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  2. Evolutionary Biology
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Nitric oxide radicals are emitted by wasp eggs to kill mold fungi

  1. Erhard Strohm  Is a corresponding author
  2. Gudrun Herzner
  3. Joachim Ruther
  4. Martin Kaltenpoth
  5. Tobias Engl
  1. University of Regensburg, Germany
  2. Max Planck Institute for Chemical Ecology, Germany
Research Article
Cite this article as: eLife 2019;8:e43718 doi: 10.7554/eLife.43718
11 figures, 3 tables, 11 data sets and 2 additional files

Figures

Paralyzed honeybees under different conditions.

(A) Brood cell of the European beewolf with two bees, one carrying an egg, in an observation cage. (B) Honeybee paralyzed by a beewolf female but immediately removed and kept in an artificial brood cell, heavily overgrown by mold fungi that have already developed conidia. Scale bar = 5 mm.

https://doi.org/10.7554/eLife.43718.003
Onset of fungal growth on paralyzed honeybees taken from Philanthus triangulum nests and kept in artificial brood cells.

The fraction of bees showing first signs of fungal growth is shown as a function of days since oviposition. (A) Honeybees that either carried an egg (dashed line) or not (solid line) (N = 22 each, hazard ratio = 0.29, 95% confidence interval: 0.13–0.64). (B) Honeybees that were either kept alone (solid line) or shared a brood cell with a bee carrying an egg (dashed line) (N = 16 each, hazard ratio = 0.39, 95% confidence interval: 0.17–0.9).

https://doi.org/10.7554/eLife.43718.004
Figure 2—source data 1

Effect of egg on fungus growth.

https://doi.org/10.7554/eLife.43718.005
Bioassay demonstrating the inhibitory effect of a beewolf egg against Aspergillus flavus.

Two areas on the agar were covered by caps of a volume similar to natural beewolf brood cells. One cap, the control (C), was empty, while the experimental cap (E) contained a fresh beewolf egg attached to the ceiling of the cap. The caps were removed and the picture was taken after 24 hr of incubation at 25°C. The control area (C) shows dense whitish fungal hyphae similar to the surroundings. However, the area that was exposed to the volatiles from a beewolf egg (E) shows bare agar, indicating that the growth of this aggressive fungus was entirely inhibited. Scale bar = 2.5 cm.

https://doi.org/10.7554/eLife.43718.006
Figure 4 with 1 supplement
Visualization of NO emission by beewolf eggs using fluorescence imaging.

(A) Honeybee from a brood cell without an egg and (B) honeybee with egg. Both bees were sprayed with a solution of the NO specific fluorescence probe DAR4M-AM. Only the droplets on the bee with the egg (B) show a bright yellow and orange fluorescence indicating the presence of NO. Images are composites of multiple pictures of the x/y plane and z-axis. Scale bar = 1 mm.

https://doi.org/10.7554/eLife.43718.007
Figure 4—figure supplement 1
Results of the Griess assay with beewolf eggs.

The left reaction vial is a control without beewolf egg, the other two vials containd eggs that were placed in the lid within 1 hr of oviposition. Vials were incubated at 25°C for 24 hr.

https://doi.org/10.7554/eLife.43718.008
Figure 5 with 1 supplement
Detection of nitric oxide (NO) in beewolf eggs.

Newly laid eggs of beewolves, Philanthus triangulum, of the cockroach wasp Ampulex compressa and of the Red Mason bee, Osmia bicornis were injected with the NO sensitive fluorescence probe DAR4M-AM. Control beewolf eggs were injected with phosphate buffer. Images were obtained by fluorescence microscopy 0, 3 and 24 hr after injection. Row (A) DAR4M-AM injected beewolf egg showing strong increase in fluorescence; (B) Buffer-injected control beewolf egg showing the level of autofluorescence; (C) DAR4M-AM injected egg of A. compressa; (D) DAR4M-AM injected egg of O. bicornis. Scale bar: 1 mm.

https://doi.org/10.7554/eLife.43718.009
Figure 5—source data 1

Eggs injected with DAR4M-AM.

https://doi.org/10.7554/eLife.43718.011
Figure 5—figure supplement 1
Detection of nitric oxide (NO) in beewolf larva.

