoThe role of yeast in the nutrition of an insectDrosophilardquoThe Journal of Biological Chemistry vol 30 pp 30ndash181 1917
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Microbiology 3Ta
ble1Identifi
catio
nanddistrib
utionof
fung
iand
bacteriaon
andin
fruitfl
iesGenBa
nkaccessionnu
mbersarefor
ITSsequ
enceso
ffun
giand16SrD
NAsequ
encesfor
bacteriaTh
elast
sixcolumns
show
numbersof
each
microorganism
isolatedwith
thefollowingprotocolsflies
thatwalkedacrosspetriplatesflies
washedin
001Tw
een80surface-sterilized
fliesand
guts
removed
from
flies
Strain
Species
GenBa
nkaccessionID
Highesthit
GenBa
nkaccessionID
identity
Externaliso
latio
nInternaliso
latio
nTo
tal119875value
Walked
Washed
Insect
Gut
Fung
usH137
Cand
idainconspicua
KU238836
Cand
idainconspicua
KT207004
100
2720
2314
840275
H35
Penicilliu
mcrustosumcom
mun
eKU
238837
Penicilliu
mcommun
eKR
012904
100
105
67
280705
H127
Issatchenkiahanoien
sisKU
238838
Issatchenkiahanoien
sisFJ153178
991
19
213
0013lowast
H66
Aspergillus
versico
lorsydowiinidu
lans
KU238839
Aspergillus
sydowii
KT989398
100
31
04
810
00
H11
Aspergillus
fumiga
tus
KU238840
Aspergillus
fumiga
tus
AB2
98709
100
21
20
50655
H37
Fusariu
msp
KU238841
Fusariu
mequiseti
HQ332532
100
11
21
50655
H44
Galactomyces
cand
idum
geotrichium
KU238842
Galactomycescand
idum
KJ579946
991
11
14
1000
H102
Pichiamem
branifa
ciens
KU238843
Pichiamem
branifa
ciens
FJ231462
993
01
04
0317
H7
Rhizopus
sp
mdashmdash
mdashmdash
03
00
30083
H24
Fusariu
mequisetioxysporum
KU238844
Fusariu
mequiseti
KJ174399
100
20
01
30564
H77
Penicilliu
mcitrin
umgriseofulvum
KU238845
Penicilliu
mcitrin
umKU
681430
980
21
03
0564
H31
Aspergillus
niger
KU238846
Aspergillus
niger
KP748369
991
11
03
0564
H46
Geotrichum
cand
idum
KU238847
Geotrichum
cand
idum
KF713518
100
11
00
20157
H69
Aspergillus
flavuso
ryzae
KU238848
Aspergillus
flavus
KU360621
100
01
00
10317
H70
Aspergillus
nigertubingensis
KU238849
Aspergillus
niger
EU64
5723
980
01
01
0317
Bacteria
B82
Klebsiella
sp
KU238850
Klebsiella
sp
FN178363
995
68
1332
0077
B5Ba
cillussp
KU238851
Bacillusp
umilu
sKU
517819
997
54
622
0670
B30
Klebsiella
oxytoca
KU238852
Klebsiella
mich
iganensis
KP717391
996
63
520
0371
B44
Klebsiella
pneumoniaevariicola
KU238853
Klebsiella
variicola
KT895843
993
14
614
0109
B22
Erwiniasp
KU238854
Uncultu
redErwiniasp
HE5
75588
993
43
111
0366
B105
Bacillusp
umilu
ssafensis
KU238855
Bacillusp
umilu
sKU
239978
992
03
49
0096
B39
Stenotrophom
onas
maltophilia
KU238856
Stenotrophom
onas
maltophilia
LT222226
992
23
29
0739
B84
Micrococcus
luteusyun
nanensis
KU238857
Micrococcusluteus
KT901825
100
40
40
810
00
B43
Alcaligenesfaecalis
KU238858
Alcaligenesfaecalis
KU179370
992
11
26
1000
B8Microbacteriu
moxydan
sKU
238859
Microbacteriu
moxydan
sKT
580637
990
10
01
0317
Drosoph
ilaD1
Drosophila
mela
nogaste
rKU
238860
Drosophila
mela
nogaste
rKP
161877
99D2
Drosophila
mela
nogaste
rKU
238861
Drosophila
mela
nogaste
rKP
161877
99D3
Drosophila
mela
nogaste
rKU
238862
Drosophila
mela
nogaste
rKP
161877
99Aste
riskrepresentssig
nificantd
ifferencesb
yCh
isqu
aretest
4 International Journal of Microbiology
did not differ significantly in frequency between external andinternal origin (119875 gt 005)
The species composition did not differ significantlybetween internal and external microbiotas either for fungi(Bray-Curtis = 068) or for bacteria (Bray-Curtis = 071)
32 Potential Opportunistic Pathogens Isolated from Dro-sophila melanogaster Bacteria and fungi isolated from Dro-sophila melanogaster included opportunistic pathogens ofhumans and animals includingKlebsiella oxytocaAlcaligenesfaecalis Microbacterium oxydans Stenotrophomonas malto-philia Aspergillus fumigatus A flavus and A niger (Table 1)Also A flavus A niger Fusarium equisetioxysporum andGeotrichum candidum are considered opportunistic plantpathogens (Table 1)
4 Discussion
41 Differences between Bacteria and Fungi Isolated fromDrosophila melanogaster This study was limited to cultur-able microorganisms which were used for experiments onprobiotics [19] However our protocol excluded the majorityof bacteria and many fungi which are nonculturable orrequire specialized media or culture conditions [25]
The richness of fungal morphospecies was higher thanthat of bacteria (Figure 1) The accumulation curves for flieslevelled off suggesting that nearly all the culturable bacterialspecies present in flies were detected but not for fungiThese results contradict a previous study where the fungalcommunities associated with different Drosophila speciessampled around theworldwere less rich that those of bacteria[26]However that study only focused on yeasts isolated fromguts of flies which constitute the vast majority of knownDrosophila-associated fungi
