1. Exploring cellular and molecular
aspects of the immune system in
Locusta migratoria
Tewodros Firdissa Duressa
Public defense
October 2015
ARENBERG DOCTORAL SCHOOL
FACULTY OF SCIENCE

2. INTRODUCTION
Book of Exodus (Old testament)
Madagascar 2013
National Geographic society ©2015
Locust range

3. INTRODUCTION
Chemical
insecticides
Biocontrol Entomopathogens
Locust pathogen Organism
Metarhizium spp. Fungi
Amphimermis spp. Nematod
H. megidis and St. feltiae Nematod
Nosema maroccams Protozoan
Scello fulgidus Wasp
Tarsonemidae spp Mite
Trichopsidea oestracea Fly
Blaesoxipha spp Fly
Entomopoxviruses Viruses

4. INTRODUCTION
Host defense mechanism
(Immunity)
Pathogen offense mechanism
(Virulence)
Virus
Bacteria
Fungi Insects
Worms

5. INSECT IMMUNITY
Pathogen
recognition
Immune
response
Infection
Injury
Exoskeleton
Innate
Adaptive
PAMP ⇔ PRR

6. INNATE IMMUNITY
Cellular response
Humoral response
- Fat body
- Anti-microbial peptides
- Prophenol oxidase
enzymatic cascade
- Lysozymes
- Coagulation factors
- Hemocytes
- Phagocytic organs
(Hematopoietic organs)

7. CELLULAR IMMUNITY
Hemocytes Phagocytic organ
(Hematopoietic organ)
HPO
Dorsal vessel
Phagocytosis Nodulation Encapsulation
Prohemocyte Plasmatocyte Granulocyte
Coagulocyte Oenocytoids Spherulocyte

8. CELLULAR IMMUNITY
Direct cellular
immune signals
Cytokine based
immune signaling
Hemocoel
⇔
Hemocyte
recruitment

9. CELLULAR IMMUNITY
Infection site
Hemocyte
proliferation
Plasmatocytes
Granulocytes
Plasmatocytes
Prohemocytes
Hematopoietic organHemocoel
⇔

10. CELLULAR IMMUNITY
Hemocoel
0
2
4
6
8
10
0 h 0,5 h 3 h 6 h 24 h 48 h 72 h
Hemocytedensity
(x1000/µL)
NaCl/Pi β-1,3-glucan
?
?
?

11. RESEARCH QUESTIONS
#1. Patterns and sites of hemocyte proliferation following an
infection (circulating hemocytes, hematopoietic organ)?
#3. Identification of cytokines involved in hemocyte recruitment
during infection (cellular response, hemocyte proliferation)?
#2. Development of a cell line (primary cell culture) using
hemocytes or cells of hematopoietic organ?
#4. Functional characterization of locust orthologue of angiotensin
converting enzyme in the locust immune system?
#1. Patterns and sites of hemocyte proliferation following an
infection (circulating hemocytes, hematopoietic organ)?
#3. Identification of cytokines involved in hemocyte
recruitment during infection (cellular response,
hemocyte proliferation)?

12. 5-bromo-2’-
deoxyuridine
(BrdU)
Histone 3
Serine 10
phosphorylation
(H3S10P)
RESULTS: HEMOCYTE PROLIFERATION
A C G T A
| | | | |
T G C A ?

13. 0
5
10
15
20
30h BrdU 30h BrdU +
6h NaCl/Pi
30h BrdU +
6h β-1,3-
glucan
%BrdUpositivehemocytes
P<0.001
DAPI BrdU Merge
Circulating hemocytes
30 h BrdU and 6 h β-1,3-glucan
RESULTS: HEMOCYTE PROLIFERATION
BrdU
BrdU +
β-1,3-glucan
Hemocyte
smear

14. 0.0
0.5
1.0
1.5
2.0
2.5
Naive 3 6
Mitoticindex
Hours post β-1,3-glucan
injection
P< 001
P<0.05H3S10PDAPI
BrdU Merge
RESULTS: HEMOCYTE PROLIFERATION
Hematopoietic organ: 30 h BrdU
DAPI H3S10P MergeBrdUBF
10 µm
10 µm
Circulating hemocytes

15. RESULTS: HEMOCYTE PROLIFERATION
dv
dv
TUNELDAPI
Hematopoietic organ
6 h β-1,3-glucan
0
10
20
30
40
50
4 d (+ 3 d) 6 d (+ 5 d)
%BrdUpositivecells
Time in days
BrdU
BrdU + β-1,3-glucan
HPO
Hematopoietic organ
4 d BrdU
Nodule
footprint
Hematopoietic organ
4 d BrdU and
3 d β-1,3-glucan
Hematopoietic organ 6 h post injection of
S. lutea (left) and C. albicans (right)

