3.1 Isolation of fungi
A total of 1.541 fungi were isolated from the hives of M. interrupta and M. seminigra. The experiments were performed during both dry and rainy season, to check the influence of seasonality on the number of CFUs. In M. interrupta, a total of 850 isolates were registered, 537 during the rainy season, and 313 during the dry season. In M. seminigra, a total of 691 isolates were found, 545 during the rainy season, and 146 during the dry one.
3.2 Morphological identification
Altogether 737 filamentous fungi were identified by morphology, for the other filamentous fungi, it was not possible to characterize them by morphology. In general, these fungi belonged to four genera of filamentous fungi: Aspergillus, Penicillium, Rhizopus and Cladosporium. Five morphotypes of Aspergillus spp. were observed, and two of them could be identified at the species level: A. niger and A. ochraceus. Isolates of A. niger were characterized by a dark colony, with a conidial and globose head composed of dark conidia, while isolates of A. ochraceus, by a yellow colony with a conidial and globose head composed of yellow conidia. The other three morphotypes of Aspergillus genus were grouped as Aspergillus sp. 1, Aspergillus sp. 2 and Aspergillus sp. 3. A single morphotype was observed for the genera, Cladosporium, Penicillium and Rhizopus both of which were not identified at the species level.
Moreover, nine isolates that were obtained from larval brood cells of both M. interrupta and M. seminigra and presented the same morphotype were identified as belonging to the species Monascus ruber.
3.3 Molecular identification
The sequencing of this region was able to identify a total of 802 isolates. They belonged to ten species, five of filamentous fungi (Aspergillus fumigatus, Aspergillus nomius, Cladosporium cladosporioides, Monascus ruber and Penicillium citrinum); and five of yeasts (Cystobasidium minutum, Debaryomyces hansenii, Starmerella apicola, Starmerella etchellsii and Wickerhamiella versatilis (Table 1). Molecular analysis corroborated the morphological identification of Aspergillus sp. at the genera level, but not at the species level since A. niger and A. ochraceus were not discriminated by their rDNA ITS1-5.8S-ITS2 sequences. Moreover, samples that were grouped as Aspergillus sp. 2 and Aspergillus sp. 3 in the morphological essays were identified as A. fumigatus and A. nomius in the molecular analysis, respectively. Aspergillus sp. 1, however, was also not discriminated at species level in this analysis. The isolates previously identified as Penicillium sp. showed a match with the species P. citrinum; and Cladosporium sp., with the species Cladosporium cladosporioides (Table 1). Also, the fungi isolated from larval brood cells were confirmed as M. ruber.
Furthermore, molecular analysis enabled the identification of agents present in the brood cells of pupae, and, thus, the comparison of isolates between castes. In M. interrupta, 129 isolates were obtained from queen cells, and 528 isolates, from worker cells. The identified taxa were A. niger, C. cladosporioides, D. hansenii, P. citrinum, and S. apicola in queen cells; Aspergillus sp. 1, A. niger, A. fumigatus, C. cladosporioides, P. citrinum, S. apicola and S. etchellsii in worker cells (Table 2). In M. seminigra, 119 isolates were found in queen cells, and 312 isolates, in worker cells. The identified taxa were: Aspergillus sp. 1, A. niger, A. nomius and W. versatilis in queen cells; A. niger, A. fumigatus, C. cladosporioides and C. minutum in worker cells (Table 2). The unidentified fungi that add up to 163 isolates make up a group of isolates in which the combination of morphological and molecular analyzes was inconsistent, the molecular analysis indicated genera incompatible with the morphological characteristics of the isolates when verified in the identification keys, in these cases, the morphological analysis was disregarded and it is not possible to define the gender (Table 2).
