1. Evolutionary Biology
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Oral transfer of chemical cues, growth proteins and hormones in social insects

  1. Adria C LeBoeuf  Is a corresponding author
  2. Patrice Waridel
  3. Colin S Brent
  4. Andre N Gonçalves
  5. Laure Menin
  6. Daniel Ortiz
  7. Oksana Riba-Grognuz
  8. Akiko Koto
  9. Zamira G Soares
  10. Eyal Privman
  11. Eric A Miska
  12. Richard Benton  Is a corresponding author
  13. Laurent Keller  Is a corresponding author
  1. University of Lausanne, Switzerland
  2. Arid Land Agricultural Research Center, USDA-ARS, United States
  3. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brasil
  4. University of Cambridge, United Kingdom
  5. Ecole Polytechnique Fédérale de Lausanne, Switzerland
  6. The University of Tokyo, Japan
  7. University of Haifa, Israel
  8. Wellcome Trust Genome Campus, United Kingdom
Research Article
Cite this article as: eLife 2016;5:e20375 doi: 10.7554/eLife.20375
6 figures, 1 table, 3 data sets and 3 additional files

Figures

Figure 1 with 2 supplements
Proteomic characterization of Camponotus floridanus trophallactic fluid.

(A) Heat map showing the percentage of total molecular weight-normalized spectra assigned to proteins from voluntary TF, forced TF, midgut, or hemolymph fluids (normalized spectral abundance factor, NSAF [Zybailov et al., 2006]). C1-C10 indicate colony of origin. Forced trophallaxis samples are pooled from 10 to 20 ants, hemolymph from 30 ants, and the contents of dissected midguts from five ants each. Voluntary and midgut samples were collected from ants of multiple colonies; multiple samples are differentiated by letters. Approximately unbiased (AU) bootstrap probabilities for 10,000 repetitions are indicated by black circles where greater or equal to 95%. (B) Trophallaxis samples from the same ants, in-colony and group-isolated. Trophallactic fluids were sampled first upon removal from the colony, then after 14 days of group isolation (20–30 individuals per group). Values were compared by spectral counting, and the dendrogram shows approximately unbiased probabilities for 10,000 repetitions. Along the right side, asterisks indicate Bonferroni-corrected t-test significance to p < 0.05 between in-colony and isolated TF. Approximately unbiased (AU) bootstrap probabilities for 10,000 repetitions are indicated by black circles where greater or equal to 95%. (C) The most abundant proteins present in TF sorted by natural-log-scaled NSAF value. The UniProt ID or NCBI ID is listed to the right. (D) A dendrogram of proteins including all proteomically observed juvenile hormone esterases (JHE)/Est-6 proteins in C. floridanus, the orthologs in D. melanogaster and A. mellifera, and biochemically characterized JHEs. Each protein name is followed by the UniProt ID. C. floridanus JHE/Est-6 proteins are listed with the fluid source where they have been found. Names are color-coded by species. Bootstrap values >95% are indicated with a black circle. JHE/Est-6 6 (E2AJL7) is identified by PEAKS software but not by Scaffold and consequently is not shown in the proteomic quantifications in panels (A–C).

https://doi.org/10.7554/eLife.20375.003
Figure 1—figure supplement 1
Proteomic characterization of C. floridanus trophallactic fluid.

Heat map showing the percentage of total molecular weight-normalized spectra assigned to proteins from voluntary TF, forced TF, midgut, or hemolymph fluids (normalized spectral abundance factor, NSAF [Zybailov et al., 2006]. C1-C10 indicate colony of origin. Forced trophallaxis samples are pooled from 10 to 20 ants, hemolymph from 30 ants, and the contents of dissected midguts from five ants each. Voluntary and midgut samples were collected from ants of multiple colonies. Approximately unbiased bootstrap probabilities for 10,000 repetitions are indicated by black circles where greater or equal to 95%. UniProt IDs are listed to the left and protein names to the right.

https://doi.org/10.7554/eLife.20375.004
Figure 1—figure supplement 2
Social isolation influences trophallactic fluid content.