Newly hatched beewolf larva, Philanthus triangulum, was injected with the NO sensitive fluorescence probe DAR4M-AM. Images were obtained by fluorescence microscopy 0, 3 and 24 hr after injection. Scale bar: 1 mm.

https://doi.org/10.7554/eLife.43718.010
Figure 6 with 1 supplement
Timing of NO emission from beewolf eggs (kept at 28°C).

The photometrically determined absorbance at 590 nm (mean ± SD) is shown as a function of time after oviposition for iodide-starch solutions successively exposed to beewolf eggs for one hour. Sample size (number of eggs measured) at each one hour interval is indicated above the x-axis.

https://doi.org/10.7554/eLife.43718.012
Figure 6—figure supplement 1
Start of NO emission (h after oviposition) as a function of temperature.

Beewolf eggs were kept at different temperatures and the onset of NO release was assessed using the color change of an iodide starch solution as monitored by a digital camera at 30 min intervals. Symbols are means ± SD (Quadratic regression: R2 = 0.98, N = 33, p<0.001; Q10 = 2.74). Source data file: Figure 6—figure supplement 1—source data 1.

https://doi.org/10.7554/eLife.43718.013
Figure 6—figure supplement 1—source data 1

Start of NO emission.

https://doi.org/10.7554/eLife.43718.015
Onset of fungal growth (time after onset of experiment) on honeybees that were not embalmed in artificial brood cells.

Brood cells were either injected with synthetic NO to a concentration of 1500ppm (solid line) or were injected with nitrogen (dashed line) (N = 20 each, hazard ratio = 0.41, 95% confidence interval: 0.198–0.845).

https://doi.org/10.7554/eLife.43718.016
Figure 7—source data 1

Effect of synthetic nitric oxide on fungus growth.

https://doi.org/10.7554/eLife.43718.017
Fungus growth on honeybees of four different treament groups.

Timing of occurrence of (A) fungal hyphae and (B) conidia on paralyzed honeybees that were (1) not embalmed by beewolf females and did not carry an egg (n = 25, colorless bee, point line), (2) embalmed but did not carry an egg (n = 68, colored bee, solid line), (3) not embalmed but carried an egg (n = 21, colorless bee with egg, dash-point line) or (4) embalmed and carried an egg (n = 21, colored bee with egg, dashed line). See Appendix 1—table 2 for hazard ratios.

https://doi.org/10.7554/eLife.43718.018
Figure 8—source data 1

Combined effect of embalming and fumigation.

https://doi.org/10.7554/eLife.43718.019
Micrograph of a longitudinal section of a beewolf egg fixed 15–16 hr after oviposition showing fixation insensitive NADPH-diaphorase activity.

Strong blue staining in the embryonic tissue indicates the presence of reduced nitroblue tetrazolium demonstrating NOS activity (c = cuticle, s = serosa, e = embryo, a = amnion, ac = amnion cavity, scale bar = 1 mm, image composed from two separate photos of the left and right parts of the egg.).

https://doi.org/10.7554/eLife.43718.020
Gene expression of NOS relative to ß-actin in beewolf eggs at different times after oviposition and in freshly hatched larvae.

Two trials were conducted, each with 25 pooled eggs or larvae per time interval. Mean ratios of NOS-mRNA to ß-Actin-mRNA are shown (with standard deviations), as determined by Q-RT-PCR.

https://doi.org/10.7554/eLife.43718.021
Figure 11 with 2 supplements
Effect of NOS inhibition on NO production.

Amount of NO and/or NO2 emanating from non-injected beewolf eggs (control; N = 14) and those injected with D-NAME (a non-inhibiting enantiomer of L-NAME, N = 9) or L-NAME (a NOS inhibiting L-arginine analog, N = 14). The photometrically determined absorbance at 590 nm is shown for iodide-starch solutions that were exposed for 24 hr to the headspace of eggs of the indicated treatment group (shown are median, quartiles and range, * indicates an outlier, included in the analysis). P-values are for Holm-corrected Mann-Whitney U-tests.

https://doi.org/10.7554/eLife.43718.023
Figure 11—figure supplement 1
Structure of the Pt-NOS gene indicating position and length of exons.