In contrast even though the fungal community is richerin species the bacteria community is more diverse in Dmelanogaster (1198671015840 fungi = 187 versus bacteria = 223) Thissuggests that the population sizes of different bacterial speciesin the flies are more equitable This is supported by twostudies in which bacterial diversity exceeds fungal diversityin Drosophila populations [2 26]
The yeast Issatchenkia hanoiensis was more abundant ininternal parts of flies than externally (119875 lt 0013) Yeasts arecommon Drosophila symbionts and some are food sourcesfor Drosophila [26] Yeast like Saccharomyces cerevisiae cansurvive passage through the digestive tract of flies because theconstituents of spore walls are more resistant than vegetativecells [27] It would be interesting to examine if I hanoiensisprovides any benefit to flies for example food source forlarvae roles in attraction ovoposition development orprotection against pathogens [5 7 28ndash31] I hanoiensis wasfirst described in 2003 from insect frass it has not previouslybeen reported from Drosophila [32]
Apart from Issatchenkia species composition internallyversus externally in flies was similar for fungi (Bray-Curtis =068) and for bacteria (Bray-Curtis = 071) This result con-trasts with a previous microbiome study where the internalbacterial communities were a reduced subset of the external
bacterial communities suggesting that flies can control themicroorganisms in the digestive tract and internal tissues [3]
42 Drosophila melanogaster as a Potential Vector of Path-ogens Drosophila melanogaster can carry opportunisticpathogens of humans [18] We isolated the Gram-negativebacterium Klebsiella oxytoca which has been reported asa causal agent of hemorrhagic colitis and Alcaligenes fae-calis was previously associated with infections in newborns[33 34] Other microorganisms isolated in this study werealso reported as emerging clinical pathogens for exampleMicrobacterium oxydans and Stenotrophomonas maltophilia[35ndash38]We also isolated three opportunistic pathogens capa-ble of causing animal and human aspergillosis Aspergillusfumigatus A flavus and A niger [39ndash42] Their presenceis not surprising because they are ubiquitous in nature withabundant airborne conidia [43]
Fruit flies as sources of contamination could represent apublic health risk especially to patients with compromisedimmune systems For example Mediterranean fruit flies(Ceratitis capitata) exposed to fecal material enriched withGFP-tagged Escherichia coli are capable of transmitting E colito intact apples in a cage model system [10] The same wasseen in D melanogaster [9]
In addition plant pathogens of agricultural concern weredocumented in the sampled flies for exampleA niger Fusar-ium equisetioxysporum and Geotrichum candidum [44ndash47]A flavus causes substantial problems in agriculture as asource of aflatoxins and frequently enters plants throughinsect-induced wounds [40 48]
Almost one hundred years ago D melanogaster com-monly found in exposed fruit in grocery stores and houseswas reported as ldquonot an efficient disease carrierrdquo [8] Thiswas based on the fact that D melanogaster is rarely attractedto excrement However the studies mentioned support ourhypothesis that flies might serve as vectors for opportunisticpathogens to humans and plants More experiments arenecessary to clarify the identity and virulence of the oppor-tunistic pathogens found in this study
5 Conclusions
The isolation of culturable microorganisms from wild Dmelanogaster suggests that its microbiota is rich diverseand distributed throughout internal and external surfacesIssatchenkia hanoiensiswas identified as common componentof the fly microbiota Other microorganisms are relatedto opportunistic human pathogens which may represent apublic health risk indicating D melanogaster is a potentialvector of disease
Conflicts of Interest
The authors report no conflicts of interest
Acknowledgments
This study was part of the doctoral thesis of Luis ARamırez-Camejo [49] and was supported by the following
International Journal of Microbiology 5
entities National Science Foundation-Center for AppliedTropical Ecology and Conservation (NSF-CRESTHRD0734826) National Institutes of Health-Supportof Continuous Research Excellence (NIH SCORE2S06GM08102) Secretarıa Nacional de Ciencia Tecnologıa eInnovacion of Panama (SENACYT ITE15-030) and SistemaNacional de Investigacion of Panama (SNI-NM2017-062)Special thanks are due to undergraduates Ana P Torres-Ocampo Ivana Serrano-Lachapel Michael Garcıa-Aliceaand Luisa Bernacet for help in the lab