Table 1. Fungi identified from hives of Melipona interrupta and Melipona seminigra. Identification by macro and micromorphology and by sequences of the rDNA ITS1-5.8S-ITS2 region were used for the identification. Identity was obtained by searching for a match with the available sequences at the GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Filo
Family
|
Identified species
(GenBank access number)
|
Identity
(%)
|
Ascomycota
|
|
|
Aspergillaceae
|
Aspergillus fumigatus (access KY926853.1)
|
99
|
|
Aspergillus nomius (access KJ634091.1)
|
100
|
|
Aspergillus niger
|
|
|
Aspergillus ochraceus
|
|
|
Aspergillus sp.
|
|
|
Penicillium citrinum (access LT558892.1)
|
99
|
|
Monascus ruber (access KY635850.1)
|
100
|
Cladosporiaceae
|
Cladosporium cladosporioides (access KX960612.1)
|
100
|
Debaryomyceteceae
|
Debaryomyces hansenii (access KY103209.1)
|
99
|
Mucoraceae
|
Rhizopus sp.
|
|
Saccharomyceteceae
|
Wickerhamiella versatilis (access KY102502.1)
|
99
|
Unclassified
|
Starmerella apicola (access EU926479.1)
|
99
|
Unclassified
|
Starmerella etchellsii (access KY102074.1)
|
99
|
Basidiomycota
|
|
|
Cystobasidiaceae
|
Cystobasidium minutum (access KT329195.1)
|
100
|
3.4 Analysis of richness, diversity, equitability and similarity indexes
Indexes of richness, diversity, equitability and similarity were compared between microenvironments (Table 2), seasons (Tables 3 and 4) and caste-specific brood cells (Tables 5 and 6) for each bee species.
Total taxa richness was of thirteen species. Among these, ten were found in M. interrupta and ten, in M. seminigra (Table 2). In M. interrupta, seven taxa consisted of filamentous fungi, while the other three were of yeasts (Table 2). However, yeasts were more abundant. S. etchellsii, which was exclusively found in M. interrupta, presented the higher number of CFUs, followed by A. niger (Supplementary Figure 1). In M. seminigra, eight taxa were of filamentous fungi and two were of yeasts (Table 2). The most predominant species was C. minutum, followed by A. niger.
In M. interrupta, diversity and equitability indexes were higher in the dry than in the rainy season (Table 3). This indicates that fungi community is more heterogenous during the dry season, whereas some taxa are dominant in the rainy season. The similarity index indicated that most of the taxa occurred in both seasons (Table 3). In contrast, diversity was lower and equitability was higher during the dry season in M. seminigra (Table 4). Also, similarity index indicated a low overlap of taxa between the seasons (Table 4).
The richness index of pupae brood cells was of eight taxa in M. interrupta. Among these, six were found in queen cells and seven, in worker cells (Table 5). Equitability and similarity indexes indicated that there were dominant taxa in each caste-associated community and that the fungi species found in queen and worker cells were similar (Table 5). In M. seminigra, the richness index of pupae brood cells was of eight taxa, five found in queen cells and four, in worker cells (Table 6). The diversity index was higher in queen cells than in worker cells. The equitability and similarity indexes indicated that there were also dominant taxa in queen and worker cells, but the overlap of taxa between castes was low (Table 6). Comparing both bee species, diversity was lower in queen cells of M. interrupta than of M. seminigra. However, it was higher in worker cells of M. interrupta than of M. seminigra (Tables 5 and 6).
Table 2. Number and relative frequency of the fungi isolated from the internal hive environment (IE), larval brood cells (L), queen brood cells (Q) and worker brood cells (W) of Melipona interrupta and Melipona seminigra.
Class/Genera
|
|
M. interrupta
|
|
|
M. seminigra
|
Total (%)
|
IE
|
L
|
Q
|
W
|
|
IE
|
L
|
Q
|
W
|
Aspergillus sp.
|
7
|
-
|
-
|
109
|
|
48
|
-
|
31
|
-
|
195 (12,6)
|
Aspergillus fumigatus
|
-
|
-
|
7
|
1
|
|
-
|
-
|
-
|
6
|
14 (0,9)
|
Aspergillus niger
|
132
|
-
|
100
|
3
|
|
72
|
-
|
8
|
43
|
358 (23,2)
|
Aspergillus nomius
|
2
|
-
|
-
|
-
|
|
12
|
-
|
64
|
-
|
78 (5,1)
|
Aspergillus ochraceus
|
-
|
-
|
-
|
-
|
|
24
|
-
|
-
|
-
|
24 (1,6)
|
Cladosporium cladosporioides
|
-
|
-
|
2
|
3
|
|
-
|
-
|
-
|
2
|
7 (0,5)
|
Cystobasidium minutum
|
-
|
-
|
-
|
-
|
|
-
|
-
|
-
|
258
|
258 (16,7)
|
Debaryomyces hansenii
|
-
|
-
|
1
|
-
|
|
-
|
-
|
-
|
-
|
1 (0,06)
|
Monascus ruber
|
-
|
1
|
-
|
-
|
|
-
|
8
|
-
|
-
|
9 (0.6)
|
Penicillium citrinum
|
1
|
-
|
6
|
46
|
|
1
|
-
|
2
|
-
|
56 (3,6)
|
Rhizopus sp.