Exon 14 (red) is missing in the NOS mRNA in beewolf eggs compared to adults. Presumed cofactor-binding domains as deduced from homologous sequences of the NOS of Anopheles stephensi (Luckhart et al., 1998; Luckhart and Li, 2001) are indicated for heme, calmodulin (CaM), FMN, FAD pyrophosphate (FAD PPi) and FAD isoalloxazine (FAD Iso), NADPH ribose, NADPH adenine, and NADPH.

https://doi.org/10.7554/eLife.43718.024
Figure 11—figure supplement 2
Consensus tree obtained from Bayesian analysis of NOS amino acid sequences from five orders of insects (distinguished by different colors), including the NOS sequences of P. triangulum eggs (lowermost entry).

Values at the nodes represent Bayesian posterior probabilities and local support values (FastTree analysis), respectively. Scale bar represents 0.1 changes per site.

https://doi.org/10.7554/eLife.43718.025

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Biological sampleEuropean beewolf,
Philanthus triangulum
Field caught or
laboratory reared F1 of field caught females
Biological sampleEmerald cockroach wasp,
Ampulex compressa
Laboratory reared
Biological sampleRed mason bee, Osmia bicornisField caught
Biological sampleAspergillus flavusStrain I: Isolated from beewolf brood cells,
Strain II: Department
of Hygiene and Microbiology of the University Hospital, Würzburg, Germany
na
Biological samplePenicillium roquefortiiDepartment of Hygiene and Microbiology of the University Hospital,
Würzburg, Germany
na
Biological sampleCandida albicansDepartment of Hygiene and Microbiology of the University Hospital,
Würzburg, Germany
na
Biological sampleTrichophyton rubrumDepartment of Hygiene and Microbiology of the University Hospital,
Würzburg, Germany
na
Sequence-based reagentAdapter + PolyT3'RACE, Molecular cloning protocolSee Supplementary file 1
Sequence-based reagentAdapter3'RACE, Molecular
cloning protocol
See Supplementary file 1
Sequence-based reagentpolyTReverse transcription protocolSee Supplementary file 1
Sequence-based reagentNOS_qPCR_F2P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_qPCR_R2P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentActin_qPCR_F1Apis mellifera, Gryllus bimaculatus,
P. triangulum, this paper
See Supplementary file 1
Sequence-
based reagent
Actin_qPCR_R1A. mellifera, G. bimaculatus, P. triangulum, this paperSee Supplementary file 1
Sequence-
based reagent
NOS860fwd2A. mellifera,
D. melanogaster, Anopheles stephensi, Rhodnius prolixus, Manduca sexta, this paper
See Supplementary file 1
Sequence-
based reagent
NOS1571rev1A. mellifera, D. melanogaster, A. stephensi, R. prolixus,
M. sexta, this paper
See Supplementary file 1
Sequence-based reagentNOS_seq_F1_degA. mellifera, Nasonia vitripennis, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_R1_degA. mellifera, N. vitripennis,
this paper
See Supplementary file 1
Sequence-based reagentNOS_seq_5-F1P. triangulum, this paperSee Supplementary file 1
Sequence-
based reagent
NOS_seq_5-R1P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_5-F2P. triangulum, this paperSee Supplementary file 1
Sequence-
based reagent
NOS_seq_5-R2P. triangulum,
this paper
See Supplementary file 1
Sequence-based reagentNOS_seq_5-F3P. triangulum,
this paper
See Supplementary file 1
Sequence-based reagentNOS_seq_5-F6P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_3-F1P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_3-R1P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_3-F2P. triangulum, this paperSee Supplementary file 1