References
[1] V Corby-Harris A C Pontaroli L J Shimkets J L BennetzenK E Habel and D E L Promislow ldquoGeographical distributionand diversity of bacteria associated with natural populations ofDrosophila melanogasterrdquo Applied and Environmental Microbi-ology vol 73 no 11 pp 3470ndash3479 2007
[2] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophila melanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007
[3] J A Chandler J Lang S Bhatnagar J A Eisen and A KoppldquoBacterial communities of diverse Drosophila species ecologi-cal context of a host-microbemodel systemrdquo PLoS Genetics vol7 no 9 p e1002272 2011
[4] N A Broderick and B Lemaitre ldquoGut-associated microbes ofDrosophila melanogasterrdquo Gut Microbes vol 3 no 4 pp 307ndash321 2012
[5] M A Ebbert J L Marlowe and J J Burkholder ldquoProtozoanand intracellular fungal gut endosymbionts in Drosophila Pre-valence and fitness effects of single and dual infectionsrdquo Journalof Invertebrate Pathology vol 83 no 1 pp 37ndash45 2003
[6] P B Morais M-A Lachance and C A Rosa ldquoSaturnisporahagleri sp nov a yeast species isolated from Drosophila flies inAtlantic rainforest in Brazilrdquo International Journal of Systematicand Evolutionary Microbiology vol 55 no 4 pp 1725ndash17272005
[7] C AnagnostouMDorsch andM Rohlfs ldquoInfluence of dietaryyeasts on Drosophila melanogaster life-history traitsrdquo Entomo-logia Experimentalis et Applicata vol 136 no 1 pp 1ndash11 2010
[8] A H Sturtevant ldquoFlies of the genus Drosophila as possibledisease carriersrdquo The Journal of Parasitology vol 5 no 2 pp84-85 1918
[9] W J Janisiewicz W S Conway M W Brown G M SapersP Fratamico and R L Buchanan ldquoFate of Escherichia coliO157H7 on fresh-cut apple tissue and its potential for trans-mission by fruit fliesrdquo Applied and Environmental Microbiologyvol 65 no 1 pp 1ndash5 1999
[10] S Sela D Nestel R Pinto E Nemny-Lavy and M Bar-Joseph ldquoMediterranean fruit fly as a potential vector of bacterialpathogensrdquo Applied and Environmental Microbiology vol 71no 7 pp 4052ndash4056 2005
[11] A Keller ldquoDrosophila melanogasterrsquos history as a human com-mensalrdquo Current Biology vol 17 no 3 pp R77ndashR81 2007
[12] Chartered Institute of Environmental Health ldquoPest control proce-dures in the food industryrdquo London pp52 2009
[13] J Landau Fruit flies disrupt surgeries at AtlantiCare hospital inGallowayrdquo Press of Atlantic City 2013 httpwwwpressof-atlanticcitycomnewspressatlanticfruit-flies-disrupt-surgeries-at-atlanticare-hospital-in-gallowayarticle 2f2a7fe6-3779-11e3-9754-001a4bcf887ahtml
[14] P Irving L Troxler T S Heuer et al ldquoA genome-wide analysisof immune responses inDrosophilardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 98 no26 pp 15119ndash15124 2001
[15] A-M Alarco A Marcil J Chen B Suter D Thomas andM Whiteway ldquoImmune-deficient Drosophila melanogaster amodel for the innate immune response to human fungalpathogensrdquoThe Journal of Immunology vol 172 no 9 pp 5622ndash5628 2004
[16] M S Lionakis andD P Kontoyiannis ldquoThe growing promise oftoll-deficient Drosophila melanogaster as a model for studyingAspergillus pathogenesis and treatmentrdquo Virulence vol 1 no 6pp 488ndash499 2010
[17] Y Tanada and H K Kaya ldquoAssociations between insects andnonpathogenicmicroorganismsrdquo in Insect Pathology AcademicPress San Diego Calif USA 1993
[18] W D Munyikombo ldquoPotential for transmission of Pseu-domonas aeruginosa and other bacterial and parasitic infectiousagents by Drosophila sp (fruit flies) as mechanical vectorsrdquoJournal of Biology Agriculture and Healthcare vol 4 no 20 pp154ndash170 2014
[19] L A Ramırez-Camejo M Garcıa-Alicea and G Maldonado-Morales ldquoProbiotics may protect Drosophila from infectionby Aspergillus flavusrdquo International Journal of PharmaceuticalSciences and Research vol 8 no 4 pp 1624ndash1632 2017
[20] A D Banjo O A Lawal andO O Adeduji ldquoBacteria and fungiisolated from housefly (Musca domestica L) larvaerdquo AfricanJournal of Biotechnology vol 4 no 8 pp 780ndash784 2005
[21] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA Genes forphylogeneticsrdquo in PCR Protocols a Guide to Methods andApplicationsW T J InnisM D GelfandH and J Sninsky Edspp 315ndash322 Academic Press Inc New York NY USA 1990
[22] M Gardes and T D Bruns ldquoITS primers with enhancedspecificity for basidiomycetesmdashapplication to the identificationof mycorrhizae and rustsrdquo Molecular Ecology vol 2 no 2 pp113ndash118 1993
