Plant alkaloid and probiotics increase resistance of honeybees to … · 2015. 8. 25. · control...
Transcript of Plant alkaloid and probiotics increase resistance of honeybees to … · 2015. 8. 25. · control...
• Nematodes H. bacteriophora and S. feltiae were
multiplied on Galleria mellonella larvae.
• Honeybees were treated with potentially immuno-
modulating substances (plant alkaloid and probiotics)
during 8 days of development.
• Bee larvae and pupae were collected and infected on
Petri dishes with the dose 1, 5, 10, 15 or 20 EPN/larva
(Fig. 4).
• 48 hours after infection larvae and pupae were scored
for mortality.
• H. bacteriophora harbouring GFP labeled P.
luminescens was used to monitor the infection (Fig. 5).
1 Department of Animal Physiology and Immunology, Institute of Experimental
Biology, Masaryk University, Kotlarska 2,
61137 Brno, Czech Republic2Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food
and Natural Resources, Czech University of Life Sciences Prague, Czech Republic
3Bee Research Institute; Libcice nad Vltavou, Czech Republic4Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the
Czech Republic, Prague, Czech Republic5Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural
Resources, Czech University of Life Sciences Prague, Czech Republic
Pavel Hyršl1, Pavel Dobeš1, Libor Vojtek1, Jakub Berka1, Jana Hurychová1, Jaroslav Havlík2,
Martin Kamler2,3, Zuzana Hroncová2, Jiří Killer4, Jan Tyl3, Dalibor Titěra2,5
Plant alkaloid and probiotics increase resistance
of honeybees to nematobacterial infection
Our research is supported by research grants from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047), MUNI/C/1406/2014 and by the Program of „Employment of Newly Graduated Doctors of
Science for Scientific Excellence“ (CZ.1.07/2.3.00/30.0009) co-financed from European Social Fund and the state budget of the Czech Republic.
Bees are used by mankind for several thousand years, but their immune system is still far from
being fully understood and moreover we still don’t have clear idea about all immune
mechanisms, which mediate bees’ response to the pathogens. These pathogens negatively
influence life of the bees and very often even their viability, causing direct impact on agriculture
and industry. Detailed knowledge of bee immunity is crucial for successful fight against bee
diseases.
We successfully used nematode infections in our previous research to study immunity of
Drosophila melanogaster and determined several genes involved in the immune reaction
against nematobacterial complex. We also previously showed that honeybee larvae are
suitable hosts for nematodes and our tripartite model (honeybee, nematodes, bacteria) can be
used as standard procedure for testing honeybees' immune response (Fig. 2). Our current aim
was to optimize the number of nematode infective juveniles (IJ) needed to set the sublethal
dose for honey bee larvae model and subsequently use it to test immune defence of larvae
treated with potentially immuno-modulating substances (plant alkaloid and probiotics).
B
Conclusions
• Bee larvae and pupae can be infected with entomopathogenic nematodes that will enable
detailed studies of their immune response.
• Infection with H. bacteriophora can be visualized using GFP labeled symbiotic bacteria.
• We optimized the natural infection for EPN Heterorhabditis bacteriophora and Steinernema
feltiae; both species show dose dependence infection (higher EPN dose resulted also in
higher amount of invaded parasites).
• As an ideal sub-lethal dose was chosen 10 IJ per larva was selected.
• Bee larvae are more susceptible to the infection than pupae.
• Larvae fed by probiotics and plant alkaloid showed higher resistance against EPN infection
which supports our immuno-stimulating hypothesis.
BODY BARRIERS
HEMOCOEL
EXTERNAL SPACE
Humoral Immunity
Cellular Immunity
invasion
regurgitation of EPB
Barrier Immunity
PRODUCTS OF EPB
• lectins
• toxins
• hydrolytic enzymes
• antibiotics
• pigments
FUNCTION IN INFECTION PROCESS
• lectin-mediated attachment
• disruption of cytoskelet, impaired hemocyte movement
• lysis of hemocytes and tissue cells
• inhibition of phenoloxidase system
• protecting niche from competitive organism
hemocytes
fat body toxicaemia, septicaemia
Entomopathogenic nematodes (EPN, Fig. 1A) Heterorhabditis bacteriophora and Steinernema
feltiae are obligate and lethal insect parasites. These EPN are symbiotically associated with
entomopathogenic bacteria Photorhabdus luminescens or Xenorhabdus bovienii respectively,
creating the highly pathogenic nematobacterial complex that is able to kill the host within 24 to
48 hours. EPN with its bacterial symbionts are able to infect a broad spectrum of insect
species including e.g. larvae of flies, moths (Fig. 1B, C) or bees. Symbiotic bacteria help to
digest host tissues and provide nutrients for themselves and developing nematodes.
Fig. 1 A: Infective juveniles (IJ) of H. bacteriophora with GFP labeled bacteria P. luminescens in the gut.
B, C: Drosophila melanogaster and Galleria mellonella larvae infected by nematobacterial complex
Heterorhabditis–Photorhabdus. Cadavers have the typical coloration caused by pigments produced by symbiotic
bacteria.
A
A B
Entomopathogenic nematodes (EPN)
Infection of honeybee larvae
A
Honeybee immunity
GFP as a tool to track the infection
Material and method
Fig. 2: For successful development within the host, EPN and their symbiotic bacteria must overcome insect
defences including both cellular and humoral immune responses.
Results
Fig. 5: To demonstrate the role of symbiotic bacteria we exchanged the natural symbiont with TT01-GFP
expressing strain. The bacteria are localized in the gut of IJ and cause septicemia after release into the insect body.
Pictures shows infected bee larvae infected in honeycomb under fluorescence (A), uninfected and infected larvae
under day light (B) and fluorescence (C).
control S. feltiae H. bacteriophora
Fig. 3
C
Larvae as well as pupaes were successfully infected by two entomopathogenic nematode
species (H. bacteriophora is more pathogenic for larvae). We observed dose dependence in
mortality (Fig. 6) and as a sublethal dose were chosen 10 IJ per larvae. Results from Petri
dishes confirmed the immuno-stimulating effect of probiotics and plant alkaloid – stronger
effect (up to 30 % decrease of mortality compared to control) was observed against S. feltiae
but even H. bacteriophora showed better defence response (10 % decrease of mortality
compared to control, Fig. 7).
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H. bacteriophora S. feltiae
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Fig. 4: Bee larvae and pupae infected by nematobacterial complex Heterorhabditis–Photorhabdus (A, B).
Melanized wounds caused by nematodes during invasion are visible in the cuticle (C).
B
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Fig. 6: Mortality of larvae (A) and pupae (B) bee larvae is dependent on dose of IJ used for infection. Bees were
infected with nematobacterial complex Heterorhabditis–Photorhabdus and Steinernema-Xenorhabdus.
Fig. 7: Immuno-stimulating effect of plant alkaloid and probiotics on bee larvae using 10 IJ of S. feltiae (A) and H.
bacteriophora (B) per larva, expressed as % of mortality ± SD; n=20 per group; tested in triplicates.
CB
B
B CA
Contact e-mail: [email protected]