|
-
|
-
|
-
|
-
|
|
10
|
-
|
-
|
-
|
10 (0.7)
|
Starmerella apicola
|
-
|
-
|
11
|
2
|
|
-
|
-
|
-
|
-
|
13 (0,8)
|
Starmerella etchellsii
|
-
|
-
|
-
|
364
|
|
-
|
-
|
-
|
-
|
364 (23,6)
|
Wickerhamiella versatilis
|
-
|
-
|
-
|
-
|
|
-
|
-
|
2
|
-
|
2 (0,1)
|
Non-identified
|
50
|
-
|
2
|
-
|
|
85
|
-
|
12
|
3
|
152 (10,0)
|
Total
|
192
|
1
|
129
|
528
|
|
252
|
8
|
119
|
312
|
1.541 (100)
|
Isolated Colonies
|
|
850
|
|
|
691
|
|
Richness (R)
|
|
10
|
|
|
11
|
|
Diversity (H')
|
|
1,3553
|
|
|
1,5907
|
|
Equitability (E)
|
|
0,3878
|
|
|
0,4461
|
|
Similarity (S')
|
|
|
0,7
|
|
Table 3. Comparison between the fungi isolated in the rainy and dry seasons from distinct hives (H1 to H5) of Melipona interrupta.
Species
|
Rainy season
|
Dry season
|
H1
|
H2
|
H3
|
H4
|
H5
|
H1
|
H2
|
H3
|
H4
|
H5
|
Aspergillus sp. 1
|
-
|
1
|
-
|
3
|
3
|
108
|
1
|
-
|
-
|
-
|
Aspergillus fumigatus
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
5
|
-
|
3
|
Aspergillus niger
|
16
|
4
|
3
|
115
|
-
|
6
|
12
|
24
|
53
|
2
|
Aspergillus nomius
|
1
|
1
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cladosporium cladosporioides
|
-
|
-
|
-
|
-
|
-
|
1
|
-
|
4
|
-
|
-
|
Debaryomyces hansenii
|
-
|
-
|
-
|
1
|
-
|
-
|
-
|
-
|
-
|
-
|
Monascus ruber
|
-
|
-
|
-
|
-
|
-
|
1
|
-
|
-
|
-
|
-
|
Penicillium citrinum
|
-
|
1
|
-
|
-
|
-
|
52
|
-
|
-
|
-
|
-
|
Starmerella apicola
|
-
|
-
|
3
|
-
|
-
|
-
|
-
|
-
|
-
|
10
|
Starmerella etchellsii
|
-
|
-
|
-
|
364
|
-
|
-
|
-
|
-
|
-
|
-
|
Non-identified
|
2
|
-
|
-
|
17
|
2
|
3
|
7
|
15
|
-
|
6
|
Total per hive
|
19
|
7
|
6
|
500
|
5
|
171
|
20
|
48
|
53
|
21
|
Total
|
537
|
313
|
Richness (R)
|
7
|
7
|
Diversity (H’)
|
0,7184
|
1,3587
|
Equitability (E)
|
0,2930
|
0,5558
|
Similarity (S’)
|
0,5714
|
Table 4. Comparison between the fungi isolated in the rainy and dry seasons from distinct hives (H1 to H5) of Melipona seminigra.
Species
|
Rainy season
|
Dry season
|
H1
|
H2
|
H3
|
H4
|
H5
|
H1
|
H2
|
H3
|
H4
|
H5
|
Aspergillus sp. 1
|
4
|
27
|
7
|
2
|
2
|
1
|
36
|
-
|
-
|
-
|
Aspergillus fumigatus
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
6
|
-
|
Aspergillus niger
|
87
|
-
|
9
|
3
|
-
|
14
|
8
|
2
|
-
|
-
|
Aspergillus nomius
|
68
|
8
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Aspergillus ochraceus
|
-
|
-
|
24
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cladosporium cladosporioides
|
-
|
-
|
2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cystobasidium minutum
|
258
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Monascus ruber
|
-
|
-
|
-
|
-
|
-
|
5
|
-
|
2
|
-
|
1
|
Penicillium citrinum
|
-
|
2
|
-
|
-
|
1
|
-
|
-
|
-
|
-
|
-
|
Rhizopus sp.