[23] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991
[24] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMole-cular Marine Biology and Biotechnology vol 3 no 5 pp 294ndash299 1994
[25] P Bayman ldquoDiversity scale and variation of endophytic fungiin leaves of tropical plantsrdquo inMicrobiol Ecology of Aerial PlantSurfaces B MJ L AK T-W TM and P T N Spencer-PhillipsEds pp 37ndash50 CABI Publishing UK 2006
[26] J A Chandler J A Eisen and A Kopp ldquoYeast communities ofdiverseDrosophila species Comparison of two symbiont groupsin the samehostsrdquoApplied andEnvironmentalMicrobiology vol78 no 20 pp 7327ndash7336 2012
[27] A E Coluccio R K Rodriguez M J Kernan and A MNeiman ldquoThe yeast spore wall enables spores to survive passagethrough the digestive tract ofDrosophilardquo PLoS ONE vol 3 no8 p e2873 2008
[28] J H Northrop ldquoThe role of yeast in the nutrition of an insectDrosophilardquoThe Journal of Biological Chemistry vol 30 pp 30ndash181 1917
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 International Journal of Microbiology
did not differ significantly in frequency between external andinternal origin (119875 gt 005)
The species composition did not differ significantlybetween internal and external microbiotas either for fungi(Bray-Curtis = 068) or for bacteria (Bray-Curtis = 071)
32 Potential Opportunistic Pathogens Isolated from Dro-sophila melanogaster Bacteria and fungi isolated from Dro-sophila melanogaster included opportunistic pathogens ofhumans and animals includingKlebsiella oxytocaAlcaligenesfaecalis Microbacterium oxydans Stenotrophomonas malto-philia Aspergillus fumigatus A flavus and A niger (Table 1)Also A flavus A niger Fusarium equisetioxysporum andGeotrichum candidum are considered opportunistic plantpathogens (Table 1)
4 Discussion
41 Differences between Bacteria and Fungi Isolated fromDrosophila melanogaster This study was limited to cultur-able microorganisms which were used for experiments onprobiotics [19] However our protocol excluded the majorityof bacteria and many fungi which are nonculturable orrequire specialized media or culture conditions [25]
The richness of fungal morphospecies was higher thanthat of bacteria (Figure 1) The accumulation curves for flieslevelled off suggesting that nearly all the culturable bacterialspecies present in flies were detected but not for fungiThese results contradict a previous study where the fungalcommunities associated with different Drosophila speciessampled around theworldwere less rich that those of bacteria[26]However that study only focused on yeasts isolated fromguts of flies which constitute the vast majority of knownDrosophila-associated fungi
In contrast even though the fungal community is richerin species the bacteria community is more diverse in Dmelanogaster (1198671015840 fungi = 187 versus bacteria = 223) Thissuggests that the population sizes of different bacterial speciesin the flies are more equitable This is supported by twostudies in which bacterial diversity exceeds fungal diversityin Drosophila populations [2 26]
The yeast Issatchenkia hanoiensis was more abundant ininternal parts of flies than externally (119875 lt 0013) Yeasts arecommon Drosophila symbionts and some are food sourcesfor Drosophila [26] Yeast like Saccharomyces cerevisiae cansurvive passage through the digestive tract of flies because theconstituents of spore walls are more resistant than vegetativecells [27] It would be interesting to examine if I hanoiensisprovides any benefit to flies for example food source forlarvae roles in attraction ovoposition development orprotection against pathogens [5 7 28ndash31] I hanoiensis wasfirst described in 2003 from insect frass it has not previouslybeen reported from Drosophila [32]
Apart from Issatchenkia species composition internallyversus externally in flies was similar for fungi (Bray-Curtis =068) and for bacteria (Bray-Curtis = 071) This result con-trasts with a previous microbiome study where the internalbacterial communities were a reduced subset of the external
bacterial communities suggesting that flies can control themicroorganisms in the digestive tract and internal tissues [3]