Trophallaxis samples from the same ants, in-colony and group-isolated. Trophallactic fluid of ant workers sampled when first removed from the colony, then after 14 days of group isolation (~20–30 individuals per group). Values were normalized using spectral counting and the dendrogram shows approximately unbiased probabilities for 10,000 repetitions. Along the right side, UniProt IDs are shown. Proteins that significantly decreased in abundance in isolation are shown in orange, and proteins that increased in abundance are shown in green (t-test p < 0.05). Asterisks indicate Bonferroni-corrected t-test significance. Names in bold italics indicate proteins that significantly decreased in abundance in voluntary samples (socially isolated, starved then fed) relative to forced TF samples (in-colony) from data shown in Figure 1A and Figure 1—figure supplement 1. Approximately unbiased (AU) bootstrap probabilities for 10,000 repetitions are indicated by black circles where greater or equal to 95% and grey circles where greater or equal to 75%.

https://doi.org/10.7554/eLife.20375.005
Trophallactic fluid of C. floridanus contains microRNAs.

Left: heatmap showing the length of reads assigned to each C. floridanus microRNA (miRNA) found in TF. MiRNAs should exhibit a consistent read size typically between 18 and 22 base pairs. Right: histogram indicating read abundance. miRNA names were assigned through homology to A. mellifera where possible. Letters were assigned for novel miRNAs. Bold miRNA names indicate miRNAs whose homologs were also observed in royal and/or worker jelly in A. mellifera (Guo et al., 2013). Source data in Figure 2—source data 1.

https://doi.org/10.7554/eLife.20375.006
Figure 3 with 1 supplement
Trophallactic fluid of C. floridanus contains cuticular hydrocarbons.

(A–B) Gas chromatography-mass spectrometry profiles in the retention time window for cuticular hydrocarbons (C28–C37), from hexane extracts of whole body (A) and from trophallactic fluid (B). Samples were extracts from whole body and trophallactic fluid for five groups of 20–38 ants. Each group of ants is from a different colony, C11-C15. Different colonies are shown in distinct colors. Source data in Figure 3—source data 1. The abundant component (peak A) found in TF samples but not on the cuticle was cholesterol, a molecule that insects cannot synthesize but must receive from their diet. Three molecules outside this window were found only in TF and not on the cuticle: 7Z-tricosene, oleic acid, ethyl oleate (Table 1). All have been reported to be pheromones in other insect species (Wang et al., 2011; Le Conte et al., 2001; Mohammedi et al., 1996; Choe et al., 2009). (C) A hierarchically clustered heatmap of the dominant peaks in the range of retention times for long-chain cuticular hydrocarbons. The dendrogram shows approximately unbiased probabilities for 10,000 repetitions. Approximately unbiased bootstrap values > 95% are indicated with black circles. Letters along the right correspond to individual peaks in (A) and (B). (D) Normalized pair-wise cross-correlation values for each TF and body hydrocarbon profile for each of the five colonies. Source data in Figure 3—source data 2. (E) Normalized pair-wise cross-correlation values between TF hydrocarbon profiles and between body hydrocarbon profiles indicate that the TF hydrocarbon profiles are significantly more similar than are body hydrocarbon profiles. Median values and interquartile ranges are shown. t-test, p<0.0003.

https://doi.org/10.7554/eLife.20375.007
Figure 3—source data 1

Peak lists for cuticular and trophallactic fluid long chain hydrocarbons form GC-MS experiments for five different colonies of C.floridanus.

C11, 20 ants, 9.5 µL of TF; C12, 38 ants, 20 µL; C13, 34 ants, 11 µL; C14, 26 ants, 11.5 µL; C15, 35 ants, 9 µL.

https://doi.org/10.7554/eLife.20375.008
Figure 3—source data 2

Cross-correlation matrix for panels 3D-E.

https://doi.org/10.7554/eLife.20375.009
Figure 3—figure supplement 1

(A) Typical GC-MS chromatogram of a TF sample from C. floridanus, showing the main hydrocarbons C28-C37 eluting after 40 min. The insert shows the region with minor hydrocarbons and other components C15-C28. (B–D) GC-MS Mass spectra of linear alkane (n-hexacosane at Rt 34.69 min, panel B) and a dimethylated alkane at Rt 43.29 min (C). Extracted MS profiles were fitted with an exponential decay equation (B, D). The proposed structure for the branched alkane based on fragments ions (141 and 309) is 9,20-dimethyl nonacosane (D). (E) RI values vs. number of carbons extracted from the NIST Chemistry WebBook library, depending on the number of ramifications: linear (black), monomethyl (red), dimethyl (blue), trimethyl (pink), tetramethyl (green) and pentamethyl (dark blue). (F) The black dots represent experimental retention times with the calculated RI index for all identified hydrocarbons of the TF sample. The red dots represent the experimental retention times and with their RI index for the linear C8-C40 alkane standard.

https://doi.org/10.7554/eLife.20375.010
Juvenile hormone passed in trophallactic fluid increases larval growth and rate of pupation in C. floridanus.

