PMID- 27004717
OWN - NLM
STAT- MEDLINE
DCOM- 20161213
LR  - 20221229
IS  - 1756-3305 (Electronic)
IS  - 1756-3305 (Linking)
VI  - 9
DP  - 2016 Mar 22
TI  - Flow cytometry analysis of the microbiota associated with the midguts of vector 
      mosquitoes.
PG  - 167
LID - 10.1186/s13071-016-1438-0 [doi]
LID - 167
AB  - BACKGROUND: The scientific interest to understand the function and structure of 
      the microbiota associated with the midgut of mosquito disease vectors is 
      increasing. The advancement of such a knowledge has encountered challenges and 
      limitations associated with conventional culture-based and PCR techniques. 
      METHODS: Flow cytometry (FCM) combined with various cell marking dyes have been 
      successfully applied in the field of ecological microbiology to circumvent the 
      above shortcomings. Here, we describe FCM technique coupled with live/dead 
      differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to 
      quantify and study other essential characteristics of the mosquito gut 
      microbiota. RESULTS: A clear discrimination between cells and debris, as well as 
      between live and dead cells was achieved when the midgut homogenate was subjected 
      to staining with 5 x 103 dilution of the SGI and 30 muM concentration of the PI. 
      Reproducibly, FCM event collections produced discrete populations including 
      non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that 
      fluoresce both in SYBR and PI, all these cell populations representing, 
      respectively, background noise, live bacterial, dead cells and inactive cells 
      with partial permeability to PI. The FCM produced a strong linear relationship 
      between cell counts and their corresponding dilution factors (R (2) = 0.987), and 
      the technique has a better precision compared to qRT-PCR. The FCM count of the 
      microbiota reached a peak load at 18 h post-feeding and started declining at 24 
      h. The present FCM technique also successfully applied to quantify bacterial 
      cells in fixed midgut samples that were homogenized in 4 % PFA. CONCLUSION: The 
      FCM technique described here offers enormous potential and possibilities of 
      integration with advanced molecular biochemical techniques for the study of the 
      microbiota community in disease vector mosquitoes.
FAU - Habtewold, Tibebu
AU  - Habtewold T
AD  - Department of Life Sciences, Imperial College London, London, UK. 
      t.habtewold@imperial.ac.uk.
AD  - Department of Comparative Physiology and Biometrics, University of Ghent, Ghent, 
      Belgium. t.habtewold@imperial.ac.uk.
FAU - Duchateau, Luc
AU  - Duchateau L
AD  - Department of Comparative Physiology and Biometrics, University of Ghent, Ghent, 
      Belgium.
FAU - Christophides, George K
AU  - Christophides GK
AD  - Department of Life Sciences, Imperial College London, London, UK.
LA  - eng
GR  - BB/K009338/1/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20160322
PL  - England
TA  - Parasit Vectors
JT  - Parasites & vectors
JID - 101462774
RN  - 0 (Fluorescent Dyes)
SB  - IM
MH  - Animals
MH  - Culicidae/*microbiology
MH  - Entomology/methods
MH  - Flow Cytometry/*methods
MH  - Fluorescent Dyes
MH  - *Gastrointestinal Microbiome
MH  - Staining and Labeling/*methods
PMC - PMC4802834
OTO - NOTNLM
OT  - Anopheles Coluzzii
OT  - Dead
OT  - Fixed cells
OT  - Flow cytometry
OT  - Live
OT  - Microbiota
OT  - Midgut homogenate
OT  - Propidium Iodide
OT  - discrimination
EDAT- 2016/03/24 06:00
MHDA- 2016/12/15 06:00
PMCR- 2016/03/22
CRDT- 2016/03/24 06:00
PHST- 2015/10/22 00:00 [received]
PHST- 2016/03/08 00:00 [accepted]
PHST- 2016/03/24 06:00 [entrez]
PHST- 2016/03/24 06:00 [pubmed]
PHST- 2016/12/15 06:00 [medline]
PHST- 2016/03/22 00:00 [pmc-release]
AID - 10.1186/s13071-016-1438-0 [pii]
AID - 1438 [pii]
AID - 10.1186/s13071-016-1438-0 [doi]
PST - epublish
SO  - Parasit Vectors. 2016 Mar 22;9:167. doi: 10.1186/s13071-016-1438-0.