42 Drosophila melanogaster as a Potential Vector of Path-ogens Drosophila melanogaster can carry opportunisticpathogens of humans [18] We isolated the Gram-negativebacterium Klebsiella oxytoca which has been reported asa causal agent of hemorrhagic colitis and Alcaligenes fae-calis was previously associated with infections in newborns[33 34] Other microorganisms isolated in this study werealso reported as emerging clinical pathogens for exampleMicrobacterium oxydans and Stenotrophomonas maltophilia[35ndash38]We also isolated three opportunistic pathogens capa-ble of causing animal and human aspergillosis Aspergillusfumigatus A flavus and A niger [39ndash42] Their presenceis not surprising because they are ubiquitous in nature withabundant airborne conidia [43]
Fruit flies as sources of contamination could represent apublic health risk especially to patients with compromisedimmune systems For example Mediterranean fruit flies(Ceratitis capitata) exposed to fecal material enriched withGFP-tagged Escherichia coli are capable of transmitting E colito intact apples in a cage model system [10] The same wasseen in D melanogaster [9]
In addition plant pathogens of agricultural concern weredocumented in the sampled flies for exampleA niger Fusar-ium equisetioxysporum and Geotrichum candidum [44ndash47]A flavus causes substantial problems in agriculture as asource of aflatoxins and frequently enters plants throughinsect-induced wounds [40 48]
Almost one hundred years ago D melanogaster com-monly found in exposed fruit in grocery stores and houseswas reported as ldquonot an efficient disease carrierrdquo [8] Thiswas based on the fact that D melanogaster is rarely attractedto excrement However the studies mentioned support ourhypothesis that flies might serve as vectors for opportunisticpathogens to humans and plants More experiments arenecessary to clarify the identity and virulence of the oppor-tunistic pathogens found in this study
5 Conclusions
The isolation of culturable microorganisms from wild Dmelanogaster suggests that its microbiota is rich diverseand distributed throughout internal and external surfacesIssatchenkia hanoiensiswas identified as common componentof the fly microbiota Other microorganisms are relatedto opportunistic human pathogens which may represent apublic health risk indicating D melanogaster is a potentialvector of disease
Conflicts of Interest
The authors report no conflicts of interest
Acknowledgments
This study was part of the doctoral thesis of Luis ARamırez-Camejo [49] and was supported by the following
International Journal of Microbiology 5
entities National Science Foundation-Center for AppliedTropical Ecology and Conservation (NSF-CRESTHRD0734826) National Institutes of Health-Supportof Continuous Research Excellence (NIH SCORE2S06GM08102) Secretarıa Nacional de Ciencia Tecnologıa eInnovacion of Panama (SENACYT ITE15-030) and SistemaNacional de Investigacion of Panama (SNI-NM2017-062)Special thanks are due to undergraduates Ana P Torres-Ocampo Ivana Serrano-Lachapel Michael Garcıa-Aliceaand Luisa Bernacet for help in the lab
References
[1] V Corby-Harris A C Pontaroli L J Shimkets J L BennetzenK E Habel and D E L Promislow ldquoGeographical distributionand diversity of bacteria associated with natural populations ofDrosophila melanogasterrdquo Applied and Environmental Microbi-ology vol 73 no 11 pp 3470ndash3479 2007
[2] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophila melanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007
[3] J A Chandler J Lang S Bhatnagar J A Eisen and A KoppldquoBacterial communities of diverse Drosophila species ecologi-cal context of a host-microbemodel systemrdquo PLoS Genetics vol7 no 9 p e1002272 2011
[4] N A Broderick and B Lemaitre ldquoGut-associated microbes ofDrosophila melanogasterrdquo Gut Microbes vol 3 no 4 pp 307ndash321 2012
[5] M A Ebbert J L Marlowe and J J Burkholder ldquoProtozoanand intracellular fungal gut endosymbionts in Drosophila Pre-valence and fitness effects of single and dual infectionsrdquo Journalof Invertebrate Pathology vol 83 no 1 pp 37ndash45 2003
[6] P B Morais M-A Lachance and C A Rosa ldquoSaturnisporahagleri sp nov a yeast species isolated from Drosophila flies inAtlantic rainforest in Brazilrdquo International Journal of Systematicand Evolutionary Microbiology vol 55 no 4 pp 1725ndash17272005
[7] C AnagnostouMDorsch andM Rohlfs ldquoInfluence of dietaryyeasts on Drosophila melanogaster life-history traitsrdquo Entomo-logia Experimentalis et Applicata vol 136 no 1 pp 1ndash11 2010
[8] A H Sturtevant ldquoFlies of the genus Drosophila as possibledisease carriersrdquo The Journal of Parasitology vol 5 no 2 pp84-85 1918
[9] W J Janisiewicz W S Conway M W Brown G M SapersP Fratamico and R L Buchanan ldquoFate of Escherichia coliO157H7 on fresh-cut apple tissue and its potential for trans-mission by fruit fliesrdquo Applied and Environmental Microbiologyvol 65 no 1 pp 1ndash5 1999