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
10
|
-
|
Wickerhamiella versatilis
|
-
|
-
|
-
|
2
|
-
|
-
|
-
|
-
|
-
|
-
|
Non-identified
|
-
|
3
|
17
|
10
|
9
|
1
|
-
|
18
|
27
|
15
|
Total per hive
|
417
|
40
|
59
|
17
|
12
|
21
|
44
|
22
|
43
|
16
|
Total
|
545
|
146
|
Richness (R)
|
8
|
5
|
Diversity (H’)
|
1,3727
|
1,3804
|
Equitability (E)
|
0,4932
|
0,7953
|
Similarity (S’)
|
0,3086
|
Table 5. Comparison between the fungi isolated from queen and worker brood cells of distinct hives (H1 to H5) of Melipona interrupta.
Species
|
Queens
|
Workers
|
H1
|
H2
|
H3
|
H4
|
H5
|
H1
|
H2
|
H3
|
H4
|
H5
|
Aspergillus sp. 1
|
-
|
-
|
-
|
-
|
-
|
107
|
-
|
-
|
2
|
-
|
Aspergillus niger
|
-
|
2
|
-
|
98
|
-
|
2
|
1
|
-
|
-
|
-
|
Aspergillus fumigatus
|
-
|
-
|
4
|
-
|
3
|
-
|
-
|
1
|
-
|
-
|
Cladosporium cladosporioides
|
1
|
-
|
1
|
-
|
-
|
-
|
-
|
3
|
-
|
-
|
Debaryomyces hansenii
|
-
|
-
|
-
|
1
|
-
|
-
|
-
|
-
|
-
|
-
|
Penicillium citrinum
|
6
|
-
|
-
|
-
|
-
|
46
|
-
|
-
|
-
|
-
|
Starmerella apicola
|
-
|
-
|
1
|
-
|
10
|
-
|
-
|
2
|
-
|
-
|
Starmerella etchellsii
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
364
|
-
|
Non-identified
|
-
|
-
|
-
|
1
|
1
|
-
|
-
|
-
|
-
|
-
|
Total per hive
|
7
|
2
|
6
|
100
|
14
|
155
|
1
|
6
|
366
|
0
|
Total
|
129
|
528
|
Richness (R)
|
6
|
7
|
Diversity (H’)
|
0,8076
|
0,8865
|
Equitability (E)
|
0,3737
|
0,3466
|
Similarity (S’)
|
0,7693
|
Table 6. Comparison between the fungi isolated from queen and worker brood cells of distinct hives (H1 to H5) of Melipona seminigra.
Species
|
Queens
|
Workers
|
H1
|
H2
|
H3
|
H4
|
H5
|
H1
|
H2
|
H3
|
H4
|
H5
|
Aspergillus sp. 1
|
-
|
27
|
1
|
1
|
2
|
-
|
-
|
-
|
-
|
-
|
Aspergillus fumigatus
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
6
|
-
|
Aspergillus niger
|
-
|
-
|
8
|
-
|
-
|
42
|
-
|
1
|
-
|
-
|
Aspergillus nomius
|
64
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cladosporium cladosporioides
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
2
|
-
|
-
|
Cystobasidium minutum
|
-
|
-
|
-
|
-
|
-
|
258
|
-
|
-
|
-
|
-
|
Penicillium citrinum
|
-
|
2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Wickerhamiella versatilis
|
-
|
-
|
-
|
2
|
-
|
-
|
-
|
-
|
-
|
-
|
Non-identified
|
-
|
-
|
4
|
7
|
1
|
-
|
-
|
2
|
-
|
1
|
Total per hive
|
64
|
29
|
13
|
10
|
3
|
300
|
0
|
5
|
6
|
1
|
Total
|
119
|
312
|
Richness (R)
|
5
|
4
|
Diversity (H’)
|
1,009
|
0,5342
|
Equitability (E)
|
0,5486
|
0,4265
|
Similarity (S’)
|
0,2222
|