Sequence-
based reagent
NOS_seq_3-R2P. triangulum, this paperSee Supplementary file 1
Sequence-based reagentNOS_seq_3-F3P. triangulum,
this paper
See Supplementary file 1
Sequence-
based reagent
NOS_seq_3-F6P. triangulum,
this paper
See Supplementary file 1
Sequence-based reagentNOS_RT_R1P. triangulum, this paperSee Supplementary file 1
Commercial assay or kitGriess assay. Merck SpectroquantMerck, Darmstadt, Germany114776
Commercial assay or kitpeqGOLD total RNA KitpeqLab, Erlangen, Germany732–2867
Commercial assay or kitGeneRacer KitInvitrogen, Carlsbad, CA, USAL1502-01
Commercial assay or kitBioScript One-Step RT-PCR-KitBioline, London, UKBIO-65033
Commercial assay or kitpeqGOLD Taq-DNA-PolymerasepeqLab, Erlangen, Germany01–1030
Commercial assay or kitSensiMixPlus SYBR MitQuantace/Bioline, London, UKQT615-05
Commercial assay or kitEpicentre MasterPure
Complete DNA and
RNA purification Kit
Epicentre, now Lucigen, Middleton, WI, USAMC85200
Commercial assay or kitinnuPREP
RNA Mini Kit
Analytik Jena,
Jena, Germany
845-KS-2040050
Commercial assay or kitPeqGOLD Mid-
Range PCR System
peqLab, Erlangen, GermanyPEQL02-3020_P
Chemical compound, drugDNase IFermentas, Lithuania
Now Thermo Fisher Scientific, Germany
EN0525
Chemical compound, drugOligo-dT primerFermentas, Lithuania
Now Thermo Fisher
Scientific, Germany
na
Chemical compound, drugL-NAME HydrochlorideAxxora Deutschland,
Lörrach, Germany
ALX-105–004 M250
Chemical compound, drugD-NAME HydrochlorideAxxora Deutschland, Lörrach, GermanyALX-105–003 G005
Chemical
compound, drug
DAR-4M AMAxxora Deutschland, Lörrach, GermanyALX-620–069 M001
Chemical compound, drug4-Nitro-m-XylolMerck, Darmstadt, Germany8415470025
Chemical compound, drugNADPH-
Tetranatriumsalz
Carl-Roth,
Karlsruhe, Germany
AE14.1
ToolsEppendorf
Microinjector with Femtotips II
Eppendorf, Hamburg, Germany930000043
ToolsAxiophot II
Fluorescence microscope
Zeiss, Jena, Germany
ToolsNikon DS-2 MvNikon, Tokyo, Japan
ToolsUvikon 860 spektrophotometerKontron, Augsburg, Germany
ToolsCryostat microtome CM3000Leica, Wetzlar, Germany
ToolsEppendorf Realplex CyclerEppendorf, Hamburg, Germany
ToolsNanoDrop TM1000peqLab, Erlangen, GermanyRRID: SCR_016517
ToolsImplen Nanophotometer ClassicImplen, Munich, Germany
ToolsBiometra T Gradient ThermocyclerAnalytik Jena, Jena, Germany
SoftwareBioEdithttp://www.mbio.ncsu.edu/BioEdit/bioedit.htmlRRID: SCR_007361
SoftwareGeneiousBiomatters, New Zealand
SoftwareSPSSIBM, Armonk, NY, USARRID: SCR_002865
SoftwareFastTreehttp://www.microbesonline.org/fasttree/RRID:SCR_015501
SoftwareMrBayeshttp://mrbayes.sourceforge.net/RRID: SCR_012067
SoftwareCombine-ZPwww.hadleyweb.pwp.blueyonder.co.uk
SoftwarePhotoshop Elements 5PSE5, Adobe Systems Inc, San José, CA, USA
SoftwareCLC genomics
workbench
Qiagen, Hilden, GermanyRRID: SCR_011853
DatabaseNCBIhttp://www.ncbi.nlm.nih.govRRID: SCR_006472
DatabasePrimer3http://primer3.ut.eeRRID: SCR_003139
DerviceSanger SequencingSeqlab, Göttingen, Germany
ServiceTranscriptome Sequencing on Illumina HiSeq TM2000Fasteris, Geneva, Switzerland
Appendix 1—table 1
Scores of fungus growth (0 = no fungus, 1 = very little fungus, 2 = little fungus, 3 = strong fungus) after 24 h of incubation at 25°C on Petri dishes inoculated with Aspergillus flavus conidia.