[10] S Sela D Nestel R Pinto E Nemny-Lavy and M Bar-Joseph ldquoMediterranean fruit fly as a potential vector of bacterialpathogensrdquo Applied and Environmental Microbiology vol 71no 7 pp 4052ndash4056 2005
[11] A Keller ldquoDrosophila melanogasterrsquos history as a human com-mensalrdquo Current Biology vol 17 no 3 pp R77ndashR81 2007
[12] Chartered Institute of Environmental Health ldquoPest control proce-dures in the food industryrdquo London pp52 2009
[13] J Landau Fruit flies disrupt surgeries at AtlantiCare hospital inGallowayrdquo Press of Atlantic City 2013 httpwwwpressof-atlanticcitycomnewspressatlanticfruit-flies-disrupt-surgeries-at-atlanticare-hospital-in-gallowayarticle 2f2a7fe6-3779-11e3-9754-001a4bcf887ahtml
[14] P Irving L Troxler T S Heuer et al ldquoA genome-wide analysisof immune responses inDrosophilardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 98 no26 pp 15119ndash15124 2001
[15] A-M Alarco A Marcil J Chen B Suter D Thomas andM Whiteway ldquoImmune-deficient Drosophila melanogaster amodel for the innate immune response to human fungalpathogensrdquoThe Journal of Immunology vol 172 no 9 pp 5622ndash5628 2004
[16] M S Lionakis andD P Kontoyiannis ldquoThe growing promise oftoll-deficient Drosophila melanogaster as a model for studyingAspergillus pathogenesis and treatmentrdquo Virulence vol 1 no 6pp 488ndash499 2010
[17] Y Tanada and H K Kaya ldquoAssociations between insects andnonpathogenicmicroorganismsrdquo in Insect Pathology AcademicPress San Diego Calif USA 1993
[18] W D Munyikombo ldquoPotential for transmission of Pseu-domonas aeruginosa and other bacterial and parasitic infectiousagents by Drosophila sp (fruit flies) as mechanical vectorsrdquoJournal of Biology Agriculture and Healthcare vol 4 no 20 pp154ndash170 2014
[19] L A Ramırez-Camejo M Garcıa-Alicea and G Maldonado-Morales ldquoProbiotics may protect Drosophila from infectionby Aspergillus flavusrdquo International Journal of PharmaceuticalSciences and Research vol 8 no 4 pp 1624ndash1632 2017
[20] A D Banjo O A Lawal andO O Adeduji ldquoBacteria and fungiisolated from housefly (Musca domestica L) larvaerdquo AfricanJournal of Biotechnology vol 4 no 8 pp 780ndash784 2005
[21] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA Genes forphylogeneticsrdquo in PCR Protocols a Guide to Methods andApplicationsW T J InnisM D GelfandH and J Sninsky Edspp 315ndash322 Academic Press Inc New York NY USA 1990
[22] M Gardes and T D Bruns ldquoITS primers with enhancedspecificity for basidiomycetesmdashapplication to the identificationof mycorrhizae and rustsrdquo Molecular Ecology vol 2 no 2 pp113ndash118 1993
[23] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991
[24] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMole-cular Marine Biology and Biotechnology vol 3 no 5 pp 294ndash299 1994
[25] P Bayman ldquoDiversity scale and variation of endophytic fungiin leaves of tropical plantsrdquo inMicrobiol Ecology of Aerial PlantSurfaces B MJ L AK T-W TM and P T N Spencer-PhillipsEds pp 37ndash50 CABI Publishing UK 2006
[26] J A Chandler J A Eisen and A Kopp ldquoYeast communities ofdiverseDrosophila species Comparison of two symbiont groupsin the samehostsrdquoApplied andEnvironmentalMicrobiology vol78 no 20 pp 7327ndash7336 2012
[27] A E Coluccio R K Rodriguez M J Kernan and A MNeiman ldquoThe yeast spore wall enables spores to survive passagethrough the digestive tract ofDrosophilardquo PLoS ONE vol 3 no8 p e2873 2008
[28] J H Northrop ldquoThe role of yeast in the nutrition of an insectDrosophilardquoThe Journal of Biological Chemistry vol 30 pp 30ndash181 1917
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Microbiology 5
entities National Science Foundation-Center for AppliedTropical Ecology and Conservation (NSF-CRESTHRD0734826) National Institutes of Health-Supportof Continuous Research Excellence (NIH SCORE2S06GM08102) Secretarıa Nacional de Ciencia Tecnologıa eInnovacion of Panama (SENACYT ITE15-030) and SistemaNacional de Investigacion of Panama (SNI-NM2017-062)Special thanks are due to undergraduates Ana P Torres-Ocampo Ivana Serrano-Lachapel Michael Garcıa-Aliceaand Luisa Bernacet for help in the lab
References
[1] V Corby-Harris A C Pontaroli L J Shimkets J L BennetzenK E Habel and D E L Promislow ldquoGeographical distributionand diversity of bacteria associated with natural populations ofDrosophila melanogasterrdquo Applied and Environmental Microbi-ology vol 73 no 11 pp 3470ndash3479 2007
