The microbiota of wild
In the early 20th century, some
By the start of the 21st century,
In this study, we isolated microorganisms from wild
Wild females of
Fungi were isolated on Potato Dextrose Agar (PDA) and bacteria on Tryptic Soy Agar (TSA); they are common and nonselective media that provide enough nutrients to encourage growth of a range of fungi and bacteria, respectively. Fungi and bacteria were isolated externally and internally from 40 flies per sample as follows: 5 flies each were placed on plates of PDA and TSA and allowed to walk on the surface for five minutes. Another 10 flies per sample were then anesthetized with CO2, placed in a microcentrifuge tube with sterile water and Tween 80 (0.01%) and mixed in a vortex for 1 minute to release microbial cells from body surfaces [
Microorganisms were isolated every day and plated in a 2 mL glass vial with PDA or TSA. Microorganisms were grouped by morphospecies based on morphological characteristics, for example, colony size, color, texture, and type of margin.
One fungal isolate from each morphospecies was cultured in Potato Dextrose Broth (PDB), filtered, and macerated in liquid nitrogen. DNA was extracted using a phenol-chloroform method [
For fungi, the nuclear ribosomal Internal Transcribed Spacer (ITS) was amplified using primers ITS 1F and ITS 4 [
Identification and distribution of fungi and bacteria on and in fruit flies. GenBank accession numbers are for ITS sequences of fungi and 16S rDNA sequences for bacteria. The last six columns show numbers of each microorganism isolated with the following protocols: flies that walked across petri plates; flies washed in 0.01% Tween 80; surface-sterilized flies; and guts removed from flies.
Strain | Species | GenBank accession ID | Highest hit | GenBank accession ID | % identity | External isolation | Internal isolation | Total |
|
||
---|---|---|---|---|---|---|---|---|---|---|---|
Walked | Washed | Insect | Gut | ||||||||
Fungus | |||||||||||
H137 |
|
KU238836 |
|
KT207004 | 100 | 27 | 20 | 23 | 14 | 84 | 0.275 |
H35 |
|
KU238837 |
|
KR012904 | 100 | 10 | 5 | 6 | 7 | 28 | 0.705 |
H127 |
|
KU238838 |
|
FJ153178 | 99 | 1 | 1 | 9 | 2 | 13 | 0.013 |
H66 |
|
KU238839 |
|
KT989398 | 100 | 3 | 1 | 0 | 4 | 8 | 1.000 |
H11 |
|
KU238840 |
|
AB298709 | 100 | 2 | 1 | 2 | 0 | 5 | 0.655 |
H37 |
|
KU238841 |
|
HQ332532 | 100 | 1 | 1 | 2 | 1 | 5 | 0.655 |
H44 |
|
KU238842 |
|
KJ579946 | 99 | 1 | 1 | 1 | 1 | 4 | 1.000 |
H102 |
|
KU238843 |
|
FJ231462 | 99 | 3 | 0 | 1 | 0 | 4 | 0.317 |
H7 |
|
— | — | — | — | 0 | 3 | 0 | 0 | 3 | 0.083 |
H24 |
|
KU238844 |
|
KJ174399 | 100 | 2 | 0 | 0 | 1 | 3 | 0.564 |
H77 |
|
KU238845 |
|
KU681430 | 98 | 0 | 2 | 1 | 0 | 3 | 0.564 |
H31 |
|
KU238846 |
|
KP748369 | 99 | 1 | 1 | 1 | 0 | 3 | 0.564 |
H46 |
|
KU238847 |
|
KF713518 | 100 | 1 | 1 | 0 | 0 | 2 | 0.157 |
H69 |
|
KU238848 |
|
KU360621 | 100 | 0 | 1 | 0 | 0 | 1 | 0.317 |
H70 |
|
KU238849 |
|
EU645723 | 98 | 0 | 0 | 1 | 0 | 1 | 0.317 |
|
|||||||||||
Bacteria | |||||||||||
B82 |
|
KU238850 |
|
FN178363 | 99 | 5 | 6 | 8 | 13 | 32 | 0.077 |
B5 |
|
KU238851 |
|
KU517819 | 99 | 7 | 5 | 4 | 6 | 22 | 0.670 |
B30 |
|
KU238852 |
|
KP717391 | 99 | 6 | 6 | 3 | 5 | 20 | 0.371 |
B44 |
|
KU238853 |
|
KT895843 | 99 | 3 | 1 | 4 | 6 | 14 | 0.109 |
B22 |
|
KU238854 |
|
HE575588 | 99 | 3 | 4 | 3 | 1 | 11 | 0.366 |
B105 |
|
KU238855 |
|
KU239978 | 99 | 2 | 0 | 3 | 4 | 9 | 0.096 |
B39 |
|
KU238856 |
|
LT222226 | 99 | 2 | 2 | 3 | 2 | 9 | 0.739 |
B84 |
|
KU238857 |
|
KT901825 | 100 | 4 | 0 | 4 | 0 | 8 | 1.000 |
B43 |
|
KU238858 |
|
KU179370 | 99 | 2 | 1 | 1 | 2 | 6 | 1.000 |
B8 |
|
KU238859 |
|
KT580637 | 99 | 0 | 1 | 0 | 0 | 1 | 0.317 |
|
|||||||||||
Drosophila | |||||||||||
D1 |
|
KU238860 |
|
KP161877 | 99 | ||||||
D2 |
|
KU238861 |
|
KP161877 | 99 | ||||||
D3 |
|
KU238862 |
|
KP161877 | 99 |
Asterisk represents significant differences by Chi square test.
EstimateS (version 9.1.0 for Mac) was used to compare the richness (Chao 1), diversity (Shannon index), and composition (Bray-Curtis index) of communities in flies (
We isolated 314 microorganisms from wild
Species richness estimated as Chao 1 was 20 and 12, in fungal and bacterial communities, respectively. Species accumulation curves showed that for bacteria sampling was sufficient, assuming that our morphospecies did not contain cryptic species. For fungi the sampling was insufficient (Figure
Species accumulation curves for fungal and bacterial morphospecies isolated from female
Only the yeast
The species composition did not differ significantly between internal and external microbiotas, either for fungi (Bray-Curtis = 0.68) or for bacteria (Bray-Curtis = 0.71).
Bacteria and fungi isolated from
This study was limited to culturable microorganisms which were used for experiments on probiotics [
The richness of fungal morphospecies was higher than that of bacteria (Figure
In contrast, even though the fungal community is richer in species, the bacteria community is more diverse in
The yeast
Apart from
Fruit flies as sources of contamination could represent a public health risk, especially to patients with compromised immune systems. For example, Mediterranean fruit flies
In addition, plant pathogens of agricultural concern were documented in the sampled flies, for example,
Almost one hundred years ago,
The isolation of culturable microorganisms from wild
The authors report no conflicts of interest.
This study was part of the doctoral thesis of Luis A. Ramírez-Camejo [