Scores are given for three areas of the petri dish that were covered with a cap under which either a 'Bee with egg', a 'Bee without egg' was placed as well as a 'Control' with no bee.

https://doi.org/10.7554/eLife.43718.030
Petri dishBee with eggBee without eggControl
1033
2022
3023
4123
5133
6033
7033
8033
9033
10033
11033
12033
Appendix 1—table 2
Hazard ratios (and 95 % confidence intervals) for the comparison of timing of the onset of fungus growth on bees of four treatment groups: bees that carried an egg and were embalmed (+/+), bees that carried no egg but were embalmed (-/+), bees that carried no egg and were not embalmed (-/-) and bees that carried an egg but were not embalmed (+/-).
https://doi.org/10.7554/eLife.43718.031
Comparison
Egg/EmbalmingEgg/EmbalmingHazard ratio95% conf. interval
+/+-/+0.070.19 - 0.03
+/+-/-0.130.36 - 0.05
+/++/-0.650.90 - 0.47
-/+-/-0.480.79 - 0.29
+/--/+0.610.45 - 0.83
+/--/-0.220.10 - 0.47

Data availability

The GenBank accession numbers for the P. triangulum NOS (Pt-NOS) gene sequence is: KJ425525, for the NOS mRNA of P. triangulum eggs: KJ425526, for the NOS mRNA in P. triangulum female antennae: KJ425527, for the transcriptome: Bioproject accession number PRJNA542283 with biosample accession number SAMN11793601. Other data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 5, 6, 6—supplement 1, 7, 8, 10, 11.

The following data sets were generated
  1. 1
    GenBank
    1. E Strohm
    2. G Herzner
    3. J Ruther
    4. M Kaltenpoth
    5. T Engl
    (2015)
    ID KJ425525.1. Philanthus triangulum nitric oxide synthase gene, complete cds, alternatively spliced.
  2. 2
    GenBank
    1. E Strohm
    2. G Herzner
    3. J Ruther
    4. M Kaltenpoth
    5. T Engl
    (2015)
    ID KJ425526.1. Philanthus triangulum nitric oxide synthase egg isoform mRNA, complete cds, alternatively spliced.
  3. 3
    GenBank
    1. E Strohm
    2. G Herzner
    3. J Ruther
    4. M Kaltenpoth
    5. T Engl
    (2015)
    ID KJ425527.1. Philanthus triangulum nitric oxide synthase adult isoform mRNA, complete cds, alternatively spliced.
  4. 4
    Genbank
    1. M Sandoval-Calderón
    2. T Nechitaylo
    3. T Engl
    4. H Vogel
    5. S Koehler
    6. M Kaltenpoth
    (2019)
    ID PRJNA542283. RNAseq reads from Philanthus triangulum and its symbiont, 'Candidatus Streptomyces philanthi'.
  5. 5
    Genbank
    1. M Sandoval-Calderón
    2. T Nechitaylo
    3. T Engl
    4. H Vogel
    5. S Koehler
    6. M Kaltenpoth
    (2019)
    ID SAMN11793601. RNA-Seq of Philanthus triangulum: female antenna rRNA depleted, polyA-enriched.
The following previously published data sets were used
  1. 1
    GenBank
    1. M Regulski
    2. T Tully
    (1995)
    ID U25117.1. Drosophila melanogaster Ca/calmodulin-dependent nitric oxide synthase (NOS) mRNA, complete cds.
  2. 2
    GenBank
    1. T Watanabe
    2. T Shiga
    3. T Yamamoto
    4. N Suzuki
    5. E Ito
    (2005)
    ID AB204558.1. Apis mellifera AmNOS mRNA for nitric oxide synthase, complete cds.
  3. 3
    GenBank
    1. S Luckhart
    2. Y Vodovotz
    3. L Cui
    (1999)
    ID AH007775.1. Anopheles stephensi nitric oxide synthase gene, complete cds.
  4. 4
    GenBank
    1. M Yuda
    (1996)
    ID U59389.1. Rhodnius prolixus salivary gland nitric oxide synthase mRNA, complete cds.
  5. 5
    GenBank
    1. A Nighorn
    2. NJ Gibson
    3. DM Rivers
    4. JG Hildebrand
    5. DB Morton
    (1998)
    ID AF062749.1. Manduca sexta nitric oxide synthase (NOS) mRNA, complete cds.
  6. 6
    GenBank
    1. et al Werren
    (2016)
    ID NM_001168232.1. Nasonia vitripennis nitric oxide synthase (Nos), mRNA.

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