[2] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophila melanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007
[3] J A Chandler J Lang S Bhatnagar J A Eisen and A KoppldquoBacterial communities of diverse Drosophila species ecologi-cal context of a host-microbemodel systemrdquo PLoS Genetics vol7 no 9 p e1002272 2011
[4] N A Broderick and B Lemaitre ldquoGut-associated microbes ofDrosophila melanogasterrdquo Gut Microbes vol 3 no 4 pp 307ndash321 2012
[5] M A Ebbert J L Marlowe and J J Burkholder ldquoProtozoanand intracellular fungal gut endosymbionts in Drosophila Pre-valence and fitness effects of single and dual infectionsrdquo Journalof Invertebrate Pathology vol 83 no 1 pp 37ndash45 2003
[6] P B Morais M-A Lachance and C A Rosa ldquoSaturnisporahagleri sp nov a yeast species isolated from Drosophila flies inAtlantic rainforest in Brazilrdquo International Journal of Systematicand Evolutionary Microbiology vol 55 no 4 pp 1725ndash17272005
[7] C AnagnostouMDorsch andM Rohlfs ldquoInfluence of dietaryyeasts on Drosophila melanogaster life-history traitsrdquo Entomo-logia Experimentalis et Applicata vol 136 no 1 pp 1ndash11 2010
[8] A H Sturtevant ldquoFlies of the genus Drosophila as possibledisease carriersrdquo The Journal of Parasitology vol 5 no 2 pp84-85 1918
[9] W J Janisiewicz W S Conway M W Brown G M SapersP Fratamico and R L Buchanan ldquoFate of Escherichia coliO157H7 on fresh-cut apple tissue and its potential for trans-mission by fruit fliesrdquo Applied and Environmental Microbiologyvol 65 no 1 pp 1ndash5 1999
[10] S Sela D Nestel R Pinto E Nemny-Lavy and M Bar-Joseph ldquoMediterranean fruit fly as a potential vector of bacterialpathogensrdquo Applied and Environmental Microbiology vol 71no 7 pp 4052ndash4056 2005
[11] A Keller ldquoDrosophila melanogasterrsquos history as a human com-mensalrdquo Current Biology vol 17 no 3 pp R77ndashR81 2007
[12] Chartered Institute of Environmental Health ldquoPest control proce-dures in the food industryrdquo London pp52 2009
[13] J Landau Fruit flies disrupt surgeries at AtlantiCare hospital inGallowayrdquo Press of Atlantic City 2013 httpwwwpressof-atlanticcitycomnewspressatlanticfruit-flies-disrupt-surgeries-at-atlanticare-hospital-in-gallowayarticle 2f2a7fe6-3779-11e3-9754-001a4bcf887ahtml
[14] P Irving L Troxler T S Heuer et al ldquoA genome-wide analysisof immune responses inDrosophilardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 98 no26 pp 15119ndash15124 2001
[15] A-M Alarco A Marcil J Chen B Suter D Thomas andM Whiteway ldquoImmune-deficient Drosophila melanogaster amodel for the innate immune response to human fungalpathogensrdquoThe Journal of Immunology vol 172 no 9 pp 5622ndash5628 2004
[16] M S Lionakis andD P Kontoyiannis ldquoThe growing promise oftoll-deficient Drosophila melanogaster as a model for studyingAspergillus pathogenesis and treatmentrdquo Virulence vol 1 no 6pp 488ndash499 2010
[17] Y Tanada and H K Kaya ldquoAssociations between insects andnonpathogenicmicroorganismsrdquo in Insect Pathology AcademicPress San Diego Calif USA 1993
[18] W D Munyikombo ldquoPotential for transmission of Pseu-domonas aeruginosa and other bacterial and parasitic infectiousagents by Drosophila sp (fruit flies) as mechanical vectorsrdquoJournal of Biology Agriculture and Healthcare vol 4 no 20 pp154ndash170 2014
[19] L A Ramırez-Camejo M Garcıa-Alicea and G Maldonado-Morales ldquoProbiotics may protect Drosophila from infectionby Aspergillus flavusrdquo International Journal of PharmaceuticalSciences and Research vol 8 no 4 pp 1624ndash1632 2017
[20] A D Banjo O A Lawal andO O Adeduji ldquoBacteria and fungiisolated from housefly (Musca domestica L) larvaerdquo AfricanJournal of Biotechnology vol 4 no 8 pp 780ndash784 2005
[21] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA Genes forphylogeneticsrdquo in PCR Protocols a Guide to Methods andApplicationsW T J InnisM D GelfandH and J Sninsky Edspp 315ndash322 Academic Press Inc New York NY USA 1990
[22] M Gardes and T D Bruns ldquoITS primers with enhancedspecificity for basidiomycetesmdashapplication to the identificationof mycorrhizae and rustsrdquo Molecular Ecology vol 2 no 2 pp113ndash118 1993
[23] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991
[24] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMole-cular Marine Biology and Biotechnology vol 3 no 5 pp 294ndash299 1994
[25] P Bayman ldquoDiversity scale and variation of endophytic fungiin leaves of tropical plantsrdquo inMicrobiol Ecology of Aerial PlantSurfaces B MJ L AK T-W TM and P T N Spencer-PhillipsEds pp 37ndash50 CABI Publishing UK 2006
