Ants play an integral role in terrestrial ecosystems. Yet, surprisingly, little is known about their behavior, feeding habits, and source of nutrition. Bacterial symbionts are known to play a key role in the diversification and ecological adaptation of numerous organisms, including ants. Previous studies have shown that “turtle ants” from the tribe
possess an abundance of symbiotic bacteria in their gut [1], and that members from the phyla
,
, and
are consistently detected in the gut despite variations in diet, population, and geographical location [2].
Thus, it is clear that isolating, identifying, and understanding the physiological characteristics of these core bacteria will help to elucidate the mechanisms by which bacterial symbionts interact with
and thereby drive their ecological adaptation and evolution.
Previously, we successfully isolated two novel bacteria belonging to
from ant guts.
Here, we present preliminary results on isolates CV41 and CAG34, cultivated from
and
, respectively
1. Physiologically characterize and determine the roles of the isolates in ant guts.
2. Systematically categorize and properly name the isolates.
2
2
.
70
60
50
40
30
20
10
0
0.0%
CV41
CAG34
1.0% 2.0% 3.0%
CO
2
Concentration
4.0% 5.0% 6.0%
Figure 1 . Effect of CO
2 concentration on generation times of CV41 and
CAG34. Under a starting headspace of 100% N
2
, Balch tubes were injected with pure CO
2 and atmospheric air to obtain a final headspace composition of 0-5% CO
2 in 0.5% increments. O
2 was maintained at 2% in each tube.
Values are expressed as mean ± SD of hours and are obtained from 3 replicates.
A B
60
50
20
10
40
30
0
0.0%
CV41
CAG34
5.0% 10.0%
O
2
Concentration
15.0% 20.0%
Figure 2 . Effect of O
2 concentration on generation times of CV41 and
CAG34. Under a starting headspace of 100% N
2
, Balch tubes were injected with atmospheric air and pure CO
2 to obtain a final headspace composition of 0, 0.5, 1, 2, 4, 8, 12, 16, or 20% O
2
. CO
2 was maintained at 1% in each tube. Values are expressed as mean ± SD of hours and are obtained from 3 replicates.
0.16
0.14
0.12
0.10
0.08
0.06
0.04
CAG34
CV41
0.02
0.00
0/0 0/1 10/1 20/1 20/0
Figure 3 . Effect of O
2 and CO
2 concentration on growth rates of CV41 and
CAG34. Under a starting headspace of 100% N
2
, Balch tubes were injected with pure CO
2 composition of 0% O
2 and atmospheric air to obtain a final headspace with 0% CO
2
(0/0), 0% O
2 with 1% CO
2
(0/1), 10%
O
2
CO
2 with 1% CO
2
(10/1), 20% O
2 with 1% CO
2
(20/1), and 20% O
2 with 0%
(20/0). Values are expressed as mean ± SD of hours and are obtained from 6 and 3 replicates for CV41 and CAG34, respectively.
C D
Figure 4 . Transmission electron micrographs of CV41(A) and CAG34 (B).
Scale bars, .200 μM (A and B); Gram-stain light micrographs of CV41(C) and CAG34 (D). Scale bars, 500 μM (C and D).
Characteristic CV41 CAG34 D. colitermitum a O. terrae b P. vermicola c R. marina d
Isolation Source Ant gut Ant gut Termite gut Paddy soil Clamworm gut Sea sponge
Cell Shape Coccus Coccus Diplococcus Coccus Coccus Coccus or
Rod
0.5-1.1
Cell Diameter ( μ m)
Color
Anaerobic Growth
Catalase
Oxidase
Motility
Temp. range ( o C) pH range
NaCl range (%)
G+C content (% mol)
0.5-0.6
Colorless
-
-
+
ND
23-37
6.9-7.3
0.5-1.5
60.5
0.5-0.6
Colorless
-
-
-
ND
23-37
6.9-7.7
0.5-1.5
60.7
0.5-0.6
Colorless
+
-
-
-
15-35
5.5-7.5
1.5
60.5
0.4-0.6
Colorless
+
-
-
+
10-37
5.5-9.0
3.0
73.7
0.6-1.0
Pale Red
+
-
-
-
8-37
5.5-9.5
1.0-7.5
52.1
Red
-
-
+
-
8-30
6.8-8.2
ND
50.9
Arabinose
Cellobiose
Galactose
Glucose
Lactose
Mannitol
Mannose
+
+
+
+
+
-
+
-
+
+
-
+
-
-
ND
-
-
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
-
+
Melibiose + ND + ND +
Table 1 . Phenotypic characteristics of isolates CV41 and CAG34 compared a with other cultivated members of phylum Verrucomicrobia .
Diplosphaera colitermitum BAA2264 T , b Opitutus terrae DSM11246 T , c Puniceicoccus vermicola IMCC1545 T , and d Rubritalea marina
DSM177716 T . +, utilized; -, not utilized; ND, not determined. Substrate usage and enzyme activity were determined using BIOLOG microplates and API 20NE strips.
Figure 5 . Maximum likelihood-based 16S rRNA gene phylogeny of CAG34 and CV41. The phylogeny is based on 1,091 shared nucleotide positions.
* = 16S rRNA gene clones from C. rohweri guts. # = 16S rRNA gene clones from C. varians guts. Branch point support is indicated by the numbers at branch nodes. Other members of the division Verrucomicrobia are given as reference species. Genbank accession numbers are shown in brackets. Scale bar = 0.2 changes per nucleotide. Phylogenetic analysis was done using phylogeny.fr software.
Growth was possible under an atmosphere consisting of 0.5-20% O
2 and up to 5% CO
2 for
CV41 and CAG34. However, CV41 could not grow in the absence of CO
2
, making it a capnophile. This suggests that the bacteria grow in atmospheric levels of O
2 than atmospheric levels of CO environment.
2 but higher in their gut
Optimum growth for CV41 and CAG34 was observed between pH 6.9-7.7 and 0.5-1.5%
NaCl (w/v). Both isolates grew optimally at 37 o C. While this growth temperature is higher than expected, it is possible that the bacteria have adapted to the warm tropical habitat of the ants.
Most cultivated
solely use sugars as substrates. Here, our isolates use sugars as well as organic acids, suggesting that both are important in the gut. CV41 and CAG34 have slightly different substrate preferences, perhaps reflective of long-term co-speciation and dietary preferences of the ants.
From comparisons of their 16S rRNA gene sequences, CV41 and CAG34 are 98% similar, suggesting, along with physiological differences, they are distinct species. Both isolates share a 93% similarity with their closest cultivated neighbor
, suggesting that CV41 and CAG34 form a novel genus.
Future work will include determining new genus and species names for the isolates, obtaining data on their membrane fatty acids, and developing eco-physiological models for these bacteria, particularly in relationship to nitrogen reducing mechanisms in
guts .
1. Russell JA, et al. (2009) Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants. Proc. Natl. Acad. Sci. U.S.A.
106: 21236 –21241.
2. Hu Y, Lukasik P, Moreau CS, Russell JA (2014) Correlates of gut community composition across an ant species (Cephalotes varians) elucidate causes and consequences of symbiotic variability. Mol. Ecol. 23:1284-1300.
Lori Keen; Alicia Withrow (MSU); Calvin College Science Division and
Harvey Rozema Student Research Fellowship