(A) JH titer in trophallactic fluid and hemolymph (n = 20; each replicate is a group of 30 workers). Source data in Figure 4—source data 1. (B) JH content of third instar larvae. Source data in Figure 4—source data 2. (C) Head width of pupae raised by workers who were fed food supplemented with JH III or solvent. General linear mixed model (GLMM) testing effect of JH on head width with colony, replicate and experiment as random factors, ***p < 9.01e−06. Source data in Figure 4—source data 3. (D) Proportion of larvae that have undergone metamorphosis when workers were fed food supplemented with JH III or solvent only. Binomial GLMM testing effect of JH on survival past metamorphosis with colony and experiment as random factors, ***p < 7.39e−06. Median values and interquartile ranges are shown in panels (A–C). Panels (C) and (D) are data from three separate experiments where effects in each were individually significant to p<0.05. Source data in Figure 4—source data 4.

https://doi.org/10.7554/eLife.20375.012
Figure 4—source data 1

Hemolymph and trophallactic fluid Juvenile hormone titers for 20 pooled samples of each fluid.

https://doi.org/10.7554/eLife.20375.013
Figure 4—source data 2

Juvenile hormone titers for 37 individual third instar larvae.

https://doi.org/10.7554/eLife.20375.014
Figure 4—source data 3

Head-width measurements for panel 4C.

https://doi.org/10.7554/eLife.20375.015
Figure 4—source data 4

Metamorphosis or death counts for panel 4D.

https://doi.org/10.7554/eLife.20375.016
Figure 5 with 1 supplement
Proteins in trophallactic fluid across social insect species.

(A) Venn diagram indicating the number of species-specific and orthologous proteins detected in TF from the indicated species, whose phylogenetic relationships are shown with black lines. (B) Heat maps showing the percentage of total molecular-weight normalized spectra in TF samples assigned to the proteins in each given species, averaged over all in-colony samples for that species. Samples sizes: C. floridanus (n = 15), C. fellah (n = 6), S. invicta (n = 3), A. mellifera (n = 6). (C) Species-specific TF proteins. The 26 TF ortholog groups found in two or three species, but not the most closely related ones (e.g., A. mellifera and S. invicta, or S. invicta and only one of the two Camponotus species) are indicated in Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.20375.017
Figure 5—source data 1

A table of all orthologous proteins and their predicted functions, identifiers, known D. melanogaster orthologs, presence of annotated secretion signals and average NSAF values when present in TF.

A complete orthology across the four species can be found in Supplementary file 3.

https://doi.org/10.7554/eLife.20375.018
Figure 5—figure supplement 1
Heat map showing the percentage of total molecular-weight normalized spectra in trophallactic fluid samples assigned to each protein in three ant species and the European honey bee, averaged over all in-colony samples for that species.

Sample sizes: C. floridanus (n = 15), C. fellah (n = 6), S. invicta (n = 3), A. mellifera (n = 6). The 26 protein ortholog groups whose presence/absence in TF was inconsistent with phylogeny (e.g., present in only C. floridanus and A. mellifera) are shown in orange. Protein names are shown on the left. Identifiers can be found in the table in Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.20375.019
Author response image 1
Heatmap comparing TF protein abundance with RNA expression of the same genes in brain, head and thorax samples from RNA-Seq datasets (Simola et al. Genome Research 2013).