16. RESULTS: HEMOCYTE PROLIFERATION
Circulating
hemocytes
Patterns and sites of hemocyte proliferation following an infection
(circulating hemocytes, hematopoietic organ)?
Hemocoel
0
2
4
6
8
10
0 h 0,5 h 3 h 6 h 24 h 48 h 72 h
Hemocytedensity
(x1000/µL)
NaCl/Pi β-1,3-glucan

17. RESEARCH QUESTION #2
Hemocoel
?
Insect cytokines
- Toll ligand (Spaëtzle)
- Unpaired 3
- Vago
- Growth blocking peptide (GBP)
Hemocyte (plasmatocyte)
+GBP
[R/R] x(5-9)Cx2Gx(4-6)Gx(1-2)C[K/R]
GBPN- -CSP
19-30 amino acid residues (≤ 3 kD)

18. Hemolymph
collection (20 mL)
Hemocyte-free
hemolymph
(Serum)
Serum fraction
( > 30 kD)
Serum fraction
( < 30 kD)
Control (-ve)
0.1x
Lm-serum < 30 kD
0.1x 0.1x
> 30 kD
RESULTS: LOCUST GBP IDENTIFICATION
Serum fractionation
(30 kD MWCO filter)

19. RESULTS: LOCUST GBP IDENTIFICATION
0 10 20 30
minutes
0
2
4
mAU(x1000)
0
40
80
%ACN
214nm,4nm (1.00)
High-Performance
Liquid Chromatography (HPLC)
Matrix-Assisted
Laser Desorption/Ionization
Mass Spectrometry
(MALDI MS)
< 30 kD Serum fractionation
Liquid chromatography-mass
spectrometry
(LC-MS/MS)
Intens(a.u.)
m/z
Von Willebrand
factor type C
Pacifastin
Locust GBP
RMIRLGDRCPAGTVQVLG
TCRRVQKVNR

20. RESULTS: LOCUST GBP IDENTIFICATION
Control
Lm GBP (1 nM)
U
S
0
50
100
0.01 0.1 1 10 100 1000
%hemocytespreading
Concentration (nM)
Control Lm GBP
P < 0.05
P < 0.0001

21. Exon Intron
Cytoplasm
Nucleus
RNA
mRNAAAA
Structure of locust GBP encoding gene
Expression profile of locust GBP encoding gene
mRNAAAA
Protein
RESULTS: LOCUST GBP IDENTIFICATION AND CHARACTERIZATION
Hemocoel
ProGBP
GBP

22. RESULTS: LOCUST GBP IDENTIFICATION AND CHARACTERIZATION
-1
0
1
2
HE FB BR VNC GL CA CC FM GT MT OV TE MAG
Relativequantities(Log10)
Immune
system
Central nervous
system
Endocrine
glands
Muscle Digestive
tract
Reproductive
system
**
**
**
****
*
Expression profile of locust GBP encoding gene

23. 0
2
4
6
8
10
0 h 0,5 h 3 h 6 h 24 h 48 h 72 h
Hemocytedensity
(x1000/µL)
NaCl/Pi β-1,3-glucan
Hemocoel
Locust
GBP
RESULTS: LOCUST GBP IDENTIFICATION AND CHARACTERIZATION
Identification of cytokines involved in hemocyte recruitment during infection (cellular response,
hemocyte proliferation)?

24. 0
2
4
6
8
10
0 h 0,5 h 3 h 6 h 24 h 48 h 72 h
Hemocytedensity
(x1000/µL)
NaCl/Pi β-1,3-glucan
FUTURE PROSPECTS
Hemocoel
Locust
GBP
Circulating
hemocytes
Function of locust GBP
- Molecular mechanisms?
- Functional studies
(hemocyte proliferation, cross-talk and energy trade-offs)?
- Solitary vs gregarious locust immunity?
Hemocytes and phagocytic/hematopoietic organ
- Other sources and hematopoietic organ as secondary roles
in hemocyte production?
- Prophylaxis (solitary vs gregarious)?
- Translational research?

25. ACKNOWLEDGMENT
Prof. Roger Huybrechts
Luc Vanden Bosch
Dr. Kurt Boonen
Ria Vanlaer
Prof. Yoichi Hayakawa
Saga University, Japan
Prof. Lieve Van Mellaert, Rega Institute, Leuven
Dr. Heiko Vogel, Max Plank Institute, Germany
Prof. Veerle M. Darras
Prof. Jozef Vanden Broeck
Prof. Lieve Moons
Prof. Dirk C. de Graaf
Dr. Niels Wynant
Dr. Sven Zels
HSV-lab members
Prof. Arckens-lab members
Project students