[26] J A Chandler J A Eisen and A Kopp ldquoYeast communities ofdiverseDrosophila species Comparison of two symbiont groupsin the samehostsrdquoApplied andEnvironmentalMicrobiology vol78 no 20 pp 7327ndash7336 2012
[27] A E Coluccio R K Rodriguez M J Kernan and A MNeiman ldquoThe yeast spore wall enables spores to survive passagethrough the digestive tract ofDrosophilardquo PLoS ONE vol 3 no8 p e2873 2008
[28] J H Northrop ldquoThe role of yeast in the nutrition of an insectDrosophilardquoThe Journal of Biological Chemistry vol 30 pp 30ndash181 1917
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 International Journal of Microbiology
[29] R C King ldquoThe effect of yeast on phosphorus uptake byDroso-philardquo The American Naturalist vol 88 no 840 pp 155ndash1581954
[30] S M Henry ldquoThe significance of microorganisms in thenutrition of insectsrdquo Transactions of the New York Academy ofSciences vol 24 no 6 Series II pp 676ndash683 1962
[31] P G Becher G Flick E Rozpedowska et al ldquoYeast not fruitvolatiles mediate Drosophila melanogaster attraction oviposi-tion and developmentrdquo Functional Ecology vol 26 no 4 pp822ndash828 2012
[32] V Nguyen Thanh D Anh Hai and M-A Lachance ldquoIssat-chenkia hanoiensis a new yeast species isolated from frass ofthe litchi fruit borer Conopomorpha cramerella Snellenrdquo FEMSYeast Research vol 4 no 1 pp 113ndash117 2003
[33] J D Sherman D Ingall J Wiener and C V Pryles ldquoAlcaligenesfaecalis infection in the newbornrdquo American Journal of Diseasesof Children vol 100 no 2 pp 212ndash216 1960
[34] C Hogenauer C Langner and E Beubler ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006
[35] K Gneiding R Frodl and G Funke ldquoIdentities of Microbac-terium spp encountered in human clinical specimensrdquo Journalof Clinical Microbiology vol 46 no 11 pp 3646ndash3652 2008
[36] W J Looney M Narita and K Muhlemann ldquoStenotrophomo-nas maltophilia an emerging opportunist human pathogenrdquoThe Lancet Infectious Diseases vol 9 no 5 pp 312ndash323 2009
[37] H I Woo J H Lee S Lee C Ki and N Y Lee ldquoCatheter-related bacteremia due toMicrobacterium oxydans identified by16S rRNA sequencing analysis and biochemical characteristicsrdquoKorean Journal of Clinical Microbiology vol 13 no 4 p 1732010
[38] J S Brooke ldquoStenotrophomonasmaltophilia an emerging globalopportunistic pathogenrdquo Clinical Microbiology Reviews vol 25no 1 pp 2ndash41 2012
[39] J-P Latge ldquoAspergillus fumigatus and aspergillosisrdquo ClinicalMicrobiology Reviews vol 12 no 2 pp 310ndash350 1999
[40] M T Hedayati A C Pasqualotto P A Warn P Bowyer andD W Denning ldquoAspergillus flavus human pathogen allergenandmycotoxin producerrdquoMicrobiology vol 153 no 6 pp 1677ndash1692 2007
[41] S J Park C R Chung Y K Rhee H B Lee Y C Lee and EY Kweon ldquoChronic pulmonary aspergillosis due to Aspergillusnigerrdquo American Journal of Respiratory and Critical Care Medi-cine vol 186 no 10 pp e16ndashe17 2012
[42] L A Ramırez-Camejo A P Torres-Ocampo J L Agosto-Rivera and P Bayman ldquoAn opportunistic human pathogen onthe fly Strains of Aspergillus flavus vary in virulence in Droso-philamelanogasterrdquoMedicalMycology vol 52 no 2 pp 211ndash2192014
[43] M A Klich ldquoBiogeography of Aspergillus species in soil andlitterrdquoMycologia vol 94 no 1 pp 21ndash27 2002
[44] W M Coutinho N D Suassuna C M Luz F A Suinaga andO R R F Silva ldquoBole rot of sisal caused by Aspergillus niger inBrazilrdquo Fitopatologia Brasileira vol 31 no 6 p 605 2006
[45] R S Goswami Y Dong and Z K Punja ldquoHost range andmycotoxin production by Fusarium equiseti isolates originatingfrom ginseng fieldsrdquo Canadian Journal of Plant Pathology vol30 no 1 pp 155ndash160 2008
[46] T B Bourret E K Kramer J D Rogers and D A Glawe ldquoIso-lation ofGeotrichum candidum pathogenic to tomato (Solanum
lycopersicum) in Washington staterdquo North American Fungi vol8 no 14 pp 1ndash7 2013
[47] L-J Ma D M Geiser R H Proctor et al ldquoFusarium patho-genomicsrdquo Annual Review of Microbiology vol 67 pp 399ndash4162013
[48] G Barros A Torres and S Chulze ldquoAspergillus flavus popu-lation isolated from soil of Argentinarsquos peanut-growing regionSclerotia production and toxigenic profilerdquo Journal of the Sci-ence of Food andAgriculture vol 85 no 14 pp 2349ndash2353 2005
[49] L A Ramırez-Camejo and P Bayman Aspergillosis in the fruitfly Drosophila melanogaster as a model system University ofPuerto Rico Rio Piedras 2015
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 201
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