Expression values are calculated as transcripts per million (TPM) reads, and normalized for presentation on the same scale as proteomic data from Figure 5 (NSAF).

https://doi.org/10.7554/eLife.20375.023

Tables

Table 1

Table of all components identified by GC-MS in TF of C. floridanus. Molecules marked with black dots (•) were found only in TF and not on the cuticle. Peak ID corresponds to Figure 3.

https://doi.org/10.7554/eLife.20375.011

Retention time

Proposed MF

Proposed structure

RI

Peak ID

13.35

C15H32

Pentadecane

1500

14.05

C16H34

4-Methyltetradecane

1600

14.34

C10H10O3

Mellein

1674

15.05

C16H34

Hexadecane

1600

16.47

C17H36

Heptadecane

1700

17.06

C20H42

7,9-Dimethylheptadecane

1710

17.83

C18H38

Octadecane

1800

19.15

C19H40

Nonadecane

1900

19.89

C21H44

7,10,11-Trimethyloctadecane

1920

19.97

C16H32O2

n-Hexadecanoic acid

1962

20.5

C18H36O2

Ethyl palmitate

1968

20.62

C20H42

Eicosane

2000

20.98

C18H36O

Octadecanal

1999

22.89

C18H32O2

Linoleic acid

2133

23.03

C18H34O2

Oleic acid

2179

23.46

C20H36O2

Ethyl-9-Cis-11-Trans-octadecadienoate

2193

23.59

C20H38O2

Ethyl oleate

2173

24.3

C22H46

Docosane

2200

26.27

C23H46

7Z-Tricosene

2296

26.45

C20H38O2

Cis-13-Eicosenoic acid

2368

29.08

C24H50

Tetracosane

2400

32.51

C25H52

Pentacosane

2500

34.69

C26H54

Hexacosane

2600

36.37

C27H56

4-Methylhexacosane

2640

36.91

C27H54

Heptacosene

2672

38.76

C28H58

9-Methylheptacosane

2740

39.19

C28H58

5-Methylheptacosane

2740

40.05

C28H58

*-Trimethylpentacosane

2610

40.63

C28H58

Octacosane

2800

40.76

C28H58

7-Methylheptacosane

2753

41.36

C29H60

5,7,11-Trimethylhexacosane

2783

41.88

C29H60

4-Methylnonacosane

2810

42.23

C29H58

Nonacosene

2875

42.47

C30H62

2,10-Dimethyloctacosane

2874

G

42.7

C29H60

Nonacosane

2900

H

42.9

C31H64

9,16-Dimethylnonacosane

2974

43.29

C31H64

9,20-Dimethylnonacosane

2974

43.36

C30H62

7-Methylnonacosane

2940

43.51

C31H64

4-Methyltriacontane

3045

43.79

C31H64

7,16-Dimethylnonacosane

2974

43.9

C31H64

2-Methyltriacontane

3045

E

43.96

C31H64

10-Methyltriacontane

3045

K

44.12

C32H66

10,11,15-Trimethylnonacosane

3023

44.42

C32H66

*-Dimethyltriacontane

3083

B

44.74

C32H66

8,12-Dimethyltriacontane

3083

Q

45.02

C33H68

*-Trimethyltriacontane

3119

R

45.3

C31H62

Hentriacontene

3100

45.45

C33H68

5,10,19-Trimethyltriacontane

3119

S

45.54

C27H46O

Cholest-5-en-3-ol / Cholesterol

3100

A

45.7

C31H64

Hentriacontane

3100

M

46

C34H70

9,13-Dimethyldotriacontane

3185

C

46.56

C33H68

5,9-Dimethyldotriacontane

3185

D

46.69

C34H70

*-Tetramethylnonacosane

3160

J

46.93

C34H70

*-Multiramified tetratriacontane

3220–3100

O

47.15

C34H70

5,9,13,17,21-Pentamethylnonacosane

3100

N

47.28

C33H68

*-Dimethylhentriacontane

3185

T

47.58

C34H70

10,14,18,22-Tetramethyldotriacontane

3160

P

48.01

C34H70

*-Tetramethyldotriacontane

3160

48.12

C35H72

11,15-Dimethyltritiacontane

3380

F

48.77

C35H72

*-Methyltetratriacontane

3440

L

49.54

C36H74

14-Methylpentatriacontane

3540

49.67

C36H74

*-Multiramifiedhexatriacontane

3420–3300

50.28

C37H76

*-Tetramethyltetratriacontane

3480

Data availability

The following data sets were generated
  1. 1
  2. 2
  3. 3

Additional files

Supplemental file 1

RNA of microorganisms present in trophallactic fluid.

https://doi.org/10.7554/eLife.20375.020
Supplemental file 2

Proteomic experiments included in this study.

https://doi.org/10.7554/eLife.20375.021
Supplemental file 3

Orthology matrix across four social insect species.

https://doi.org/10.7554/eLife.20375.022

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